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Ecdysteroid-Dioxolanes-as-MDR-Modulators-in Cancer

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Inferential analysis, International Global Work in Pharmaceutical, Pharmaceutical Analytics, Technology Transfer: Biotech and Pharmaceutical, Translational Science, tagged Cancer - General, Multi-drug resistance, novel therapeutic approach on December 24, 2013| Leave a Comment »

Larry H Bernstein, MD, Reviewer and Content Advisor
Stephen Williams, PhD, Cancer Editor
http://pharmaceuticalintelligence.com/2013-12-24/larryhbern/
Ecdysteroid-Dioxolanes-as-MDR-Modulators-in Cancer

This article is a presentation on drug research and development in cancer therapeutics
introducing structure activity relationships of a novel class of oncotherapeutic drugs –
ecdysteroid dioxolanes as MDR modulators. Ecdysteroids are the molting hormones
of insects, and they have nonsteroidal activity in mammals. However, they have been
found to have an effect on certain derivatives on the ABCB1 transporter mediated
multidrug resistance (MDR) of a transfected murine leukemia cell line. The following
study focused on the apolar dioxolane derivatives of 20-hydroxyecdysone.

Synthesis and Structure-Activity Relationships of
Novel Ecdysteroid Dioxolanes as MDR Modulators in Cancer

Ana Martins 1,2,†,*, József Csábi 3,†, Attila Balázs 4, Diána Kitka 1,
Leonard Amaral 5, József Molnár 1, András Simon 6, Gábor Tóth 6
and Attila Hunyadi 3,*

1 Department of Medical Microbiology and Immunobiology,
University of Szeged, Szeged Hungary;
2 Unidade de Parasitologia e Microbiologia Médica, Institute of
Hygiene and Tropical Medicine, Universidade Nova de Lisboa,
Lisbon, Portugal
3 Institute of Pharmacognosy, Faculty of Pharmacy, University
of Szeged, Szeged, Hungary;
Ubichem Research Ltd., Budapest, Hungary;
4 Center for Malaria and Other Tropical Diseases (CMDT),
Institute of Hygiene and Tropical Medicine, Universidade Nova
de Lisboa, Lisbon,
Portugal;
5 Department of Inorganic and Analytical Chemistry, Budapest
University of Technology and Economics, Budapest, Hungary;
*correspondence; E-Mails: martins.a@pharm.u-szeged.hu (A.M.);
hunyadi.a@pharm.u-szeged.hu (A.H.);

Molecules 2013, 18, 15255-15275;
http://dx.doi.org/10.3390/molecules181215255

Keywords:
ecdysteroids; 20-hydroxyecdysone; acetonide; dioxolane;
stereochemistry; cancer; multi-drug resistance;
P-glycoprotein; ABCB1 transporter; efflux pump

Abstract:
Ecdysteroids, molting hormones of insects, can exert several mild,

non-hormonal bioactivities in mammals,

including humans. In a previous study, we found a significant effect of

derivatives on the ABCB1 transporter mediated multi-drug resistance

of a transfected murine leukemia cell line. In this paper, we present

a structure-activity relationship study of the apolar dioxolane
derivatives of 20-hydroxyecdysone.

Semi-synthesis and bioactivity of a total of 32 ecdysteroids,
including 20 new compounds, is presented, supplemented

with their complete 1H- and 13C-NMR signal assignment.

1. Introduction

Ecdysteroids represent a large family of steroid hormones that play a crucial role in
arthropods’physiology. The most abundant representative of these compounds,

20-hydroxyecdysone (20E), regulates
- the reproduction,
- embryogenesis,
- diapause and
- molting of arthropods [1].

Their role in plants is still to be fully understood, but it had been suggested that
they have importance in

several plants as defensive agents against non-adapted herbivores [2].

An estimated 5%–6% of the terrestrial plant species

accumulate detectable levels of ecdysteroids, among which
- Ajuga, Serratula and Silene spp.,
containing high amounts of these compounds, are
- good sources of ecdysteroids of herbal origin [3].

Ecdysteroids generally retain the cholesterol-originated side-chain, typically

contain 27–29 carbon atoms and
- are substituted with 4–8 hydroxyl groups.
their A/B ring junction is usually cis, and
- a characteristic 7-en-6-one

(α,β-unsaturated ketone) chromophore group is present in their B-ring [4].

Due to their significantly different structure as compared to the
vertebrate steroid hormones, these compounds

have no hormonal effects in humans [5].

On the other hand, a number of beneficial

metabolic effects are attributed to them [4–6], which has
encouraged the production and worldwide marketing of food supplements,
mainly containing the isolated ecdysteroid compound 20E [6].

In our recent studies, we found that certain ecdysteroid derivatives significantly

decrease the resistance of a multi-drug resistant (MDR) murine leukemia cell line
expressing the human ABCB1 transporter to doxorubicin,
- a chemotherapeutic agent and a
- substrate of the ABCB1 transporter, and

we discussed the possible mechanisms that might be involved in this activity [7].
Based on the observed structure-activity relationships

of the isolated and semi-synthesized ecdysteroids,
20-hydroxyecdysone 2,3;20,22-diacetonide (1)

was chosen as the most promising lead. Although

the acetonide moiety is generally utilized as
a protecting group for vicinal diols
(which needs a strong acidic environment for removal),
and it is also an important structural element of certain drugs,

such as triamcinolone acetonide (

not a pro-drug for triamcinolone but

has),
having

different pharmacological and pharmacokinetical properties [8].
Based on our previous work,

we have synthesized

additional dioxolane derivatives and
thoroughly discussed their structure elucidation and stereochemistry [9].

the study reported herein we present the

synthesis,
structure and
MDR-modulating activity

of 32 ecdysteroid dioxolanes, including 20 new derivatives, and

provide insights on
- their structure-activity relationships

2. Results and Discussion

2.1. Semi-Synthesis

Having a common protecting group of vicinal diols, the acetonide,

32 compounds containing one or two dioxolane rings were
synthesized from 20-hydroxyecysone
with various aldehydes and ketones
- in the presence of phosphomolybdic acid.

A summary of the reactions performed and their product structures are presented
in Figure 1.

Figure 1. Semi-synthetic transformations of 20E
and structures of the products obtained.

Substituents of the reagent oxo-compound (X1/X2 and X3/X4)

typically correspond to R1/R2 and R2/R3, respectively,

except for compounds 18 and 19, where the reagent was methyl-ethyl ketone. C-15
was obtained as a side product in the synthesis of 23.1H- and 13C-NMR data of the
new compounds are presented in Tables 1–3. To facilitate the comparison between

the NMR signals of structurally analogous hydrogen and carbon atoms
- in the different dioxolane compounds,

we applied a special numbering system

for the central atoms (C-28 and C-29) of
- the 2,3- and 20,22-dioxolane structures.

Compounds containing similar number of carbon atoms are presented
in one table, and

compounds with the highest structural similarity are presented
in neighbouring columns.

Table 1. 1H- and 13C-NMR shifts of compounds 4, 6, 22, 30, 33, 9 and 10; in ppm, in methanol-d4.
(go to source)

Table 2. 1H- and 13C-NMR shifts of compounds 16–20 and 26–27; in ppm, in methanol-d4.
(go to source)

Table 3. 1H- and 13C-NMR shifts of compounds 11–14; 31 and 32; in ppm, in methanol-d4.
(go to source)

As published before [9], the 20,22-diol moiety of 20E

is more reactive than the 2,3-diol, probably
due tothe free rotation of the 20,22-bond of 20E that
allows the 20,22-dioxolane ring to form with less strain.

This allowed us to selectively obtain the 20,22-mono-dioxolane derivatives 2–14,
or, depending on the amount of reagent and the reaction time, the

2,3;20,22-bis-homo-dioxolanes 17 and 21–25.

By utilizing the 20,22-monodioxolane ecdysteroids, another aldehyde or ketone

could be coupled to position 2,3, resulting in
several bis-hetero-dioxolane derivatives 26–33.

For this, however, gradually decreasing reactivity with the increase of

the size of the reagent was a limiting factor: larger aldehydes or ketones
(mainly those containing a substituted aromatic ring)
could not be coupled at the 2,3-position.

The 2,3-monodioxolane derivatives also appeared to be present

as minor side-products of the reactions,

and as a consequence of their low amount, only one such compound (C-15) was isolated
and studied. To selectively obtain this kind of a compound (16) in a more reasonable
yield, another, three-step approach was successfully applied:

after protecting the 20,22-diol with phenylboronic acid,
the 2,3-acetonide could be prepared, and
removal of the 20,22 protecting group

afforded the desired 2,3-monoacetonide in a one-pot procedure.

In the case of the reactions with aldehydes or asymmetric ketones, the new

C-28 and C-29 central atoms of the dioxolane rings are stereogenic centers
two possible diastereomers can be formed at both diols.

Their configuration was elucidated by

two-dimensional ROESY or
selective one-dimensional ROESY experiments,

e.g., in the doubly substituted dioxolane derivative 22
(R1 = R4 = n-Bu, R2 = R3 = H) the unambiguous differentiation of the

1H and 13C signals of the two n-butyl groups

was achieved in the following way (see Figure 2).

Figure 2. Stereostructure of 22.

Red arrows indicate the detected ROESY steric proximities, the blue numbers
give the characteristic 1H, and the black numbers the 13C chemical shifts.

Assignment of the H-C(28) atoms (δ = 4.93/105.9 ppm) was supported by

the H-2/C-28 and H-3/C-28 HMBC correlations, and
that of H-C(29) (δ = 4.91/105.6 ppm) by the H-22/C-29 cross peak

The selective ROESY experiment irradiating at 4.93 ppm

showed contacts with the Hα-2 and Hα-3 atoms
proving the α position of the R2 = H atom.

The ROESY response obtained irradiating H = R3 signal (δ = 4.91) on H-22 (δ = 3.64 ppm)

revealed their cis arrangement and the R configuration around C-29.
assignments of the signals of the two n-butyl groups R1 and R4
- were achieved by selective TOCSY experiments
  (irradiation at δ = 4.93 and 4.91, respectively).

In case of the C-28-epimers, typically an approximately 1:1 yield was obtained, and a good
separation was achieved by simple chromatographic methods (see below). On the other hand,
possibly due to steric reasons,

the longer chain of the reagent was highly selective in the α-position
- in the 20,22-dioxolane moiety.

This selectivity was, however, decreased in cases

when larger moieties were present in the reagent,

such as substituted aromatic rings, resulting in the appearance of the other epimers
These epimer pairs (compounds 11-12 and 13-14) required high-performance liquid
chromatography (HPLC) for their successful separation. C-10 was isolated by HPLC
as a minor product from the preparation of C-9; this compound, considering

the vicinal coupling constant of the olefinic hydrogen atoms
(J = 11.8 Hz) contains a Z double bond,

and most likely originated from an impurity in the trans-cinnamic aldehyde reagent used.
C-18 and C-19 were the only cases where one of the dioxolane rings was formed with

the elimination of ethanol instead of water, losing an
ethyl group from the reagent methyl ethyl ketone.

2.2. Anti-Proliferative Effect of Ecdysteroid Derivatives on
PAR and MDR Mouse Lymphoma Cells

The anti-proliferative activity of the derivatives was determined by

incubation of each of the cell lines with

Inhibitory concentrations (IC50) were calculated and are presented in Table 4.

Table 4. IC50 values of the ecdysteroid derivatives and fluorescence activity ratio (FAR)

values determined in presence of 2 and 20 μM of compound. IC50—inhibitory concentration
(concentration of compound that inhibits 50% of cell growth); IC50 values are presented as
the average of 3 independent experiments ± the standard error of the mean (SEM);
*— the compound showed cytotoxicity at this concentration and it was not possible to
calculate the FAR value; FAR values of the positive control verapamil (20.4 μM) and the negative
control DMSO (0.2%) were 5.73 and 0.72, respectively.

As seen from the table, several compounds

exert much lower anti-proliferative activity on the MDR cell line
as compared to the parental one,

while other compounds show similar activities on both cell lines.

2.3. Inhibition of the ABCB1 Pump of MDR Mouse Lymphoma Cells
(Rhodamine 123 Accumulation Assay)

Accumulation of rhodamine 123 by MDR mouse lymphoma cells
was evaluated by flow cytometry

in the presence of the newly described compounds
in order to study their capacity to inhibit the ABCB1 pump and
therefore prevent the efflux of the dye,

which was consequentially retained inside the MDR cell.
Parental mouse lymphoma cells were used as control

for dye retention inside the cell
while MDR cells alone do not retain rhodamine 123 at
the concentration employed.

The efflux pump inhibitor (EPI) verapamil was used as positive control.
All the compounds were dissolved in DMSO, which was also evaluated for

any effect on the retention of the fluorochrome.

DMSO concentration in the assay was 0.2%. For each compound,

the fluorescence activity ratio (FAR), which measures
the amount of rhodamine 123 accumulated by the cell
in presence of the compound was calculated as follows:

FAR = (FLMDRtreated/FLMDRuntreated)/(FLPARtreated/FLPARuntreated) (1)

where FL is the mean of the fluorescence. The obtained results are shown by Table 4.

As seen from the table, the compounds

showed marked differences according to their capacity to inhibit the efflux of rhodamine 123 in this bioassay:

from the practically inactive (compounds 5, 7, 9, 10, 12 and 20) to the very strong (compounds 6, 8, 14 and 25),
various activities were observed.
Most interestingly, these results did not always conform to those obtained from the combination studies, for example,
no significant differences can be observed

between the combination indices of compounds 20 and 25, and
compound 3, very weak in this assay, was able to act in a rather significant synergism with doxorubicin (see below).

These observations seem to support our initial theory, that

these compounds are not or not exclusively acting as EPIs

but other mechanisms may also be involved in their activity [7].2.4. Combination Studies: Effect of Ecdysteroid Derivatives on the Activity of Doxorubicin on MDR Mouse Lymphoma Cells

Effect of the newly synthesized derivatives was evaluated on checkerboard 96-cell plates with different concentrations of
doxorubicin and compound after 48 h of incubation of the cells, similarly to our previous approach [7]. Combination
indices for the different constant ratios of compound vs.doxorubicin were determined by using the CompuSyn software to plot four to five data points to each ratio. CI values were calculated by means of the median-effect equation [10], where

CI < 1, CI = 1, and CI > 1 represent

synergism,
additive effect (i.e., no interaction), and
antagonism, respectively.

The CI values are presented on Table 5. Combination index plots (or Fa-CI plots, where Fa is the fraction affected) were
also generated for each compound using serial deletion analysis in order to determine variability of the data [10]. An example
of Fa-CI plot is given by Figure 3 for compounds 1, 5 and 15.

Figure 3. Fraction affected (Fa) vs. combination index (CI) value plot for compounds 5 and 15, in comparison with the original lead compound 1.

Table 5.

Combination index (CI) values at different drug ratios (compound vs. doxorubicin, respectively) at 50, 75 and 90% of growth inhibition (ED50, ED75
and ED90, respectively); CIavg— weighted average CI value; CIavg = (CI50 + 2CI75 + 3CI90)/6. CI < 1, CI = 1, and CI > 1 represent

synergism,
additivity, and
antagonism, respectively.

Dm, m, and r represent antilog of the x-intercept, slope, and

linear correlation coefficient of the median-effect plot, respectively.

As seen from Table 5, all compounds acted synergistically with doxorubicin and their behavior followed our previous observation,

Error bars represent 95% confidence intervals by means of serial deletion analysis performed with the CompuSyn software.
The 2,3-mono-dioxolane derivative 15 represents significantly

stronger synergism with doxorubicin than the corresponding 20,22-dioxolane derivative 5 at practically all activity levels, and above Fa = 0.7 (which, in case of cancer, matters the most [10])
it is also stronger than compound 1.
- in case of all ecdysteroids there seems to be an “ideal” compound vs. doxorubicin ratio
  - where the strongest synergistic effect occurs.

Based on the variability of the mono-, homo-di- and hetero-di-substituted compounds, as well as

that of the coupled substituents at R1–R4, several novel structure-activity relationships (SARs) were observed.

According to this, we followed our previous approach [7]—for each compound, the strongest activity by

means of the weighted average CI values was primarily considered for comparison,

regardless of the compound vs. doxorubicin ratio where this activity was found.

the 2,3-dioxolane moiety is far more important for a strong activity, than the one at positions 20,22. compound 15, monosubstituted at position 2,3, was the only ecdysteroid derivative that was able to exert a stronger activity at its best ratio than our original lead, the diacetonide
compound 1 (Figure 3).
A very interesting SAR was revealed by comparing the activity of the C-28 and C-29 epimer pairs:
at C-28, the larger substituent needs to take the α‐position (24 vs. 25), while at C-29 the β-position
for a stronger activity (cf. 11 vs. 12 and 13 vs. 14).
As concerns the 20,22-monodioxolanes, increasing the length of the side chains coupled to C-29 lead to a significant increase in the synergistic activity with doxorubicin

till the length of three carbon atoms (compound 3), however a longer alkyl substituent (compound 4)

appeared to be less preferable. Introducing larger aromatic groups did not lead to a breakthrough, although further substituents on the aromatic ring (compounds 11, 13) were able to increase activity as compared to the
case when a non-substituted phenyl group was present (compound 7).Addition of a β-methyl group to C-29 could significantly improve the activity as compared to that of

the 29α-phenyl substituted derivative (cf. 8 vs. 7, respectively).

The observed structure-activity relationships are summarized in Figure 4.

Figure 4. SAR summary for compounds 1–33.

“Greater than” symbols denote stronger synergistic activities, i.e., lower weighted average CI values
when applied together with doxorubicin.

3. Experimental

3.1 General Information

The starting material 20E (90%, originated from the roots of Cyanotis arachnoidea) was purchased
from Shaanxi KingSci Biotechnology Co., Ltd. (Shanghai, China), and further purified by crystallization
from ethyl acetate–methanol (2:1, v/v), so that purity of 20E utilized for the semi-syntheses was 97.8%,
by means of HPLC-DAD, maximum absorbance within the range of 220–400 nm. Mono- and disubstituted
ecdysteroid dioxolanes were synthesized as published before [9]. Briefly, the starting compound was
reacted with the aldehyde or ketone to be coupled to positions 20,22 and/or 2,3 in the presence of
phosphomolybdic acid (Lach-Ner, Neratovice, Czech Republic) at room temp. for 5–60 min depending on the target compound. The reaction was terminated by neutralizing the pH with a 5% aqueous solution of NaHCO3 (Merck, Munich, Germany), methanol was evaporated until only water was present, and the
product(s) were extracted with methylene chloride. Column chromatography (CC), rotational planar
chromatography (RPC) and/or crystallization was used for purification, as detailed below. Solvent system
compositions are given in v/v%. For RPC, a Chromatotron device (Harrison Research, Palo Alto)
was used. The separation was monitored with thin layer chromatography (TLC) on silica gel 60 F254
(0.25 μm, Merck). HPLC purification of compounds 9–14 was performed on a gradient system of two
Jasco PU2080 pumps connected to a Jasco MD-2010 Plus photodiode – array detector, on a Zorbax
XDB-C8 column (5 μm, 9.6 × 250 mm) at a flow rate of 3 mL/min. Mass spectra were recorded on an
API 2000 triple quadrupole tandem mass spectrometer (AB SCIEX, Foster City, CA) in positive mode with
atmospheric pressure chemical ionization (APCI) ion source except for compound 29 which was measured
with electron-spray ionization (ESI). 1H- (500.1) and 13C- (125.6) MHz NMR spectra were recorded at room temperature on an Avance 500 spectrometer (Bruker, Billerica, MA). For the examples of compounds
3, 5, 7, 8, 15, 21, 23–25, 28 and 29, structure elucidation of ecdysteroid dioxolanes by comprehensive one-
and two-dimensional NMR methods has recently been discussed in detail elsewhere, including experimental
details for the aforementioned compounds [9]. Regarding the new compounds, amounts of approximately
1–10 mg were dissolved in 0.1 mL of methanol-d4 and transferred to a 2.5 mm Bruker MATCH NMR sample
tube. Chemical shifts are given on the δ-scale and are referenced to the solvent (MeOH-d4: δC = 49.1 and δH =
3.31 ppm). Pulse programs of all experiments (1H, 13C, DEPTQ, DEPT-135, sel-TOCSY, sel-ROE, sel-NOE,
gradient-selected (gs) 1H, 1H-COSY, edited gs-HSQC, gs-HMBC, ROESY) were taken from the Bruker software
library. Most 1H assignments were accomplished using general knowledge of chemical shift dispersion with
the aid of the proton-proton coupling pattern (1H-NMR spectra).

3.2. Semi-Synthesis and Purification of Monosubstituted Ecdysteroid Dioxolane Derivatives 2–16

20E was dissolved in methanol (10 mL, Merck) to a final concentration of 100 mM or 25 mM in case of compounds
9, 10, 13, 14, and the corresponding reagent (3: butyraldehyde, 4: valeraldehyde, 5: 3-pentanone, 6: methyl isobutyl
ketone, 10 equivalents each; 7: benzaldehyde, 5 g; 8: acetophenone, 6 g; 9, 10: cinnamaldehyde, 11, 12: vanillin, 13, 14:
4-benzyloxybenzaldehyde, 10 equivalents each; 15: 3-pentanone, 100 equivalents; (compound 15 was obtained from the
synthesis of 25, see below) was added to the solution. Phosphomolybdic acid (1.00 g) was added (except in the case of the synthesis of 9 and 10, when 0.50 g were added) and the mixture was stirred at room temp. for 10 min (except for 7: 5 min, 8: 60 min, 15: 30 min). In the case of compound 16, 20E was dissolved in methanol (10 mL) to a final
concentration of 100 mM, and after adding phenylboronic acid (1 equivalent), the mixture was stirred for 30 min.
Acetone (500 equivalents) and phoshomolybdic acid (0.5 g) were added to the mixture, and after 1 h stirring a solution
of NaOH and H2O2 was added in order to remove the phenyl-boronate group. Then, the reaction was worked up as
described above. Compounds 3, 4, 7, 8, a mixture of 9-10, and compounds 15 and 16 were obtained from RPC on silica
gel with appropriate solvent systems of ethyl acetate-ethanol-water (3, 4) or cyclohexane-ethyl acetate (7, 8, 9-10, 15, 16).
The purification of compounds 11-12 and 13-14 started with CC by using solvent systems of ethyl acetate-ethanol-water.
Isomer pairs 9-10, 11-12 and 13-14 were isolated by RP-HPLC (9, 10: 75% CH3OH aq., 3 mL/min; 11, 12: 70% CH3OH
aq., 3 mL/min; 13, 14: 80% CH3OH aq., 3 mL/min). Compounds 2, 5 and 6 were recrystallized from acetonitrile without
chromatographic purification. The yields were:
2 (236.6 mg, 45.43%), 3 (116.2 mg, 21.7%), 4 (142.8 mg, 26.0%), 5 (183.5 mg, 33.4%), 6 (71.9 mg, 25.2%), 7 (292.5 mg, 51.4%),
8 (196.8 mg, 33.8%), 9 (27.0 mg, 18.5%), 10 (13.9 mg, 9.4%), 11 (156.3 mg, 25.4%), 12 (67.0 mg, 10.9%), 13 (67.3 mg, 39.9%),
14 (33.7 mg, 20.0%), 15 (27.4 mg, 5.0%), 16 (13.3 mg, 10.2%).

3.3. Semi-Synthesis and Purification of Disubstituted Ecdysteroid Derivatives 17–25 in One-Step

20E (17–20: 200 mg; 21–25: 480 mg) was dissolved in methyl-ethyl ketone (20 mL, compounds 17–20) or methanol (10 mL)
and the reagent was added to the solution (21: butyraldehyde, 100 equivalents, 22: valeraldehyde, 100 equivalents, 23: 3-pentanone,
100 equivalents, 24, 25: benzaldehyde, 5 g). Phosphomolybdic acid was added (17–20: 20 mg; 21–25: 0.50 g), and the mixture was
stirred at room temperature for 5 (17–20, 24–25) or 30 (21–23) min. The reactions were worked up as described above, and the
products were isolated by RPC using the appropriate n-hexane-acetone (17–20) or cyclohexane-ethyl acetate-ethanol (21–25) solvent
systems. As a side-product of the reaction of 20E with methyl ethyl ketone, 20 was obtained as a 20,22-onodioxolane derivative. The
yields were:
17 (15.5 mg, 6.3%), 18 (4.9 mg, 2.1%), 19 (8.4 mg, 3.6%), 20 (4.46 mg, 2.0%) 21 (242.4 mg, 41.2%), 22 (134.5 mg, 21.8%),
23 (42.3 mg, 6.9%), 24 (36.1 mg, 5.5%), 25 (43.8 mg, 6.7%).

3.4. Semi-Synthesis and Purification of Disubstituted Ecdysteroid Derivatives 26–33 in Two-Steps

Previously obtained 20,22-monosubstituted compounds (2, 20.7 mg; 3, 40.0 mg; 5, 40.7 mg; 6, 50.0 mg; 7, 57.0 mg; 8, 87.3 mg; 2, 104.0 mg)
were dissolved in methyl ethyl ketone (2 mL, 26 and 27) or in methanol (5 mL) and the reagent (28–32: acetone, 500 equivalents; 33: butyraldehyde,
500 equivalents) was added to the solution. Phosphomolybdic acid (26, 27: 20 mg; 28–32: 0.5 g) was added to the solution, and the mixture was
stirred at room temperature for 5 (26, 27) or 60 (28–33) min. The reactions were terminated and the products were purified as described above for
the disubstituted derivatives. The yields were: 26 (5.1 mg, 23.1%), 27 (5.1 mg, 23.1%), 28 (10.9 mg, 25.4%), 29 (15.8 mg, 36.2%), 30 (15.5 mg, 28.9%),
31 (24.8 mg, 40.6%), 32 (38.7 mg, 41.5%), 33 (53.0 mg, 46.2%).

3.5. Further Experimental Data for the New Compounds

(see Archival supplement)

3.6. Preparation of the Compounds for the Bioassays

Each compound was dissolved in 99.5% DMSO (Sigma, Munich, Germany). In each protocol DMSO was always tested
as solvent control and no activity was observed.

3.7. Cell Lines

Two mouse lymphoma cell lines were used in this work: a parental (PAR) cell line, L5178 mouse T-cell lymphoma cells (ECACC
catalog no.87111908, U.S. FDA, Silver Spring, MD); and a multi-drug resistant (MDR) cell line derived from PAR by transfection
with pHa MDR1/A retrovirus [11]. MDR cell line was selected by culturing the infected cells with 60 μg/L colchicine. Both cell
lines were cultured in McCoy’s 5A medium supplemented with 10% heat inactivated horse serum, L-glutamine, and antibiotics
(penicillin and streptomycin) at 37 °C and 5% CO2 atmosphere [12].

Medium, horse serum, and antibiotics were purchased from Difco (Detroit, MI).

3.8. Anti-proliferative Assay

Anti-proliferative activities on PAR and MDR cell lines were performed as described before [7]. Briefly, 6 × 103 cells/well were
incubated with serial dilutions of each compound (n = 3) in McCoy’s 5 A medium for 72 h at 37 °C, 5% CO2. Then, MTT (Sigma) [13]
was added to each well at a final concentration of 0.5 mg/mL per well) and after 4 h of incubation, 100 μL of SDS 10% (Sigma) in
0.01 M HCl was added to each well. Plates were further incubated overnight and optical density at 540 and 630 nm using an ELISA
reader (Multiskan EX, Thermo Labsystem, Milford, MA). Fifty percent inhibitory concentrations (IC50) were calculated using non-linear regression curve fitting of log(inhibitor) vs. response and variable slope with a least squares (ordinary) fit of GraphPad Prism 5
software (GraphPad Software, San Diego, CA,).

3.9. Inhibition of ABCB1 Pump of MDR Mouse Lymphoma Cells (Rhodamine 123 Accumulation Assay)

Inhibition of ABCB1 was evaluated using rhodamine 123, a fluorescent dye, which retention inside the cells was evaluated by flow cytometry (14).
Briefly, 2 × 106 cells/mL were treated with 2 and 20 μM of each compound. After 10 min incubation, rhodamine 123 (Sigma) was added to a final
concentration of 5.2 μM and the samples were incubated at 37 °C in water bath for 20 min. Samples were centrifuged (2,000 rpm, 2 min) and washed
twice with phosphate buffer saline (PBS, Sigma). The final samples were re-suspended in 0.5 mL PBS and its fluorescence measured with a Partec CyFlow flow cytometer (Partec, Münster, Germany). Verapamil (Sanofi-Synthelabo, Budapest, Hungary) at 20.4 μM was used as positive control.

3.10. Combination Assays

The combined activity of doxorubicin (Teva, Budapest, Hungary) and the ecdysteroids was determined using the checkerboard microplate method, as
described before [7]. Briefly, 5 × 104 cells/well were incubated with doxorubicin and the compound to be tested for 48 h at 37 °C under 5% CO2. Cell
viability rate was determined through MTT staining, as described above. The interaction was evaluated using the CompuSyn software (CompuSyn Inc.,
Paramus, NJ) at each constant ratio of compound vs. doxorubicin (M/M), and combination index (CI) values were obtained for 50%, 75%, and 90% of
growth inhibition.

4. Conclusions

In the present study, we have prepared 32 semi-synthetic derivatives of 20-hydroxy- ecdysone,following our previously observed structure-activity relationships on the strong synergistic activity of ecdysteroid dioxolanes with doxorubicin on a murine MDR cancer cell line expressing the human ABCB1
transporter. By utilizing the different reactivity of the 2,3 and 20,22 vicinal diol moieties, various bis-homo- and bis-hetero-dioxolanes were synthesized,
as well as several 20,22- and two 2,3-monodioxolane derivatives. In addition to these, two epimer pairs were also obtained. Twenty compounds are reported for the first time; their chemical structures were thoroughly investigated by comprehensive 1 and 2D-NMR methods, based on which complete signal
assignments are provided. The compounds showed mild to very strong synergistic effects with doxorubicin against the aforementioned MDR cancer cell line, and the diversity of the substituents allowed us to observe several new structure-activity relationships. Among these, the importance of the 2,3-dioxolane substitution and the observations concerning the role of stereochemistry at C-28 and C-29 are the most interesting results. Apparently, ecdysteroids can be engineered to become strong MDR modulators only by decreasing the polarity at the A-ring, while the polar side-chain can be kept, providing the possibility for designing such compounds with a reasonable water solubility and high drug-likeness.

Considering the high importance of the 2,3-dioxolane group in our compounds and the fact that exactly this part is the most sensitive to an acidic environment,
per os application of these compounds requires an appropriate formulation; development of such delivery systems is currently in process, investigation on their activity against MDR cancer xenografts is going to be reported in the near future.

Acknowledgments

The authors acknowledge the support from the European Union co-funded by the European Social Fund (TÁMOP 4.2.2/B-10/1-2010-0012,
TÁMOP 4.2.2.A-11/1/KONV-2012-0035) and the Fundação para a Ciência e a Tecnologia (FCT), Portugal (PEsT-OE/SAU/UI0074/2011). A. Martins was supported by the grant SFRH/BPD/81118/2011, FCT, Portugal. The work presented here was performed within the framework
of COST Action CM1106, Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells. The authors thank Nikoletta Jedlinszki for
the mass spectroscopic measurements, Imre Ocsovszki, supported by the grant TÁMOP-4.2.1/B-09/KONV-2010-0005, for the flow cytometry measurements and Ibolya Hevérné Herke for the semi-synthetic preparation and purification of compounds 17–20.

The authors declare no conflict of interest.

References

1. Karlson, P. Mode of Action of Ecdysones. In Invertebrate Endocrinology and Hormonal Heterophylly; Burdette, W.B., Ed.; Springer:
Berlin/Heidelberg, Germany, 1974; pp. 43–54.

2. Zeleny, J.; Havelka, J.; Sláma, K. Hormonally mediated insect-plant relationships: Arthropod populations associated with ecdysteroid-containing plant, Leuzea carthamoides (Asteraceae). Eur J. Entomol. 1997, 94, 183–198.

3. Dinan, L. A strategy for the identification of ecdysteroid receptor agonists and antagonists from plants. Eur. J. Entomol. 1995, 92, 271–283.

4. Tóth, N.; Hunyadi, A.; Báthori, M.; Zádor, E. Phytoecdysteroids and vitamin D analogues—Similarities in structure and mode of action.
Curr. Med. Chem. 2010, 17, 1974–1994.

5. Báthori, M.; Tóth, N.; Hunyadi, A.; Márki, Á.; Zádor, E. Phytoecdysteroids and anabolic- androgenic steroids. Structure and
effects on humans. Curr. Med. Chem. 2008, 15, 75–91.

6. Dinan, L. The Karlson lecture. Phytoecdysteroids: What use are they? Arch. Arch. Insect Biochem. Physiol. 2009, 72, 126–141.

7. Martins, A.; Tóth, N.; Ványolós, A.; Béni, Z.; Zupkó, I.; Molnár, J.; Báthori, M.; Hunyadi, A. Significant activity of ecdysteroids on
the resistance to doxorubicin in mammalian cancer cells expressing the human ABCB1 transporter J. Med. Chem. 2012, 55, 5034–5043.

8. Möllmann, H.; Rohdewald, P.; Schmidt, E.W.; Salomon, V.; Derendorf, H. Pharmacokinetics of triamcinolone acetonide and its
phosphate ester. Eur. J. Clin. Pharmacol. 1985, 29, 85–89.

9. Balázs, A.; Hunyadi, A.; Csábi, J.; Jedlinszki, N.; Martins, A.; Simon, A.; Tóth, G. 1H- and 13C-NMR investigation of 20-hydroxyecdysone
dioxolane derivatives, a novel group of MDR modulator agents. Magn. Reson. Chem. 2013, 51, 830−836.

10. Chou, T.-C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies.
Pharmacol. Rev. 2006, 58, 621−681.

11. Pastan, I.; Gottesman, M.M.; Ueda, K.; Lovelace, E.; Rutherford, A.V.; Willingham, M.C. A retrovirus carrying an MDR1 cDNA confers
multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc. Natl. Acad. Sci. USA 1988, 85, 4486−4490.

12. Choi, K.; Frommel, T.O.; Stern, R.K.; Perez, C.F.; Kriegler, M.; Tsuruo, T.; Roninson, I.B. Multidrug resistance after retroviral transfer of
the human MDR1 gene correlates with P-glycoprotein density in the plasma membrane and is not affected by cytotoxic selection.

13. Proc. Natl. Acad. Sci. USA 1991, 88, 7386−7390. 13. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63.

Sample Availability: Samples of the compounds 1–33 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article

Archival Supplement

29α-Butyl-20,22-O-methylidene-20-hydroxyecdysone (4): white needle-like crystals; mp 197–199 °C; for 1H- and 13C-NMR data, see
Table 1; APCI-MS, m/z (Irel, %): 549 [M+H]+, 531 [M+H-H2O]+, 445, 427, 409.

29α-I-butyl-29β-methyl-20,22-O-methylidene-20-hydroxyecdysone (6): white needle-like crystals; mp 198–199 °C; for 1H- and 13C-NMR
data, see Table 1; APCI-MS, m/z (Irel, %): 563 [M+H]+, 545 [M+H-H2O]+, 445, 427, 409.

29α-E-ethenylbenzyl-20,22-O-methylidene-20-hydroxyecdysone (9): white solid; mp. 161–163 °C; for 1H- and 13C-NMR data, see Table 1,
in addition to this, the vicinal coupling constant of the olefinic hydrogen atoms (J = 16.0 Hz) proved the E configuration of the double bond;
APCI-MS, m/z (Irel, %): 595 [M+H]+, 577 [M+H-H2O]+, 445, 427, 409.

29α-Z-ethenylbenzyl-20,22-O-methylidene-20-hydroxyecdysone (10): white solid; mp 138–140 °C; for 1H- and 13C-NMR data, see
Table 1; APCI-MS, m/z (Irel, %): 595 [M+H]+, 577 [M+H-H2O]+, 445, 427, 409.

29α-(3-Methoxy-4-hydroxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (11): white solid; mp 163–165 °C; for 1H- and 13C-NMR
data, see Table 3; APCI-MS, m/z (Irel, %): 615 [M+H]+, 597 [M+H-H2O]+, 445, 427, 409.

29β-(3-Methoxy-4-hydroxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (12): white solid; mp 157–159 °C; for 1H- and 13C-NMR
data, see Table 3; APCI-MS, m/z (Irel, %): 615 [M+H]+, 597 [M+H-H2O]+, 445, 427, 409.

29α-(4-Benzyloxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (13): white solid; mp 144–146 °C; for 1H- and 13C-NMR data, see
Table 3; APCI-MS, m/z (Irel, %): 675 [M+H]+, 445, 427, 409.

29β-(4-Benzyloxyphenyl)-20,22-O-methylidene-20-hydroxyecdysone (14): white solid; mp 139–141 °C; for 1H- and 13C-NMR data, see
Table 3; APCI-MS, m/z (Irel, %): 675 [M+H]+, 445, 427, 409.

20-Hydroxyecdysone 2,3-acetonide (16): white solid; mp 124–126 °C; for 1H- and 13C-NMR data, see Table 2; APCI-MS, m/z (Irel, %):
553 [M+H+MeOH]+, 535, 503, 485, 467, 409.

28α,29α-Diethyl-28β,29β-dimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (17): white solid; mp 98–100 °C; for 1H- and
13C-NMR data, see Table 2; APCI-MS, m/z (Irel, %): 589 [M+H]+, 571 [M+H-H2O]+, 499, 481, 409.

28α,29α-Dimethyl-28β-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (18): white solid; mp 99–101 °C; for 1H- and 13C-
NMR data, see Table 2; APCI-MS, m/z (Irel, %): 561 [M+H]+, 543 [M+H-H2O]+, 499, 481, 409.

28β,29β-Dimethyl-29α-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (19): white solid; mp 79–81 °C; for 1H- and 13C-
NMR data, see Table 2; APCI-MS, m/z (Irel, %): 561 [M+H]+, 543 [M+H-H2O]+, 471, 453, 409.

29α-Ethyl-29β-methyl-20,22-O-methylidene-20-hydroxyecdysone (20): white solid; mp 140–142 °C; for 1H- and 13C-NMR data, see
Table 2; APCI-MS, m/z (Irel, %): 535 [M+H]+, 517 [M+H-H2O]+, 445, 427, 409.

28β,29α-Dibutyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (22): transparent crystals; mp 186–187 °C; for 1H- and 13C-NMR
data, see Table 1; APCI-MS, m/z (Irel, %): 617 [M+H]+, 599 [M+H-H2O]+, 513, 495, 409.

28β,29,29-Trimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (26): white solid; mp 100–102 °C; for 1H- and 13C-NMR data,
see Table 2; APCI-MS, m/z (Irel, %): 547 [M+H]+, 517, 499, 467, 409.

28α,29,29-Trimethyl-28β-ethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (27) white solid; mp 360–362 °C; for 1H- and 13C-
NMR data, see Table 2; APCI-MS, m/z (Irel, %): 575 [M+H]+, 557 [M+H-H2O]+, 499, 481, 409.

28,28,29β-Trimethyl-29α-i-buthyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (30): transparent solid; mp 114–115 °C; for 1H-
and 13C-NMR data, see Table 1; APCI-MS, m/z (Irel, %): 603 [M+H]+, 585 [M+H-H2O]+, 485, 467, 409.

28,28-Dimethyl-29α-phenyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (31): white solid; mp 114–117 °C; for 1H- and 13C-NMR
data, see Table 3; APCI-MS, m/z (Irel, %): 641 [M+H+MeOH]+, 623, 517, 485, 467, 409.

28,28,29β-Trimethyl-29α-phenyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (32): white solid; mp 126–128 °C; for 1H- and 13C-
NMR data, see Table 3; APCI-MS, m/z (Irel, %): 623 [M+H]+, 605 [M+H-H2O]+, 485, 467, 409.

28β-Propyl-29,29-dimethyl-2,3;20,22-bis-O-methylidene-20-hydroxyecdysone (33): transparent solid; mp 109–111 °C; for 1H- and 13C-
NMR data, see Table 1; APCI-MS, m/z (Irel, %): 575 [M+H]+, 557 [M+H-H2O]+, 499, 481.

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Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker

Posted in Anemia, Best evidence, Diabetes Mellitus, Folate and B12, Frontiers in Cardiology and Cardiovascular Disorders, Hematology, Hematopoiesis, Inferential analysis, Iron deficiency, Myelodysplasia, Myelofibrosis, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Nephrology, Neutropenia, Neutrophilia, Nutrition, Pharmacotherapy of Cardiovascular Disease, Platelet count disorder, Translational Effectiveness, Translational Science, tagged Aviva Lev-Ari, BNP, CAD, CHF, comorbidities - type 2 DM, Ejection Fraction, Erythropoietin, erythropoietin (EPO), Heart Failure, Hypertension, IV Iron (Fe), Journal of the American College of Cardiology, Larry H. Bernstein, myocardial infarction, N-terminal prohormone of brain natriuretic peptide on December 10, 2013| Leave a Comment »

Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker

Co-Author of the FIRST Article: Larry H. Bernstein, MD, FCAP

Reviewer and Curator of the SECOND and of the THIRD Articles: Larry H. Bernstein, MD, FCAP

and

Article Architecture Curator: Aviva Lev-Ari, PhD, RN

This article presents Advances in the Treatment using Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe) for IMPROVEMENT of Severe and Resistant Congestive Heart Failure and its resultant Anemia. The Leading Biomarker for Congestive Heart Failure is an Independent Predictor identified as an Elevated N-terminal proBNP.

FIRST ARTICLE

Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3, LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6, John F. Heitner, MD, FACC7
1Cardiology Fellow, 2 Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology, Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript) Poster Presentation

SECOND ARTICLE

The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7

http://content.onlinejacc.org/article.aspx?articleid=1127229

THIRD ARTICLE

The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6

http://content.onlinejacc.org/article.aspx?articleid=1126474

Perspective

This THREE article sequence is related by investigations occurring by me, a very skilled cardiologist and his resident, and my premedical student at New York Methodist Hospital-Weill Cornell, in Brooklyn, NY, while a study had earlier been done applying the concordant discovery, which the team in Israel had though was knowledge neglected. There certainly was no interest in the problem of the effect of anemia on the patient with severe congestive heart failure, even though erythropoietin was used widely in patients with end-stage renal disease requiring dialysis, and also for patients with myelofibrosis. The high cost of EPO was only one factor, the other being a guideline to maintain the Hb concentration at or near 11 g/dl – not higher. In the first article, the authors sought to determine whether the amino terminal pro– brain type natriuretic peptide (NT-pro BNP) is affected by anemia, and to determine that they excluded all patients who had renal insufficiency and/or CHF, since these were associated with elevated NT-proBNP. It was already well established that this pro-peptide is secreted by the heart with the action as a urinary sodium retention hormone on the kidney nephron, the result being an increase in blood volume. Perhaps the adaptation would lead to increased stroke volume from increased venous return, but that is not conjectured. However, at equilibrium, one would expect there to be increased red cell production to maintain the cell to plasma volume ratio, thereby, resulting in adequate oxygen exchange to the tissues. Whether that is always possible is uncertain because any reduction in the number of functioning nephrons would make the kidney not fully responsive at the Na+ exchange level, and the NT-pro BNP would rise. This introduces complexity into the investigation, requiring a removal of confounders to establish the effect of anemia.

The other two articles are related studies by the same group in Israel. They surmised that there was evidence that was being ignored as to the effect of anemia, and the treatment of anemia was essential in addition to other treatments. They carried out a randomized trial to determine just that, a benefit to treating the anemia. But they also conjectured that an anemia with a Hb concentration below 12 g/dl has an deleterious effect on the targeted population. Treatment by intermittent transfusions could potentially provide the added oxygen-carrying capacity, but the fractionation of blood, the potential for transfusion-transmitted disease and transfusion-reactions, combined with the need for the blood for traumatic blood loss made EPO a more favorable alternative to packed RBCs. The proof-of-concept is told below. Patients randomized to receive EPO at a lower than standard dose + iron did better.

Introduction

In this article, Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker we provides a summary of three articles on the topic and we shading new light on the role that Anemia Hb < 12 g% plays as a Biomarker for CHF and for

prediction of elevated BNP, known as an indicator for the following Clinical Uses:

Clinical Use

Rule out congestive heart failure (CHF) in symptomatic individuals
Determine prognosis in individuals with CHF or other cardiac disease
Maximize therapy in individuals with heart failure by the use of Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe)

Evaluation of BNP and NT-proBNP Clinical Performance
Study	Sensitivity(%)	Specificity(%)	PPV(%)	NPV(%)
Diagnose impaired LVEF³
BNP	73	77	70	79
NT-proBNP	70	73	61	80
Diagnose LV systolic dysfunction after MI²
BNP	68	69	56	79
NT-proBNP	71	69	56	80
Diagnose LV systolic dysfunction after MI¹²
BNP	94	40	NG	96
NT-proBNP	94	37	NG	96
Prognosis in newly diagnosed heart failure patients: prediction of mortality/survival¹
BNP	98	22	42	94
NT-proBNP	95	37	47	93
Prognosis post myocardial infarction: prediction of mortality²
BNP	86	72	39	96
NT-proBNP	91	72	39	97
Prognosis post myocardial infarction: prediction of heart failure²
BNP	85	73	54	93
NT-proBNP	82	69	50	91
PPV, positive predictive value; NPV, negative predictive value; LVEF, left ventricular ejection fraction; NG, not given.

Reference Range

BNP:	< 100 pg/mL¹³
proBNP, N-terminal:	≤ 300 pg/mL

The NT-proBNP reference range is based on EDTA plasma. Other sample types will produce higher values.

Interpretive Information

Symptomatic patients who present with a BNP or NT-proBNP level within the normal reference range are highly unlikely to have CHF. Conversely, an elevated baseline level indicates the need for further cardiac assessment and indicates the patient is at increased risk for future heart failure and mortality.BNP levels increase with age in the general population, with the highest concentrations seen in those greater than 75 years of age.¹⁴ Heart failure is unlikely in individuals with a BNP level <100 pg/mL and proBNP level ≤300 pg/mL. Heart failure is very likely in individuals with a BNP level >500 pg/mL and proBNP level ≥450 pg/mL who are <50 years of age, or ≥900 pg/mL for patients ≥50 years of age. Patients in between are either hypertensive or have mild ischemic or valvular disease and should be observed closely.¹⁵BNP is increased in CHF, left ventricular hypertrophy, acute myocardial infarction, atrial fibrillation, cardiac amyloidosis, and essential hypertension. Elevations are also observed in right ventricular dysfunction, pulmonary hypertension, acute lung injury, subarachnoid hemorrhage, hypervolemic states, chronic renal failure, and cirrhosis.NT-proBNP levels are increased in CHF, left ventricular dysfunction, myocardial infarction, valvular disease, hypertensive pregnancy, and renal failure, even after hemodialysis.Although levels of BNP and NT-proBNP are similar in normal individuals, NT-proBNP levels are substantially greater than BNP levels in patients with cardiac disease due to increased stability (half-life) of NT-proBNP in circulation. Thus, results from the two tests are not interchangeable.

References

Cowie MR and Mendez GF. BNP and congestive heart failure. Prog Cardiovasc Dis. 2002;44:293-321.
Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin. New neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation. 1998:97:1921-1929.
Hammerer-Lercher A, Neubauer E, Muller S, et al. Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction. Clin Chim Acta. 2001;310:193-197.
McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection of left-ventricular systolic dysfunction. Lancet. 1998;351:9-13.
Mukoyama Y, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans: Evidence for an exquisite dual natriuretic peptide system, ANP and BNP. J Clin Invest. 1991;87:1402-1412.
Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol. 1997;47:287-296.
Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet. 1994;343:440-444.
Kohno M, Horio T, Yokokawa K, et al. Brain natriuretic peptide as a cardiac hormone in essential hypertension. Am J Med. 1992;92:29-34.
Bettencourt P, Ferreira A, Pardal-Oliveira N, et al. Clinical significance of brain natriuretic peptide in patients with postmyocardial infarction. Clin Cardiol. 2000;23:921-927.
Jernberg T, Stridsberg M, Venge P, et al. N-terminal pro brain natriuretic peptide on admission for early risk stratification of patients with chest pain and no ST-segment elevation. J Am Coll Cardiol. 2002;40:437-445.
Richards AM, Troughton RW. Use of natriuretic peptides to guide and monitor heart failure therapy. Clin Chem. 2012;58:62-71.
Pfister R, Scholz M, Wielckens K, et al. The value of natriuretic peptides NT-pro-BNP and BNP for the assessment of left-ventricular volume and function. A prospective study of 150 patients.Dtsch Med Wochenschr. 2002;127:2605-2609.
Siemens ADVIA Centaur^® BNP directional insert; 2003.
Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain natriuretic peptide concentration: impact of age and gender. J Am Coll Cardiol. 2002;40:976-982.
Weber M, Hamm C. Role of B-type natriuretic peptid (BNP) and NT-proBNP in clinical routine.Heart. 2006;92:843-849.

SOURCE

B-type Natriuretic Peptide and proBNP, N-terminal

http://www.questdiagnostics.com/testcenter/testguide.action?dc=TS_BNP_proBNP

FIRST ARTICLE

Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3, LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6, John F. Heitner, MD, FACC7
1Cardiology Fellow, 2 Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology, Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript) Poster Presentation:

Anemia as an Independent Predictor of Elevated N-Terminal proBNP Levels in
Patients without Evidence of Heart Failure and Normal Renal Function.
Haq SA, Alam ME, Bernstein L, Banko LB, Saul BI, Lee LY, Sacchi TJ, Heitner JF.

Table 1. Patient Characteristics

Variable	No Anemia(n=138)	Anemia(n=80)
Median Age (years)	63	76
Men (%)	35	33
Creatinine (mg/dl)	0.96	1.04
Hemoglobin (g/dl)	13.7	10.2
LVEF (%)	67	63
Median NT-proBNP (pg/ml)	321.6	1896.0

A series of slide showing the determination of the representation of normal NT-proBNP range
after removal of patient confounders.

ABSTRACT

Introduction

N-terminal proBNP (NT-proBNP) has emerged as a primary tool for diagnosing congestive heart failure (CHF). Studies have shown that the level of

NT-proBNP is affected by renal insufficiency (RI) and age, independent of the diagnosis of CHF.

There is some suggestion from recent studies that

anemia may also independently affect NT-proBNP levels.

Objective

To assess the affect of anemia on NT-proBNP independent of CHF, RI, and age.

Methods

We evaluated 746 consecutive patients presenting to the Emergency Department (ED) with shortness of breath and underwent evaluation with serum NT-proBNP.

All patients underwent a trans-thoracic echocardiogram (TTE) and clinical evaluation for CHF. Patients were included in this study if they had a normal TTE (normal systolic function, mitral inflow pattern and left ventricular (LV) wall thickness) and no evidence of CHF based on clinical evaluation. Patients were excluded if they had RI (creatinine > 2 mg/dl) or a diagnosis of sepsis. Anemia was defined using the World Health Organization (W.H.O.) definition of

hemoglobin (hgb) < 13 g/dl for males and hgb < 12 g/dl for females.

Results

Of the 746 consecutive patients, 218 patients (138 anemia, 80 no anemia) met the inclusion criteria. There was a markedly significant difference between

NT- proBNP levels based on the W.H.O. diagnosis of anemia.

Patients with anemia had a

mean NT- proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p=0.0001).

There was a markedly

significant difference in patients who had a hgb > 12 (median 295 pg/ml) when compared to
both patients with an hgb of 10.0 to 11.9 (median 2,102 pg/ml; p = 0.0001) and
those with a hgb < 10 (median 2,131 pg/ml; p = 0.001).

Linear regression analysis comparing hgb with log NT-proBNP was statistically significant (r = 0.395; p = 0.0001). MANOVA demonstrated that

elevated NT- proBNP levels in patients with anemia was independent of age.

Conclusion

This study shows that NT-proBNP is associated with anemia independent of CHF, renal insufficiency, sepsis or age.

INTRODUCTION

B-type natriuretic peptide (BNP) is secreted from the myocardium in response to myocyte stretch. 1-2 BNP is released from the myocytes as a 76 aminoacid N-terminal fragment (NT-proBNP) and a 32-amino acid active hormone (BNP). 3 These peptides have emerged as a primary non-invasive modality for the diagnosis of congestive heart failure (CHF). 4- 7 In addition, these peptides have demonstrated prognostic significance in patients with invasive modality for the diagnosis of

congestive heart failure (CHF). 4- 7
heart failure 8-9,
stable coronary artery disease 10, and
in patients with acute coronary syndromes. 11-14

Studies have shown that the level of NT- proBNP is affected by

age and renal insufficiency (RI) independent of the diagnosis of CHF. 15,16

There is some suggestion from the literature that

anemia may also independently affect NT-proBNP levels. 17-20

Willis et al. demonstrated in a cohort of 209 patients without heart failure that anemia was associated with an elevated NT- proBNP. 17 Similarly, in 217 patients undergoing cardiac catheterization, blood samples were drawn from the descending aorta prior to contrast ventriculography for BNP measurements and

anemia was found to be an independent predictor of plasma BNP levels. 18

The objective of this study is to assess the effect of anemia on NT-proBNP independent of CHF, sepsis, age or renal insufficiency.

METHODS

Patient population

The study population consisted of 746 consecutive patients presenting to the emergency room who underwent NT-proBNP evaluation for the evaluation of dyspnea. Transthoracic echocardiogram (TTE) was available on 595 patients. Patients were included in this study if they had a normal TTE, which was defined as normal systolic function (left ventricular ejection fraction [LVEF] > 45%), normal mitral inflow pattern and normal LV wall thickness. CHF was excluded based on thorough clinical evaluation by the emergency department attending and the attending medical physician. Patients with disease states that may affect the NT- proBNP levels were also excluded:

left ventricular systolic dysfunction (LVEF < 45%),
renal insufficiency defined as a creatinine > 2 mg/dl and
sepsis (defined as positive blood cultures with two or more of the following systemic inflammatory response syndrome (SIRS) criteria: heart rate > 90 beats per minute;
body temperature < 36 (96.8 °F) or > 38 °C (100.4 °F);
hyperventilation (high respiratory rate) > 20 breaths per minute or, on blood gas, a PaCO2 less than 32 mm Hg;
white blood cell count < 4000 cells/mm3 or > 12000 cells/mm³ (< 4 x 109 or > 12 x 109 cells/L), or greater than 10% band forms (immature white blood cells). 21

The study population was then divided into two groups, anemic and non- anemic. Anemia was defined using the world health organization (W.H.O.) definition of hemoglobin (hgb) < 13 g/dl for males and < 12 g/dl for females.The data was also analyzed by dividing the patients into three groups based on hgb levels i.e. hgb > 12, hgb 10 to 11.9 and hgb < 10.

Baseline patient data

Patient’s baseline data including age, gender, ethnicity, hemoglobin (hgb), hematocrit (hct), creatinine, NT- proBNP were recorded from the electronic medical record system in our institution. Chemistry results were performed on the Roche Modular System (Indianapolis, IN), with the NT- proBNP done by chemiluminescence assay. The hemogram was performed on the Beckman Coulter GenS. All TTE’s were performed on Sonos 5500 machine. TTE data collected included LVEF, mitral inflow pattern and LV wall thickness assessment.

Statistical analysis

The results are reported in the means with p < 0.05 as the measure of significance for difference between means. Independent Student’s t-tests were done comparing NT proBNP and anemia. Univariate ANOVAs and multivariate ANOVA (MANOVA) with post hoc tests using the Bonferroni method were used to compare NT- proBNP levels with varying ranges of hgb and age using SPSS 13.0 (SPSS, Chicago, IL). A linear regression analysis was performed using SYSTAT. Calculations included Wilks’Lamda, Pillai trace and Hotelling-Lawley trace. A GOLDMineR® plot was constructed to estimate the effects of age and anemia on NT- proBNP levels. The GOLDMineR® effects plot displays the odds-ratios for predicted NT-proBNP elevation versus the predictor values. Unlike the logistic regression, the ordinal regression, which the plot is derived from, can have polychotomous as well as dichotomous values, as is the case for NT-proBNP.

RESULTS

Of the 746 consecutive patients, 218 patients met the inclusion criteria (fig 1). Baseline characteristics of patients are listed in table 1. The median age for anemic patients was 76 years and 63 years for patients without anemia. One third of patients in both groups were men. The mean hemoglobin for

anemic patients was 10.2 g/dl as compared to 13.7 g/dl for non-anemic patients.
The mean LVEF of patients with anemia was 64% as compared to 67% for non-anemic patients.

Based on the WHO definition of anemia, 138 patients were determined to be anemic while 80 patients were diagnosed as non-anemic. There was a markedly significant difference between NT-proBNP levels based on the WHO diagnosis of anemia.

Patients with anemia had a

mean NT-proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p = 0.0001).

Of the 218 patients in the study, 55 patients had a hgb of < 10 g/dl. Analysis using

hgb < 10 g/dl for anemia demonstrated a statistically significant difference in the NT-proBNP values.

Patients with a hgb < 10 g/dl had a mean NT- proBNP of 5,130 pg/ml

compared to 2,882 pg/ml in patients with a hgb of > 10 g/dl (p = 0.01)

The groups were also divided into three separate categories of hgb for subset analysis:

hgb > 12 g/dl,
hgb 10 to 11.9 g/dl and
hgb < 10 g/dl.

There was a markedly significant difference in

the NT- ProBNP levels of patients who had a hgb > 12 g/dl (median 295 pg/ml) when
compared to those with a hgb range of 10.0 g/dl to 11.9 g/dl (median 2,102 pg/ml) (p = 0.0001),

and also a significant difference in

NT- proBNP levels of patients with a hgb > 12 g/dl (median 295 pg/ml) when
compared to a hgb of < 10 g/dl (median 2,131 pg/ml) (p = 0.001).

However, there was no statistically significant difference in NT-proBNP levels of patients with hgb 10 g/dl to 11.9 g/dl

when compared to those with a hgb of < 10 g/dl (p = 1.0).

A scatter plot comparing hgb with log NT-proBNP and fitting of a line to the data by ordinary least squares regression was significant (p = 0.0001) and demonstrated

a correlation between anemia and NT-proBNP levels (r = 0.395) (fig. 2).

MANOVA demonstrated that elevated NT- proBNP levels in patients with anemia was independent of age (Wilks’ Lambda [p = 0.0001]). In addition, using GOLDMineR® plots (figure 3a and 3b) with a combination of age and hb scaled as predictors of elevated NT-proBNP,

both age and hgb were required as independent predictors.

What about the effect of anemia? The GOLDminer analysis of ordinal regression was carried out in a database from which renal insufficiency and CHF were removed. The anemia would appear to have an independent effect on renal insufficiency. Figure 4 is a boxplot comparison of NT – proBNP, the age normalized function NKLog (NT- proBNP)/eGFR formed from taking 1000*Log(NT- proBNP) divided by the MDRD at eGFR exceeding 60 ml/min/m2 and exceeding 30 ml/min/m2. The transformed variable substantially makes the test independent of age and renal function. The boxplot shows the medians, 97.5, 75, 25 and 2.5 percentiles. There appears to be no significance in the NKLog(NT pro-BNP)/MDRD plot. Table II compares the NT-proBNP by WHO criteria at eGFR 45, 60 and 75 ml/mln/m2 using the t-test with unequal variance assumed, and the Kolmogorov-Smirnov test for nonparametric measures of significance. The significance at 60 ml/min/m2 is marginal and nonexistent at 75 ml/min/m2. This suggests that the contribution from renal function at above 60 ml/min2 can be ignored. This is consistent with the findings using the smaller, trimmed database, but there is an interaction between

anemia, and
eGFR at levels below 60 ml/min/m2

DISCUSSION

The findings in this study indicate that

anemia was associated with elevated NT-proBNP levels independent of CHF, renal insufficiency, sepsis or age.

These findings have been demonstrated with NT-proBNP in only one previous study. Wallis et al. demonstrated that after adjusting for age, sex, BMI, GFR, LVH and valvular disease;

only age,
valvular disease and
low hemoglobin

were significantly associated with increased NT-proBNP. 18.

In our study, CHF was excluded based on both a normal TTE and a thorough clinical evaluation. In the only other study directly looking at NT- proBNP levels in anemic patients without heart failure

only 25% of patients had TTEs, with one patient having an LVEF of 40%. 17

BNP, the active molecule released after cleavage along with NT- proBNP, has also been studied in relation to blood hemoglobin levels. 18 In 263 patients undergoing cardiac catheterization blood samples were drawn from the descending aorta prior to contrast ventriculography to determine the value of BNP. Anemia was present in 217 patients. Multivariate linear regression model adjusting for

age,
gender,
body mass index,
history of myocardial infarction,
estimated creatinine clearance, and
LVEF

found hgb to be an independent predictor of BNP levels.

In our study, patients with anemia were slightly older than those without anemia. However, both MANOVA and GOLDMineR® plot demonstrated that

elevated NT-proBNP levels in patients with anemia was independent of age.

Other studies have found that BNP is dependent on renal insufficiency and age. Raymond et al. randomly selected patients to complete questionnaires regarding CHF and

then underwent pulse and blood pressure measurements,
electrocardiogram (ECG),
echocardiography and
blood sampling. 15

A total of 672 subjects were screened and 130 were determined to be normal, defined as

no CHF or ischemic heart disease,
normal LVEF,
no hypertension,
diabetes mellitus,
lung disease, and
not on any cardiovascular drugs.

They found

older age,
increasing dyspnea,
high plasma creatinine and a
LVEF < 45%

to be independently associated with an elevated NT-proBNP plasma level by multiple linear regression analysis. In another study, McCullough et al. evaluated the patients from the Breathing Not Properly Multinational Study

looking at the relationship between BNP and renal function in CHF. 16

Patients were excluded if they were on hemodialysis or had a estimated glomerular filteration rate (eGFR) of < 15 ml/min. They found that the BNP levels correlated significantly with the eGFR, especially in patients without CHF, suggesting

chronic increased blood volume and
increased left ventricular wall tension as a possible cause. 16

Our study was designed to exclude patients with known diseases such as CHF and renal insufficiency in order to demonstrate

the independent effect of anemia on elevated NT-proBNP levels.

The mechanism for elevated NT-proBNP levels in patients with anemia is unknown. Some possible mechanisms that have been reported in the literature include

hemodilution secondary to fluid retention in patients with CHF 18,
decreased oxygen carrying capacity with accompanying tissue hypoxia which
stimulates the cardio-renal compensatory mechanism leading to increased release of NT-proBNP. 17

The findings from our study suggest that

NT-proBNP values should not be interpreted in isolation of hemoglobin levels and
should be integrated with other important clinical findings for the diagnosis of CHF.

Further studies are warranted

to assess the relationship between anemia and plasma natriuretic peptides, and
possibly modify the NT-proBNP cutoff points for diagnosing acutely decompensated CHF in patients with anemia.

CONCLUSION

This study shows that elevated NT-proBNP levels are associated with anemia independent of

CHF,
renal insufficiency,
sepsis and
age.

NT-proBNP levels should be interpreted with caution in patients who have anemia.

REFERENCES

1. Brunea BG, Piazza LA, de Bold AJ. BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria.Am J Physiol 1997; 273:H2678-86.

2. Wiese S, Breyer T, Dragu A, et al. Gene expression of brain natriuretic peptide in isolated atrial and ventricular human myocardium: influence of angiotensin II and diastolic fiber length. Circ 2000; 102:3074-79.

3. de Lemos JA, McGuire DK, Drazner MH. B-type natriuretic peptide in cardiovascular disease. Lancet 2003; 362:316-22.

4. Dao Q, Krishnaswamy P, Kazanegra R, et al. Utility of B-type natriuretic peptide in the diagnosis of congestive heart failure in an urgent care setting. J Am Coll Cardiol 2001; 37:379-85.

5. Morrison LK, Harrison A, Krishnaswamy P, Kazanegra R, Clopton P, Maisel A. Utility of rapid natriuretic peptide assay in differentiating congestive heart failure from lung disease in patients presenting with dyspnea.
J Am Coll Cardiol 2003; 39:202-09.

6. Maisel AS, Krishnaswamy P, Nowak RM, et al. Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002; 347:161-67.

7. Januzzi JL, Camargo CA, Anwaruddin S, et al. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study. Am J Cardiol 2005; 95:948-954.

8. Tsutamoto T, Wada A, Meada K, et al. Attenuation of compensation of endogenous cardiac natriuretic peptide system in chronic heart failure: prognostic role of plasma brain natriuretic peptide concentration in patients with chronic symptomatic left ventricular dysfunction.
Circulation 1997; 96(2): 509-16.

9. Anand IS, Fisher LD, Chiang YT, et al. Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in the Valsartan Heart Failure Trial (Val-HEFT). Circulation 2003; 107:1278-1283.

10. Omland T, Richards AM, Wergeland R and Vik-Mo H. B-type natriuretic peptide and long term survival in patients with stable coronary artery disease.
Am J Cardiol 2005; 95:24-28.

11. Omland T, Aakvaag A, Bonarjee VV. et al. Plasma brain natriuretic peptide as an indicator of left ventricular systolic dysfunction and long term prognosis after acute myocardial infarction. Comparison with plasma atrial natriuretic peptide and N-terminal proatrial natriuretic peptide.
Circulation 1996; 93:1963-1969.

12. de Lemos JA, Morrow DA, Bently JH, et al. The prognostic value of B-type natriuretic peptide in patients with acute coronary syndromes. N Engl J Med 2001; 345:1014-1021.

13. Richards AM, Nicholls MG, Espiner EA, et al. B-type natriuretic peptides and ejection fraction for prognosis after myocardial infarction. Circulation 2003; 107:2786-2792.

14. Sabatine MS, Morrow DA, de Lemos JA, et al. Multimarker approach to risk stratification in non-ST elevation acute coronary syndromes: simultaneous assessment of troponin I, C-reactive protein and B-type natriuretic peptide.
Circulation 2002; 105:1760-1763.

15. Raymond I, Groenning BA, Hildebrandt PR, Nilsson JC, Baumann M, Trawinski J, Pedersen F. The influence of age, sex andother variables on the plasma level of N-terminal pro brain natriureticpeptide in a large sample of the general population. Heart 2003; 89:745-751.

16. McCollough PA, Duc P, Omland T, McCord J, Nowak RM, Hollander JE, et al. B-type natriuretic peptide and renal function in the diagnosis of heartfailure: an analysis from the Breathing Not Properly Multinational Study.
Am J Kidney Dis 2003; 41:571-579.

17. Willis MS, Lee ES, Grenache DG. Effect of anemia on plasma concentrations of NT-proBNP.
Clinica Chim Acta 2005; 358:175-181.

18. Wold Knudsen C, Vik-Mo H, Omland T. Blood hemoglobin is an independent predictor of B-type natriuretic peptide.
Clin Sci 2005; 109:69-74.

19. Tsuji H, Nishino N, Kimura Y, Yamada K, Nukui M, et al. Haemoglobin level influences plasma brain natriuretic peptide concentration. Acta Cardiol 2004;59:527-31.

20. Wu AH, Omland T, Wold KC, McCord J, Nowak RM, et al. Relationship of B-type natriuretic peptide and anemia in patients withand without heart failure: A substudy from the Breathing Not Properly(BNP) Multinational Study.
Am J Hematol 2005; 80:174-80.

22. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, et al. Definitions for sepsis and organ failure and guidelines for theuse of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. Chest. 1992;101(6):1644-55.

Table Legends

Table I. Clinical characteristics of the study population

Table II. Comparison of NT- proBNP means under WHO criteria at different GFR

Table I

Variable	No Anemia(n=80)	Anemia(n=138)
Median age (years)	63	76
Gender
Men (%)	27 (34)	47 (34)
Women (%)	53 (66)	91 (66)
Weight (kg)	82.9	80.1
Chest Pain	21 (26)	3 (2)

Hemoglobin (g/dl)	13.7	10.2
Hematocrit (%)	40.5	30.5
Mean Corpuscular Volume	97	87
Creatinine (mg/dl)	0.99	1.07
Median NT-proBNP (pg/ml)	321	1896

Medical History
HTN (%)	12 (15)	51 (37)
Prior MI (%)	11 (14)	5 (4)
ACS (%)	16 (20)	3 (2)
CAD (%)	2 (1)	3 (2)
DM (%)	18 (22)	11 (8)

Medication
Clopidogrel	58 (72)	15 (11)
Beta Blockers	68 (85)	27 (20)
Ace Inhibitors	45 (56)	18 (13)
Statins	57 (71)	17 (12)
Calcium Channel Blocker	17 (21)	8 (6)

LVEF (%)	67	64

HTN: Hypertension CAD: Coronary Artery Disease
MI: Myocardial Infarction DM: Diabetes Mellitus
ACS: Acute Coronary Syndrome LVEF: Left Ventricular Ejection Fraction

Table II

GFR	WHO	Mean	P (F)	N	NPar
> 45	0	3267	0.022 (4.33)	661
	1	4681
> 60*	0	2593	0.031 (5.11)	456	0.018
	1	4145
> 60r	0	786	0.203 (3.63)	303	0.08
	1	3880
> 75	0	2773	> 0.80	320	0.043
	1	3048

*AF, valve disease and elevated troponin T included
r AF, valve disease and elevated troponin T removed

FIGURE LEGENDS

FIGURE 1. Study population flow chart. (see poster)
FIGURE 2. Relationship between proBNP and hemoglobin. (see above)
FIGURE 3. NT-proBNP levels in relation to anemia (see above)

Supplementary Material

Table based on LatentGOLD Statistical Innovations, Inc., Belmont, MA., 2000: Jeroen Vermunt & Jay Magidson)

4-Cluster Model

Number of cases 408
Number of parameters (Npar) 24

Chi-squared Statistics
Degrees of freedom (df)          71             p-value
L-squared (L²) 80.2033    0.21
X-squared    80.8313 0.20
Cressie-Read 76.6761 0.30
BIC (based on L²) -346.5966
AIC3 (based on L²) -132.7967
CAIC (based on L²) -417.5966

Model for Clusters
Intercept Cluster1 Cluster2 Cluster3 Cluster4 Wald p-value
————– 0.1544 0.1434 0.0115 -0.3093 1.1981 0.75
Cluster Size 0.2870 0.2838 0.2487 0.1805
(across)

LogNTpr
< 1.5 0.0843 0.2457 0.0006 0.0084
1.6-2.5 0.6179 0.6458 0.0709 0.2809
2.5-3.5 0.2848 0.1067 0.5319 0.5883
> 3.5 0.0130 0.0018 0.3966 0.1224

MDRD
> 90 0.1341 0.7919 0.0063 0.6106
61-90 0.6019 0.2040 0.1633 0.3713
41-60 0.2099 0.0041 0.3317 0.0175
< 41 0.0542 0.0001 0.4987 0.0006

age
under 51 0.0668 0.5646 0.0568 0.0954
51-70 0.3462 0.3602 0.3271 0.3880
over 70 0.5870 0.0752 0.6161 0.5166

WHO
No anemia 0.7518 0.6556 0.2041 0.0998
Anemia 0.2482 0.3444 0.7959 0.9002

——— Cluster1 Cluster2 Cluster3 Cluster4
Overall 0.2870 0.2838 0.2487 0.1805
(down)

LogNTpro
< 1.5 0.2492 0.7379 0.0013 0.0116
1.6-2.5 0.4163 0.4243 0.0427 0.1167
2.6-3.5 0.2296 0.0887 0.3723 0.3095
> 3.5 0.0328 0.0023 0.7982 0.1666

MDRD
> 90 0.1001 0.5998 0.0043 0.2958
61-90 0.5198 0.1716 0.1136 0.1950
41-60 0.3860 0.0055 0.5847 0.0238
< 41 0.1205 0.0002 0.8785 0.0008

age
< 51 0.0720 0.7458 0.0910 0.0912
51-70 0.3036 0.3084 0.2013 0.1867
over 70 0.3773 0.0409 0.3633 0.2186

WHO
No anemia 0.4589 0.3957 0.1076 0.0378
Anemia 0.1342 0.1844 0.3742 0.3073

SECOND ARTICLE

The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7

http://content.onlinejacc.org/article.aspx?articleid=1127229

OBJECTIVES

This is a randomized controlled study of anemic patients with severe congestive heart failure (CHF) to assess the effect of correction of the anemia on cardiac and renal function and hospitalization.

BACKGROUND

Although mild anemia occurs frequently in patients with CHF, there is very little information about the effect of correcting it with erythropoietin (EPO) and intravenous iron.

METHODS

Thirty-two patients with moderate to severe CHF (New York Heart Association [NYHA] class III to IV)

who had a left ventricular ejection fraction (LVEF) of 40% despite maximally tolerated doses of CHF medications and

whose hemoglobin (Hb) levels were persistently between 10.0 and 11.5 g% were randomized into two groups.

Group A (16 patients) received subcutaneous EPO and IV iron to increase the level of Hb to at least 12.5 g%. In Group B (16 patients) the anemia was not treated. The doses of all the CHF medications were maintained at the maximally tolerated levels except for oral and intravenous (IV) furosemide, whose doses were increased or decreased according to the clinical need.

RESULTS

Over a mean of 8.2 +/- 2.6 months,

four patients in Group B and none in Group A died of CHF-related illnesses.
The mean NYHA class improved by 42.1% in A and worsened by 11.4% in B.
The LVEF increased by 5.5% in A and decreased by 5.4% in B.
The serum creatinine did not change in A and increased by 28.6% in B.
The need for oral and IV furosemide decreased by 51.3% and 91.3% respectively in A and increased by 28.5% and 28.0% respectively in B.
The number of days spent in hospital compared with the same period of time before entering the study decreased by 79.0% in A and increased by 57.6% in B.

CONCLUSIONS

When anemia in CHF is treated with EPO and IV iron, a marked improvement in cardiac and patient function is seen,

associated with less hospitalization and renal impairment and less need for diuretics. (J Am Coll Cardiol 2001;37:1775– 80)

Anemia of any cause is known to be capable of causing congestive heart failure (CHF) (1). In patients hospitalized with CHF the

mean hemoglobin (Hb) is about 12 g% (2,3),

which is considered the lower limit of normal in adults (4). Thus, anemia appears to be

common in CHF. Recently, in 142 patients in our special CHF outpatient clinic, we found that

as the CHF worsened, the mean Hb concentration decreased, from 13.7 g% in mild CHF (New York Heart Association [NYHA] class I) to 10.9 g% in severe CHF (NYHA 4), and
the prevalence of a Hb 12 g% increased from 9.1% in patients with NYHA 1 to 79.1% in those with NYHA 4 (5).

The Framingham Study has shown that anemia is an

independent risk factor for the production of CHF (6).

Despite this association of CHF with anemia,

its role is not mentioned in the 1999 U.S. guidelines for the diagnosis and treatment of CHF (7), and
many studies consider anemia to be only a rare contributing cause of hospitalization for CHF (8,9).

Recently, we performed a study in which the anemia of severe CHF that was resistant to maximally tolerated doses of standard medications

was corrected with a combination of subcutaneous (sc) erythropoietin (EPO) and intravenous iron (IV Fe) (5).

We have found this combination to be safe, effective and additive

in the correction of the anemia of chronic renal failure (CRF) in both
the predialysis period (10) and the dialysis period (11).

The IV Fe appears to be more effective than oral iron (12,13). In our previous study of CHF patients (5), the treatment resulted in

improved cardiac function,
improved NYHA functional class,
increased glomerular ﬁltration rate,
a marked reduction in the need for diuretics and
a 92% reduction in the hospitalization rate

compared with a similar time period before the intervention. In the light of these positive results, a prospective randomized study was undertaken

to determine the effects of the correction of anemia in severe symptomatic CHF resistant to maximally tolerated CHF medication.

Abbreviations and Acronyms

CABG	coronary artery bypass graft
CHF	congestive heart failure
CRF	chronic renal failure
EPO	erythropoietin
%Fe Sat	percent iron saturation
GFR	glomerular ﬁltration rate
Hb	hemoglobin
Hct	hematocrit
IU	international units
IV	intravenous
LVEF	left ventricular ejection fraction
NYHA	New York Heart Association
PA	pulmonary artery
sc	subcutaneous
SOLVD	Studies Of Left Ventricular Dysfunction

MATERIALS AND METHODS

Patients.Thirty-two patients with CHF were studied. Before the study, the patients were treated for least six months in the CHF clinic with

maximally tolerated doses of angiotensin-converting enzyme inhibitors, the beta-blockers bisoprolol or carvedilol, aldospirone, long-acting nitrates, digoxin and oral and intravenous (IV) furosemide.

In some patients these agents could not be given because of contraindications and in others they had to be stopped because of side effects. Despite this maximal treatment

the patients still had severe CHF (NYHA classIII), with fatigue and/or shortness of breath on even mild exertion or at rest. All had levels of
Hb in the range of 10 to 11.5 g% on at least three consecutive visits over a three-week period.
All had a LVEF of 40%.

Secondary causes of anemia including hypothyroidism, and folic acid and vitamin B12 deﬁciency were ruled out and

there was no clinical evidence of gastrointestinal bleeding.

The patients were randomized consecutively into two groups:

Group A, 16 patients, was treated with sc EPO and IV Fe to achieve a target Hb of at least 12.5 g%.
Group B, 16 patients, did not receive the EPO and IV Fe.

Treatment protocol for correction of anemia.

All patients in Group A received the combination of sc EPO and IV Fe. The EPO was given once a week at a starting dose of 4,000 international units (IU) per week and
the dose was increased to two or three times a week or decreased to once every few weeks as necessary

to achieve and maintain a target Hb of 12.5 g%.

The IV Fe (Venofer-Vifor International, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every two weeks

until the serum ferritin reached 400 g/l or
the %Fe saturation (%Fe Sat is serum iron/total iron binding capacity 100) reached 40% or
the Hb reached 12.5g%.

The IV Fe was then given at longer intervals as needed to maintain these levels.

Investigations.

Visits to the clinic were at two- to three week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before then,

potassium and ferritin and %Fe Sat were performed on every visit.
blood pressure was measured by an electronic device on every visit.
LVEF was measured initially and at four- to six-month intervals by MUGA radioisotope ventriculography.

This technique measures

the amount of blood in the ventricle at the end of systole and at the end of diastole, thus giving
a very accurate assessment of the ejection fraction.

It has been shown to be an accurate and reproducible method of measuring the ejection fraction (14). Hospital records were reviewed at the end of the intervention period to compare

the number of days hospitalized during the study with
the number of days hospitalized during a similar period

when the patients were treated in the CHF clinic before the initial randomization and entry into the study.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study. The mean follow-up for patients was 8.2 +/- 2.7 months (range 5 to 12 months). The study was done with the approval of the local ethics committee.Statistical analysis.

An analysis of variance with repeated measures (over time) was performed to compare the two study groups (control vs. treatment) and

to assess time trend and the interactions between the two factors.
A separate analysis was carried out for each of the outcome parameters.
The Mann-Whitney test was used to compare the change in NYHA class between two groups.

All the statistical analysis was performed by SPSS (version 10).

RESULTS

The mean age in Group A (EPO and Fe) was 75.3 +/- 14.6 years and in group B was 72.2 +/- 9.9 years. There were 11 and 12 men in Groups A and B, respectively.
Before the study the two groups were similar in

cardiac function,
comorbidities,
laboratory investigations and
medications

(Tables 1, 2 and 3), except for IV furosemide (Table 3),

which was higher in the treatment group. The mean NYHA class of Group A before the study was 3.8 0.4 and was 3.5 0.5 in Group B. The contributing factors to CHF in Groups A and B, respectively, are seen in Table 1 and were similar.

Table 1. Medical Conditions and Contributing Factors to Congestive Heart Failure in the 16 Patients Treated for the Anemia and in the 16 Controls

Table 2. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP blood pressure; Fe Sat iron saturation; Hb hemoglobin; IV intravenous; NS not stated; Std Dev. standard deviation.

The main contributing factors to CHF were considered to be

ischemic heart disease (IHD) in 11 and 10 patients respectively,
hypertension in two and two patients,
valvular heart disease in twoand two patients, and
idiopathic cardiomyopathy in one and two patients, respectively.

A signiﬁcant change after treatment was observed in the two groups in the following parameters:

IV furosemide,
days in hospital,
Hb,
ejection fraction,
serum creatinine and
serum ferritin.

In addition, the interaction between the study group and time trend was signiﬁcant in all measurements except for blood pressure and %Fe Sat. This interaction indicates that

the change over time was signiﬁcantly different in the two groups.

Table 3. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

We find in the comparisons of Tables 2 and 3:

before treatment the level of oral furosemide was higher in the control group (136.2 mg/day) compared with the treatment group (132.2 mg/day).
after treatment, while the dose of oral furosemide of the treated patients was reduced to 64.4 mg/day

the dose of the nontreated patients was increased to 175 mg/day.

The same results of improvement in the treated group and deterioration in the control group are expressed in the following parameters:

IV furosemide, days in hospital,
Hb,
ejection fraction and
serum creatinine.

The NYHA class was

3.8 +/- 0.4 before treatment and 2.2 +/- 0.7 after treatment in Group A (delta mean = – 1.6) and
3.5 +/- 0.7 before treatment and 3.9 +/- 0.3 after treatment in Group B. (delta mean = 0.4)

The improvement in NYHA class was signiﬁcantly higher (p < 0.0001) in the treatment group compared with the control group (Table 4).

Table 4. Changes from Baseline to Final New York Heart Association (NYHA) Class
Initial minus ﬁnal

The improvement in NYHA class was statistically higher (p < 0.0001) in the treatment group compared with control.

There were no deaths in Group A and four deaths in Group B.

Case 1: A 71-year-old woman with severe mitral insufﬁciency and severe pulmonary hypertension (a pulmonary artery [PA] pressure of 75 mm Hg) had persistent NYHA 4 CHF and died during mitral valve surgery seven months after onset of the study. She was hospitalized for 21 days in the seven months before randomization and for 28 days during the seven months after randomization.

Case 2:

A 62-year-old man with a longstanding history of hypertension complicated by IHD, coronary artery bypass graft (CABG) and atrial ﬁbrillation had persistent NYHA 4 CHF and a PA pressure of 35 mm Hg, and died from pneumonia and septic shock eight months after onset of the study. He was hospitalized for seven days in the eight months before randomization and for 21 days during the eight months after
randomization.

Case 3:
A 74-year old man with IHD, CABG, chronic obstructive pulmonary disease, a history of heavy smoking and diabetes had persistent NYHA 4 CHF and a PA pressure of 28 mm Hg, and died of pulmonary edema and cardiogenic shock nine months after onset of the study. He was hospitalized for 14 days in the nine months before randomization and for 41 days during the nine months after randomization.

Case 4:
A 74-year-old man with a history of IHD, CABG, diabetes, dyslipidemia, hypertension and atrial ﬁbrillation, had persistent NYHA 4 CHF and a PA pressure of 30 mm Hg, and died of pneumonia and septic shock six months after onset of the study. He was hospitalized for ﬁve days in the six months before randomization and for 16 days during the nine months after randomization.

DISCUSSION

Main ﬁndings.

The main ﬁnding of the present study is that the correction of

even mild anemia in patients with symptoms of very severe CHF despite being on maximally tolerated drug therapy
resulted in a signiﬁcant improvement in their cardiac function and NYHA functional class.

There was also a large

reduction in the number of days of hospitalization compared with a similar period before the intervention.
all this was achieved despite a marked reduction in the dose of oral and IV furosemide.

In the group in whom the anemia was not treated, four patients died during the study. In all four cases

the CHF was unremitting and contributed to the deaths.

In addition, for the group as a whole,

the LVEF, the NYHA class and the renal function worsened.

There was also need for

increased oral and IV furosemide as well as increased hospitalization.

Study limitations.

The major limitations of this study are

the smallness of the sample size and
the fact that randomization and treatment were not done in a blinded fashion.

Nevertheless, the two groups were almost identical in

cardiac, renal and anemia status;
in the types and doses of medication they were taking before and during the intervention and
in the number of hospitalization days before the intervention.

Although the results of this study, like those of our previous uncontrolled study (5), suggest that

anemia may play an important role in the mortality and morbidity of CHF,
a far larger double-blinded controlled study should be carried out to verify our ﬁndings.

Anemia as a risk factor for hospitalization and death in CHF.

Our results are consistent with a recent analysis of 91,316 patients hospitalized with CHF (15). Anemia was found to be a stronger predictor of

the need for early rehospitalization than was hypertension, IHD or the presence of a previous CABG.

A recent analysis of the Studies Of Left Ventricular Dysfunction (SOLVD) (16) showed that

the level of hematocrit (Hct) was an independent risk factor for mortality.

During a mean follow-up of 33 months the mortality was

22%, 27% and 34% for those with a Hct of 40, 35 to 40 and 35 respectively.

The striking response of our patients to

correction of mild anemia suggests that the failing heart may be very susceptible to anemia.

It has, in fact, been found in both animal (17) and human studies (17–19) that

the damaged heart is more vulnerable to anemia and/or ischemia than is the normal heart.

These stimuli may result in a more marked reduction in cardiac function than occurs in the normal heart and may explain why, in our study,

the patients were so resistant to high doses of CHF medications and
responded so well when the anemia was treated.

Our ﬁndings about the importance of anemia in CHF are not surprising when one considers that, in dialysis patients,

anemia has been shown to be associated with an increased prevalence and incidence of CHF (20) and that
correction of anemia in these patients is associated with improved
- cardiac function (21,22),
- less mortality (23,24) and
- fewer hospitalizations (23,25).

Effect of improvement of CHF on CRF.

Congestive heart failure can cause progressive renal failure (26,27). Renal ischemia is found very early on

in patients with cardiac dysfunction (28,29), and
chronic ischemia may cause progression of renal failure (30). Indeed, the development of
CHF in patients with essential hypertension has been found to be one of the most powerful predictors of
the eventual development of end-stage renal disease (31).

Patients who develop CHF after a myocardial infarction experience a

fall in the glomerular ﬁltration rate (GFR) of about 1 ml/min/month if the CHF is not treated (32).

In another recent analysis of the SOLVD study, treating the CHF with

both angiotensin-converting enzyme inhibitors and beta-blockers resulted in better preservation of the renal function than did
angiotensin-converting enzyme inhibitors alone (26),

suggesting that the more aggressive the treatment of the CHF, the better the renal function is preserved. In the present study, as in our previous one (5), we found that the deterioration of GFR was prevented with

successful treatment of the CHF, including correction of the anemia, whereas
the renal function worsened when the CHF remained severe.

All these ﬁndings suggest that early detection and treatment of CHF and systolic and/or diastolic dysfunction from whatever cause could prevent

the deterioration not only of the cardiac function
but of the renal function as well.

This ﬁnding has very broad implications in the prevention of CRF, because most patients with advanced CRF have

either clinical evidence of CHF or at least some degree of systolic dysfunction (33).

Systolic and/or diastolic dysfunction can occur early on in many conditions, such as

essential hypertension (34),
renal disease of any cause (35,36) or
IHD, especially after a myocardial infarction (37).

The early detection and adequate treatment of this cardiac dysfunction, including correction of the anemia, could prevent this cardiorenal insufﬁciency. To detect cardiac dysfunction early on, one would need at least an echocardiogram and MUGA radio-nucleotide ventriculography. These tests should probably be done not only in patients with signs and symptoms of CHF, but in all patients where CHF or systolic and/or diastolic dysfunction are suspected, such as those with a history of heart disease or suggestive changes of ischemia or hypertrophy on the electrocardiogram, or in patients with hypertension or renal disease.

Other positive cardiovascular effects of EPO treatment.

Another possible explanation for the improved cardiac function in this study may be the direct effect that EPO itself has on improving cardiac muscle function (38,39) and myocardial cell growth (39,40) unrelated to its effect of the anemia. In fact EPO may be crucial in the formation of the heart muscle in utero (40). It may also improve endothelial function (41). Erythropoietin may be superior to blood transfusions not only because adverse reactions to EPO are infrequent, but also because

EPO causes the production and release of young cells from the bone marrow into the blood.

These cells have an oxygen dissociation curve that is shifted to the right of the normal curve, causing the release of

much greater amounts of oxygen into the tissues than occurs normally (42).

On the other hand, transfused blood consists of older red cells with an oxygen dissociation curve that is

shifted to the left, causing the release of much less oxygen into the tissues than occurs normally (42).

The combination of IV Fe and EPO. The use of IV Fe along with EPO has been found to have an additive effect,

increasing the Hb even more than would occur with EPO alone while at the same time
allowing the dose of EPO to be reduced (10 –13).
The lower dose of EPO will be cost-saving and also reduce the chances of hypertension developing (43).

We used iron sucrose (Venofer) as our IV Fe medication because, in our experience, it is extremely well tolerated (10,11) and

has not been associated with any serious side effects in more than 1,200 patients over six years.

Implications of treatment of anemia in CHF. The correction of anemia is not a substitute for the well-documented effective therapy of CHF but seems to be an important, if not vital, addition to the therapy. It is surprising, therefore, that judging from the literature on CHF,

such an obvious treatment for improving CHF is so rarely considered.

We believe that correction of the anemia will have an important role to play in

the amelioration of cardiorenal insufﬁciency, and that this improvement will have
signiﬁcant economic implications as well.

Acknowledgments

The authors thank Rina Issaky, Miriam Epstein, Hava Ehrenfeld and Hava Rapaport for their secretarial assistance.
Reprint requests and correspondence: Dr. D. S. Silverberg, Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv, 64239, Israel.

THIRD ARTICLE

The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6

http://content.onlinejacc.org/article.aspx?articleid=1126474

OBJECTIVES

This study evaluated the prevalence and severity of anemia in patients with congestive heart failure (CHF) and

the effect of its correction on cardiac and renal function and hospitalization.

BACKGROUND

The prevalence and signiﬁcance of mild anemia in patients with CHF is uncertain, and the role of erythropoietin with intravenous iron supplementation in treating this anemia is unknown.

METHODS

In a retrospective study, the records of the 142 patients in our CHF clinic were reviewed to ﬁnd

the prevalence and severity of anemia (hemoglobin [Hb]12 g).

In an intervention study, 26 of these patients, despite maximally tolerated therapy of CHF for at least six months, still had had severe CHF and were also anemic. They were treated with

subcutaneous erythropoietin and intravenous iron sufﬁcient to increase the Hb to 12 g%.

The doses of the CHF medications, except for diuretics, were not changed during the intervention period.

RESULTS

The prevalence of anemia in the 142 patients increased with the severity of CHF,

reaching 79.1% in those with New York Heart Association class IV.

In the intervention study, the anemia of the 26 patients was treated for a mean of 7.2 5.5 months.

The mean Hb level and mean left ventricular ejection fraction increased signiﬁcantly.
The mean number of hospitalizations fell by 91.9% compared with a similar period before the study.
The New York Heart Association class fell signiﬁcantly,
as did the doses of oral and intravenous furosemide.
The rate of fall of the glomerular ﬁltration rate slowed with the treatment.

CONCLUSIONS

Anemia is very common in CHF and its successful treatment is associated with a signiﬁcant improvement in

cardiac function,
functional class,
renal function and
in a marked fall in the need for diuretics and hospitalization.

Abbreviations and Acronyms

ACE	Angiotensin-converting enzyme
CHF	congestive heart failure
COPD	chronic obstructive pulmonary disease
CRF	chronic renal failure
CVA	cerebrovascular accident
EPO	erythropoietin
Fe	iron
g%	grams Hb /100 ml blood
GFR	glomerular ﬁltration rate
Hb	hemoglobin
Hct	hematocrit
IV	intravenous
LVEF	left ventricular ejection fraction
LVH	left ventriculr hypertrophy
NYHA	New York Heart Association
%Fe Sat	percent iron saturation
sc	subcutaneous
TNF	tumor becrosis factor
ACE	Angiotensin-converting enzyme
CHF	congestive heart failure
COPD	chronic obstructive pulmonary disease
CRF	chronic renal failure
CVA	cerebrovascular accident
EPO	erythropoietin
Fe	iron
g%	grams Hb /100 ml blood
GFR	glomerular ﬁltration rate
Hb	hemoglobin
Hct	hematocrit
IV	intravenous
LVEF	left ventricular ejection fraction
LVH	left ventriculr hypertrophy
NYHA	New York Heart Association
%Fe Sat	percent iron saturation
sc	subcutaneous
TNF	tumor becrosis factor

The mean hemoglobin (Hb) in patients with congestive heart failure (CHF) is about 12 g Hb per 100 ml blood (g%) (1–3), which is considered to be the lower limit of normal in adult men and postmenopausal women (4). Thus, many patients with CHF are anemic, and

this anemia has been shown to worsen as the severity of the CHF progresses (5,6).

Severe anemia of any cause can produce CHF, and treatment of the anemia can improve it (7). In patients with chronic renal failure (CRF) who are anemic,

treatment of the anemia with erythropoietin (EPO) has improved many of the abnormalities seen in CHF,
reducing left ventricular hypertrophy (LVH) (8 –10),
preventing left ventricular dilation (11) and,
- in those with reduced cardiac function, increasing the left ventricular ejection fraction (LVEF)(8 –10),
- the stroke volume (12) and
- the cardiac output (12).

In view of the high prevalence of anemia in CHF, it is surprising that we could ﬁnd no studies in which EPO was used in the treatment of the anemia of CHF, and the use of EPO is not included in U.S. Public Health Service guide-lines of treatment of the anemia of CHF (13). In fact, anemia has been considered

only a rare contributing factor to the worsening of CHF, estimated as contributing to
no more than 0% to 1.5% of all cases (14 –16).

Perhaps for this reason, recent guidelines for the prevention and treatment of CHF do not mention treatment of anemia at all (17). If successful treatment of anemia could improve cardiac function and patient function in CHF,

this would have profound implications, because,
despite all the advances made in the treatment of CHF, it is still a major and steadily increasing cause of hospitalizations, morbidity and mortality (18 –20).

The purpose of this study is to examine

the prevalence of anemia (Hb 12 g%) in patients with different levels of severity of CHF and
to assess the effect of correction of this anemia in severe CHF patients
resistant to maximally tolerated doses of CHF medication.

A combination of subcutaneous (SC) EPO and intravenous (IV) iron (Fe) was used. We have found this combination to be additive in improving the anemia of CRF (21,22).

METHODS

Patients.

The medical records of the 142 CHF patients being treated in our special outpatient clinic devoted to CHF were reviewed to determine the prevalence and severity of anemia and CRF in these patients. These patients were referred to the clinic either from general practice or from the various wards in the hospital.

Intervention study.

Despite at least six months of treatment in the CHF clinic,

26 of the above patients had persistent, severe CHF (New York Heart Association [NYHA] class III),
had a Hb level of 12 g% and were on
- angiotensin-converting enzyme [ACE] inhibitors, the
- alpha-beta-blocker carvedilol,
- long-acting nitrates,
- digoxin,
- aldactone and
- oral and IV furosemide.

These 26 patients participated in an intervention study. The mean age was 71.76 8.12 years. There were 21 men and 5 women. They all had a

LVEF below 35%,
persistent fatigue and
shortness

of breath on mild to moderate exertion and often at rest, and had
required hospitalizations at least once during their follow-up in the CHF clinic for pulmonary edema.

In 18 of the 26 patients, the CHF was associated with ischemic heart disease either

alone in four patients, or
with hypertension in six,
diabetes in four,
the two together in three, or with
valvular heart disease in one.

Of the remaining eight patients,

four had valvular heart disease alone and
four had essential hypertension alone.

Secondary causes of anemia including

gastrointestinal blood loss (as assessed by history and by three negative stool occult blood examinations),
folic acid and vitamin B12 deﬁciency and
hypothyroidism were ruled out.

Routine gastrointestinal endoscopy was not carried out. The study passed an ethics committee.

Table 1. Initial Characteristics of the 142 Patients With CHF Seen in the CHF Clinic

Age, yearsMale/female, %Associated conditionsDiabetesHypertensionDyslipidemiaSmoking

Main cardiac diagnosis
Ischemic heart disease

Dilated CMP

Valvular heart disease

Hypertension

LVEF, %

Left atrial area (n 15 cm2)

Pulmonary artery pressure (15 mm Hg)

Previous hospitalizations/year

Serum Na, mEq/liter

Serum creatinine, mg%

Hemoglobin, g%

70.1 +/- 11.1

79/21

28%

64%

72%

40%

74%

15%

32.5 +/- 12.2

31.3 +/- 10.3

43.1 +/-14.9

3.2 +/- 1.5

139.8 +/- 4.0

1.6 +/- 1.1

11.9 +/- 1.5

CMP cardiomyopathy; LVEF left ventricular ejection fraction; NYHA New York Heart Association class.

Correction of the anemia.

All patients received the combination of SC EPO and IV Fe. The EPO was given once a week at a starting dose of 2,000 IU per week subcutaneously, and the dose was increased or decreased as necessary to achieve and maintain a target Hb of 12 g%. The IV Fe (Venofer-Vifor International, St. Gallen, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every week until the serum ferritin reached 400 g/liter or the percent Fe saturation (%Fe Sat: serum iron/total iron binding capacity 100) reached 40% or until the Hb reached 12 g%. The IV Fe was then given at longer intervals as needed to maintain these levels.

Medication dose.

Except for oral and IV furosemide therapy, the doses of all the other CHF medications, which were used in the maximum tolerated doses before the intervention, were kept unchanged during the intervention period.

Duration of the study.

The study lasted for a mean of 7.2 5.5 months (range four to 15 months).

Investigations.

Visits were at weekly intervals initially and then at two- to three-week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before the intervention study.

A complete blood count, serum creatinine, serum ferritin and % Fe Sat were performed on every visit.
An electronic device measured the blood pressure on every visit.
The LVEF was measured by a multiple gated ventricular angiography heart scan initially and at four- to six-month intervals.

Hospital records were reviewed to compare the number of hospitalizations during the time the patients were treated for the anemia with the number of hospitalizations

during a similar period of time that they were treated in the CHF clinic
before the anemia was treated.

Clinic records were reviewed to evaluate the types and doses of CHF medications used

before and during the study.

Period of time that they were treated in the CHF clinic before the anemia was treated.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study. The glomerular ﬁltration rate (GFR) was calculated from the serum creatinine by the formula: 1/serum creatinine in mg% x 100 GFR in ml/min. The rate of change of the GFR before and during the intervention period was calculated by comparing the change in GFR per month in the year before the intervention with that during the intervention.

Statistical analysis.

Mean standard deviation was calculated. One-way analysis of variance (ANOVA) was performed to compare parameter levels between the four NYHA groups. Hochberg’s method of multiple comparisons (23) was used for pair-wise comparison between two groups. A p value of less than 0.05 was considered statistically signiﬁcant. In the intervention study, the signiﬁcance of the difference between the initial values and those at the end of the study for the individual parameters in the 26 treated patients was assessed by paired student’s t test; p < 0.05 was considered statistically signiﬁcant. All the statistical analysis was performed by the SPSS program (Version 9, Chicago, Illinois).

RESULTS

CHF: the whole study group.

The clinical, biochemical and hematological characteristics of the 142 patients seen in the clinic are shown in Tables 1 and 2.

Sixty-seven patients (47%) had severe CHF as judged by a NYHA class of IV (Table 2).
Seventy- nine of the 142 patients (55.6%) were anemic (Hb 12 g%).

The mean Hb level fell progressively from 13.73 +/- 0.83 g% in class I NYHA to 10.90 +/- 1.70 g% in class IV NYHA (p 0.01). The percentage of patients with Hb 12 g% increased from 9.1% in class I to 79.1% in class IV.
Fifty eight patients (40.8%) had CRF as deﬁned as a serum creatinine 1.5 mg%. The mean serum creatinine increased from 1.18 +/_ 0.38 mg% in class I NYHA, to 2.0 +/- 1.89 mg% in class IV NYHA, p 0.001. The percentage of patients with an elevated serum creatinine ( 1.5 mg%) increased from 18.2% in class I to 58.2% in class IV.

The mean ejection fraction fell from 37.67 +/- 15.74% in class I to 27.72 +/- 9.68% (p 0.005) in class IV.

Table 2. LVEF and Biochemical and Hematological Parameters by NYHA Class in 142 Patients With CHF

NYHA Class I II III IV Signiﬁcantly Different Pairs*

*p 0.05 at least between the two groups by pair-wise comparison between groups.

†p 0.05 at least between the groups by ANOVA.

No. of patients	11	26	38	67
(total 142) (%)	(7.7)	(18.3)	(26.8)	(47.2)
Hb, g%†	13.73 (0.83)	13.38 (1.26)	11.95 (1.48)	10.90 (1.70)	1–3, 1–4, 2–3, 2–4
Serum creatinine,	1.18	1.22	1.32	2.00	1–2, 1–3, 1–4
mg%†	(0.38)	(0.29)	(0.38)	(1.89)
LVEF, %†	37.67 (15.74)	32.88 (12.41)	32.02 (10.99)	27.72 (9.68)	1–4, 2–4
Hb 12 g%, (%)	1 (9.1)	5 (19.2)	20 (52.6)	53 (79.1)
Serum creatinine	2	5	12	39
1.5 mg%, (%)	(18.2)	(19.2)	(31.6)	(58.2)

The intervention study: medications.

The percentage of patients receiving each CHF medication before and after the intervention period and the reasons for not receiving them are seen in Table 3.

Table 3. Number (%) of the 26 Patients Taking Each Type of Medication Before and During the Intervention Period and the Reason Why the Medication Was Not Used

Medication No. of Patients (%) Reason for Not Receiving the Medications (No. of Patients)
BP blood pressure; CRF chronic renal failure; IV intravenous.

The main reason for not receiving:

1) ACE inhibitors was the presence of reduced renal function;
2) carvedilol was the presence of chronic obstructive pulmonary disease (COPD);
3) nitrates was low blood pressure and aortic stenosis and
4) aldactone was hyperkalemia.

Table 4. Mean Dose of Each Medication Initially and at the End of the Intervention Period in the 26 Patients

No. of Patients    Initial Dose ^ Final Dose^
Carvedilol (mg/day) 20 26.9 15.5 28.8 14.5
Captopril (mg/day) 7 69.6 40.0 70.7 40.4
Enalapril (mg/day) 13 25.7 12.5 26.9 12.6
Digoxin (mg/day)    25 0.10 0.07 0.10 0.07
Aldactone (mg/day) 19 61.2 49.2     59.9 47.1
Long-acting nitrates 23 53.2 13.2 54.1 14.4
Oral furosemide (mg/day) 26 200.9 120.4 78.3 41.3*
IV furosemide (mg/month)     26 164.7 178.9    19.8 47.0*
*p  0.05 at least vs. before by paired Student’s t test.
^ +/-

The mean doses of the medications are shown in Table 4.

The mean dose of oral furosemide was 200.9 +/- 120.4 mg/day before and 78.3 +/- 41.3 mg/day after the intervention (p 0.05). The dose of IV furosemide was 164.7 +/- 19.8, 178.9 mg/month before and 7.0 mg/month after the intervention (p 0.05).

The doses of the other CHF medications were almost identical in the two periods.

Clinical results.

DEATHS.

There were three deaths during the intervention period. An 83-year-old man died after eight months of respiratory failure after many years of COPD, a 65-year-old man at eight months of a CVA with subsequent pneumonia and septic shock and a 70-year-old man at four months of septicemia related to an empyema that developed after aortic valve replacement.

HEMODIALYSIS.

Three patients, a 76-year-old man, an 85-year-old woman and a 72-year-old man, required chronic hemodialysis after six, 16 and 18 months, respectively. The serum creatinines of these three patients at onset of the anemia treatment were 4.2, 3.5 and 3.6 mg%, respectively. All three had improvement in their NYHA status but

their uremia worsened as the renal function deteriorated, demanding the initiation of dialysis.

In no cases, however, was pulmonary congestion an indication for starting dialysis.

Functional results (Table 5).

During the treatment period, the NYHA class fell from a mean of 3.66 +/- 0.47 to 2.66 +/- 0.70 (p 0.05), and

24 had some improvement in their functional class.

The mean LVEF increased from 27.7 +/- 4.8 to 35.4 +/- 7.6% (p 0.001), an increase of 27.8%.

Compared with a similar period of time before the onset of the anemia treatment, the mean number of hospitalizations fell from 2.72 +/- 1.21 to 0.22 +/- 0.65 per patient (p 0.05), a decrease of 91.9%.

No signiﬁcant changes were found in the mean systolic/diastolic blood pressure.

Hematological results (Table 5).

The mean hematocrit (Hct) increased from 30.14 +/- 3.12%) to 35.9 +/- 4.22% (p < 0.001).
The mean Hb increased from 10.16 +/- 0.95 g%) to 12.10 +/- 1.21 g% (p < 0.001).
The mean serum ferritin increased from 177.07 +/- 113.80 g/liter to 346.73 +/- 207.40 g/liter (p 0.005).
The mean serum Fe increased from 60.4 +/- 19.0 g% to 74 +/- .80 20.7 g% (p 0.005).
The mean %Fe Sat increased from 20.05 6.04% to 26.14 =/- 5.23% (p 0.005).
The mean dose of EPO used throughout the treatment period was 5,227 +/- 455 IU per week, and
the mean dose of IV Fe used was 185.1 +/- 57.1 mg per month.

In four of the patients, the target Hb of 12 g% was maintained despite stopping the EPO for at least four months.

Renal results (Table 5).

The changes in serum creatinine were not signiﬁcant. The estimated creatinine clearance fell at a rate of 0.95 + 1.31 ml/min/month before the onset of treatment of the anemia and increased at a rate of 0.85 + 2.77 ml/min/month during the treatment period.

Table 5. The Hematological and Clinical Data of the 26 CHF Patients at Onset and at the End of the Intervention Period

————–                                         Initial ^    Final^
Hematocrit, vol% 30.14 3.12 35.90 4.22*
Hemoglobin, g% 10.16 0.95 2.10 1.21*
Serum ferritin, g/liter 177.07 113.80 346.73 207.40*
Serum iron, g%    60.4 19.0 74.8 20.7*
% iron saturation    20.5 6.04 26.14 5.23*
Serum creatinine, mg% 2.59 0.77 2.73 1.55
LVEF, %    27.7 4.8 35.4 7.6*
No. hospitalizations/patient    2.72 1.21     0.22 0.65*
Systolic BP, mm Hg 127.1 19.4 128.9 26.4
Diastolic BP, mm Hg 73.9 9.9 74.0 12.7
NYHA (0–4) 3.66 0.47 2.66 0.70*
*p  0.05 at least vs before by paired Student’s t test.   ^ +/-
BP  blood pressure; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association.

DISCUSSION

The main ﬁndings in the present study are that anemia is common in CHF patients and becomes progressively more prevalent and severe as CHF progresses. In addition, for patients with resistant CHF, the treatment of the associated anemia causes a marked improvement in their

functional status,
ejection fraction and
GFR.

All these changes were associated with a markedly

The effect of anemia on the ischemic myocardium.

We used the IV Fe together with EPO to avoid the Fe deﬁciency caused by the use of EPO alone (38,39).
The Fe deﬁciency will cause

a resistance to EPO therapy and
increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may increase the blood pressure excessively (41). The IV Fe reduces the dose of EPO needed to correct the anemia, because

the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42).

Oral Fe, however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain why

the blood pressure did not increase signiﬁcantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

The Effect of Anemia Correction on Renal Function.

Congestive heart failure is often associated with some degree of CRF (1–3,27–29), and

this is most likely due to renal vasoconstriction and ischemia (28,29).

When the anemia is treated and the cardiac function improves,

an increase in renal blood ﬂow and glomerular ﬁltration is seen (7,28).

In the present study, renal function decreased as the CHF functional class worsened (Table 2). The rate of deterioration of renal function was slower during the intervention period. Treatment of anemia in CRF has been associated with

a rate of progression of the CRF that is either unchanged (30) or is slowed (31–33).

It is possible, therefore, that adequate treatment of the anemia in CHF may, in the long term, help slow down the progression of CRF.

Possible Adverse Effects of Correction of the Anemia.

There has been concern, in view of the recent Amgen study (34), that correction of the Hct to a mean 42% in hemodialysis patients might increase cardiovascular events in those receiving EPO compared with those maintained at a Hct of 30%. Although there is much uncertainty about how to interpret this study (35), there is a substantial body of evidence that shows

correction of the anemia up to a Hb of 12 g% (Hct 36%) in CRF on dialysis is safe and desirable (35–38), and
results in a reduction in mortality, morbidity and in the number and length of hospitalizations.

The same likely holds true for the anemia of CHF with or without associated CRF. Certainly, our patients’ symptoms were strikingly improved, as was their cardiac function (LVEF) and need for hospitalization and diuretics. It remains to be established

if correction of the anemia up to a normal Hb level of 14 g% might be necessary in order to further improve the patient’s clinical state.

The Role of Fe Deﬁciency and its Treatment in the Anemia of CHF.

We used the IV Fe together with EPO to avoid the Fe deﬁciency caused by the use of EPO alone (38,39). The Fe deﬁciency will cause

a resistance to EPO therapy and increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may

increase the blood pressure excessively (41).

The IV Fe reduces the dose of EPO needed to correct the anemia, because the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42). Oral Fe, however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain

why the blood pressure did not increase signiﬁcantly in any patient.

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Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

Posted in Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Diabetes Mellitus, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Inferential analysis, Metabolomics, Molecular Genetics & Pharmaceutical, Nephrology, Pharmaceutical Analytics, Pharmacologic toxicities, Proteomics, Systemic Inflammatory Response Related Disorders, Translational Effectiveness, Translational Science, tagged cardiovascular complications, Diabetes mellitus type 2, gene expression, Genome Research, genomic drug development, Heart Failure, New England Journal of Medicine, Obesity, Personalized medicine, prediabetes, selective inhibitors, stage 3 & 4 renal disease on December 8, 2013| Leave a Comment »

Larry H Bernstein, MD, FCAP, Reviewer and Curator

http://pharmaceuticalintelligence.com/2013-12-08/larryhbern/Developments-in-the-Genomics-and-Proteomics-of-Type-2-Diabetes-Mellitus-and-Treatment-Targets

Researchers Solve a Mystery about Type 2 Diabetes Drug

AB SCIEX TripleTOF® and QTRAP® technologies support breakthrough medical study.

Published: Friday, November 22, 2013

Researchers from St. Vincent’s Institute of Medical Research in Melbourne, Australia, in collaboration with researchers at McMaster University in Canada, are reportedly the first to discover how the type 2 diabetes drug metformin actually works, providing a molecular understanding that could lead to the development of more effective therapies. Mass spectrometry technologies from AB SCIEX played a critical role in the analysis that led to this breakthrough finding. The research is published in this month’s issue of the journal Nature Medicine.

Doctors have known for decades that metformin helps treat type 2 diabetes. However, questions had lingered for more than 50 years whether this drug, which is available as a generic drug,

worked to lower blood glucose in patients by directly working on the glucose.

People with type 2 diabetes have high blood sugar levels and have trouble converting sugar in their blood into energy because of low levels of insulin. For treating this condition, metformin is considered the most widely prescribed anti-diabetic drug in the world.

Until now, no one had been able to explain adequately how this drug lowers blood sugar. According to this new study, the drug works by reducing harmful fat in the liver. People who take metformin reportedly often have a fatty liver, which is frequently caused by obesity.

“Fat is likely a key trigger for pre-diabetes in humans,” said Professor Bruce Kemp, PhD, the Head of Protein Chemistry and Metabolism at St. Vincent’s Institute of Medical Research. “Our study indicates that

metformin doesn’t directly reduce sugar metabolism, as previously suspected, but instead
reduces fat in the liver, which in turn allows insulin to work effectively.”

The breakthrough in pinning down how the drug functions began with the researchers making

genetic mutations to the genes of two enzymes, ACC1 and ACC2,

in mice, so they could no longer be controlled. What happened next surprised the researchers:

the mice didn’t get fat as expected,

but Associate Professor Gregory Steinberg, PhD at McMaster University noticed that

the mice had fatty livers and a pre-diabetic condition.

Then the researchers put the mice on

a high fat diet and they became fat, while metformin
did not lower the blood sugar levels of the mutant mice.

The findings are expected to help researchers better directly target the condition, which affects over 100 million people around the world, according to published reports. It is also believed that with the mystery of metformin solved, the application of the drug could go beyond just diabetes and potentially be used to treat other medical conditions.

“AB SCIEX mass spectrometry solutions help researchers explore big questions and conduct breakthrough studies, such as this remarkable type 2 diabetes study,” said Rainer Blair, President of AB SCIEX. “In order to understand disease at the molecular level, researchers need the sensitive detection and reproducible quantitation provided by AB SCIEX tools. We enable the research community to solve biological mysteries and rethink the possibilities to transform health.”

For the research conducted by the Australian and Canadian researchers, the analysis at the molecular level was optimized on AB SCIEX instrumentation, including the AB SCIEX TripleTOF® 5600 and the AB SCIEX QTRAP® 5500 system.

The TripleTOF system, with its high-speed, high-quality MS/MS capabilities,

was used for the discovery of key proteins and phosphopeptides.

The QTRAP system, with its high sensitivity MRM (multiple reaction monitoring) capabilities,

was used for quantitation of metabolites, including nucleotides and malonyl-CoA.

Bardoxolone Methyl in Type 2 Diabetes and Stage 4 Chronic Kidney Disease

D de Zeeuw, T Akizawa, P Audhya, GL Bakris, M Chin, ….,and GM Chertow, for the BEACON Trial Investigators

November 9, 2013 http://dx.doi.org/10.1056/NEJMoa1306033

http://www.nejm.org/doi/full/10.1056/NEJMoa1306033

Type 2 diabetes mellitus is the most important cause of progressive chronic kidney disease in the developed and developing worlds. Various therapeutic approaches to slow progression, including

restriction of dietary protein,
glycemic control, and
control of hypertension,

have yielded mixed results.1-3 Several randomized clinical trials have shown that

inhibitors of the renin–angiotensin–aldosterone system significantly reduce the risk of progression,4-6 although
the residual risk remains high.7

None of the new agents tested during the past decade have proved effective in late-stage clinical trials.8-12

Oxidative stress and impaired antioxidant capacity intensify

with the progression of chronic kidney disease.13

In animals with chronic kidney disease,

oxidative stress and inflammation
are associated with impaired activity of the nuclear 1 factor (erythroid-derived 2)–related factor 2 (Nrf2) transcription factor.

The synthetic triterpenoid bardoxolone methyl and its analogues are the most potent known activators of the Nrf2 pathway. Studies involving humans,14 including persons with type 2 diabetes mellitus and stage 3b or 4 chronic kidney disease, have shown that

bardoxolone methyl can reduce the serum creatinine concentration for up to 52 weeks.15

We designed the Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes Mellitus: the Occurrence of Renal Events (BEACON) trial to test the hypothesis that

treatment with bardoxolone methyl reduces the risk of end-stage renal disease (ESRD) or death from cardiovascular causes

among patients with type 2 diabetes mellitus and stage 4 chronic kidney disease.

Methods

Study Design and Oversight

The BEACON trial was a phase 3, randomized, double-blind, parallel-group, international, multicenter trial of

once-daily administration of bardoxolone methyl (at a dose of 20 mg in an amorphous spray-dried dispersion formulation), as compared with placebo.

Participants were receiving background conventional therapy that included

inhibitors of the renin–angiotensin–aldosterone system,
insulin or other hypoglycemic agents, and, when appropriate,
other cardiovascular medications.

The trial design and the characteristics of the trial participants at baseline have been described previously.16,17

Reata Pharmaceuticals sponsored the trial. The trial was jointly designed by employees of the sponsor and the academic investigators who were members of the steering committee. The steering committee, which was led by the academic investigators and included members who were employees of the sponsor, supervised the trial design and operation. An independent data and safety monitoring committee reviewed interim safety data every 90 days or on an ad hoc basis on request. The sponsor collected the trial data and transferred them to independent statisticians at Statistics Collaborative. The sponsor also contracted a second independent statistical group (Axio Research) to support the independent data and safety monitoring committee. The trial protocol was approved by the institutional review board at each participating study site. The protocol and amendments are available with the full text of this article at NEJM.org. The steering committee takes full responsibility for the integrity of the data and the interpretation of the trial results and for the fidelity of the study to the protocol. The first and last authors wrote the first draft of the manuscript. All the members of the steering committee made the decision to submit the manuscript for publication.

Study Population

Briefly, we included adults with

type 2 diabetes mellitus and
an estimated glomerular filtration rate (GFR) of 15 to <30 ml per minute per 1.73 m2 BSA.

Persons with poor glycemic control,
uncontrolled hypertension, or
a recent cardiovascular event (≤12 weeks before randomization) or
New York Heart Association class III or IV heart failure were excluded.

Additional inclusion and exclusion criteria are listed in Table S1 in the Supplementary Appendix, available at NEJM.org. All the patients provided written informed consent.

Randomization and Intervention

Randomization was stratified according to study site with the use of variable-sized blocks. The steering committee, sponsor, investigators, and trial participants were unaware of the group assignments. After randomization,

patients received either bardoxolone methyl or placebo.

The prescription of all other medications was at the discretion of treating physicians, who were encouraged to adhere to published clinical-practice guidelines. Patients underwent event ascertainment and laboratory testing according to the study schema shown in Figure S1 in the Supplementary Appendix. Ambulatory blood-pressure monitoring was performed in a substudy that included 174 patients (8%).

The statistical analysis plan defined the study period as the number of days from randomization to a common study-termination date. In the case of patients who were still receiving the study drug on the termination date, data on vital events were collected for an additional 30 days.

Outcomes

The primary composite outcome was ESRD or death from cardiovascular causes. We defined ESRD as

the need for maintenance dialysis for 12 weeks or more or kidney transplantation.

If a patient died before undergoing dialysis for 12 weeks, the independent events-adjudication committee adjudicated whether the need for dialysis represented ESRD or acute renal failure. Patients who declined dialysis and who subsequently died were categorized as having had ESRD. All ESRD events were adjudicated. Death from cardiovascular causes was defined as death due to either cardiovascular or unknown causes.

The trial had three prespecified secondary outcomes —

first, the change in estimated GFR as calculated with the use of the four-variable Modification of Diet in Renal Disease study equation, with serum creatinine levels calibrated to an isotope-dilution standard for mass spectrometry;
second, hospitalization for heart failure or death due to heart failure; and
third, a composite outcome of nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, or death from cardiovascular causes.

The events-adjudication committee, whose members were unaware of the study assignments, evaluated whether

ESRD events,
cardiovascular events,
neurologic events, and
deaths

met the prespecified criteria for primary and secondary outcomes (described in detail in the Supplementary Appendix).

Statistical Analysis

We calculated that we needed to enroll 2000 patients on the basis of the following assumptions:

a two-sided type I error rate of 5%, an event rate of 24% for the primary composite outcome in the placebo group during the first 2 years of the study,
a hazard ratio of 0.68 (bardoxolone methyl vs. placebo) for the primary composite outcome,
discontinuation of the study drug by 13.5% of the patients in the bardoxolone methyl group each year, and
a 2.5% annual loss to follow-up in each group.

Under these assumptions, if 300 patients had a primary composite outcome, the statistical power would be 85%.

We collected and analyzed all outcome data in accordance with the intention-to-treat principle. We calculated Kaplan–Meier product-limit estimates of

the cumulative incidence of the primary composite outcome.

We computed hazard ratios and 95% confidence intervals with the use of Cox proportional-hazards regression models with adjustment for

the baseline estimated GFR and urinary albumin-to-creatinine ratio.

We performed analogous analyses for secondary time-to-event outcomes. Given the abundance of early adverse events, we also report discrete cumulative incidences at 4 weeks and 52 weeks.

For longitudinal analyses of estimated GFR, we performed mixed-effects regression analyses using

study group,
time,
the interaction of study group with time,
estimated GFR at baseline,
the interaction of baseline estimated GFR with time, and
urinary albumin-to-creatinine ratio as covariates, and
we compared the means between the bardoxolone methyl group and the placebo group.

We adopted similar approaches when examining the effects of treatment on other continuous measures assessed at multiple visits. Since the between-group difference in the primary composite outcome was not significant,

secondary and other outcomes with P values of less than 0.05 were considered to be nominally significant.

Statistical analyses were performed with the use of SAS software, version 9.3 (SAS Institute). Additional details of the statistical analysis are provided in the Supplementary Appendix.

Results

Patients

From June 2011 through September 2012, a total of 2185 patients underwent randomization, including 1545 (71%) in the United States, 334 (15%) in the European Union, 133 (6%) in Australia, 87 (4%) in Canada, 46 (2%) in Israel, and 40 (2%) in Mexico. Figure S2 in the Supplementary Appendix shows the disposition of the study participants.

As shown in Table 1Table 1Baseline Characteristics of the Patients in the Intention-to-Treat Population., the patients were diverse with respect to age, sex, race or ethnic group, and region of origin;

diabetic retinopathy and neuropathy were common conditions among the patients,
as was overt cardiovascular disease.

See Table S2 in the Supplementary Appendix for a more detailed description of the characteristics of the patients at baseline; Figure S3 in the Supplementary Appendix shows the distribution of baseline estimated GFR and urinary albumin-to-creatinine ratio.

Drug Exposure

The median duration of exposure to the study drug was 7 months (interquartile range, 3 to 11) among patients randomly assigned to bardoxolone methyl and

8 months (interquartile range, 5 to 11) among those randomly assigned to placebo.

Figure S4 in the Supplementary Appendix shows the time to discontinuation of the study drug. Table S3 in the Supplementary Appendix shows the reasons that patients discontinued the study drug and the reasons that patients discontinued the study.

The median duration of follow-up was 9 months in both groups.

Outcomes

Primary Composite Outcome

A total of 69 of 1088 patients (6%) randomly assigned to bardoxolone methyl and 69 of 1097 (6%) randomly assigned to placebo had a primary composite outcome (hazard ratio in the bardoxolone methyl group vs. the placebo group, 0.98; 95% confidence interval [CI], 0.70 to 1.37; P=0.92) (Figure 1AFigure 1Kaplan–Meier Plots of the Time to the First Event of the Primary Outcome and Its Components.).

Death from cardiovascular causes occurred in 27 patients randomly assigned to bardoxolone methyl and in 19 randomly assigned to placebo (hazard ratio, 1.44; 95% CI, 0.80 to 2.59; P=0.23) (Figure 1B).
ESRD developed in 43 patients randomly assigned to bardoxolone methyl and in 51 randomly assigned to placebo (hazard ratio, 0.82; 95% CI, 0.55 to 1.24; P=0.35) (Figure 1C).

One patient in each group died from cardiovascular causes after the development of ESRD. The mean (±SD) estimated GFR

before the development of ESRD was 18.1±8.3 ml per minute per 1.73 m^2 in the bardoxolone methyl group and
14.9±4.0 ml per minute per 1.73 m2 in the placebo group.

Secondary Outcomes

During the study period, 96 patients in the bardoxolone methyl group had heart-failure events (93 patients with at least one hospitalization due to heart failure and 3 patients who died from heart failure without hospitalization),

as compared with 55 in the placebo group (55 patients with at least one hospitalization due to heart failure and
no patients who died from heart failure without hospitalization) (hazard ratio, 1.83; 95% CI, 1.32 to 2.55; P<0.001) (Figure 2AFigure 2Kaplan–Meier Plots of the Time to the First Event of the Discrete Secondary Outcomes.).

A total of 139 patients in the bardoxolone methyl group, as compared with 86 in the placebo group, had

a composite outcome event of nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, or death from cardiovascular causes (hazard ratio, 1.71; 95% CI, 1.31 to 2.24; P<0.001) (Figure 2B).

Incidences of Composite Outcomes and Rates of Death from Any Cause

The cumulative incidences of the primary composite outcome and of the two secondary composite outcomes at 4 weeks and at 52 weeks are shown in Table S4 in the Supplementary Appendix. The rates of death from any cause are shown in Figure S5 in the Supplementary Appendix. From the time of randomization to the end of follow-up, 75 patients died: 44 patients in the bardoxolone methyl group and 31 in the placebo group (hazard ratio, 1.47; 95% CI, 0.93 to 2.32; P=0.10). The causes of death are listed in Table S5 in the Supplementary Appendix.

Estimated GFR

Patients randomly assigned to placebo had a significant mean decline in the estimated GFR from the baseline value (−0.9 ml per minute per 1.73 m2; 95% CI, −1.2 to −0.5), whereas those randomly assigned to bardoxolone methyl had a significant mean increase from the baseline value (5.5 ml per minute per 1.73 m2; 95% CI, 5.2 to 5.9). The difference between the two groups was 6.4 ml per minute per 1.73 m2 (95% CI, 5.9 to 6.9; P<0.001) (Figure 3AFigure 3Estimated Glomerular Filtration Rate (GFR), Body Weight, and Urinary Albumin-to-Creatinine Ratio.).

Physiological Variables

Physiological variables are shown in Table S6 in the Supplementary Appendix. The mean body weight remained stable in the placebo group

but declined steadily and substantially in the bardoxolone methyl group (Figure 3B).

There was a significantly smaller decrease from baseline in mean systolic blood pressure in the bardoxolone methyl group than in the placebo group (between-group difference, 1.5 mm Hg [95% CI, 0.5 to 2.5]), and

the mean diastolic blood pressure increased from baseline in the bardoxolone methyl group whereas it decreased in the placebo group (between-group difference, 2.1 mm Hg [95% CI, 1.6 to 2.6]).

Blood-pressure results from the substudy in which ambulatory blood-pressure monitoring was performed were similar in direction but were more pronounced (between-group difference of 7.9 mm Hg [95% CI, 3.8 to 12.0] in systolic blood pressure and 3.2 mm Hg [95% CI, 1.3 to 5.2] in diastolic blood pressure).

Heart rate also increased significantly in the bardoxolone methyl group, as compared with the placebo group (between-group difference, 3.8 beats per minute; 95% CI, 3.2 to 4.4).

Other Laboratory Variables

Data on laboratory variables are shown in Table S7 in the Supplementary Appendix.

The urinary albumin-to-creatinine ratio increased significantly in the bardoxolone methyl group, as compared with the placebo group (Figure 3C).
Serum magnesium, albumin, hemoglobin, and glycated hemoglobin levels decreased significantly in the bardoxolone methyl group, as compared with the placebo group.
The level of B-type natriuretic peptide increased significantly by week 24 in the bardoxolone methyl group, as compared with the placebo group.

Adverse Events

The rates of serious adverse events are summarized in Table 2Table 2Most Commonly Reported Serious Adverse Events in the Intention-to-Treat Population. Serious adverse events occurred more frequently in the bardoxolone methyl group than in the placebo group (717 events in 363 patients vs. 557 events in 295 patients). There were 11 neoplastic events in the bardoxolone methyl group and 10 in placebo group. The most commonly reported adverse events are summarized in Table S8 in the Supplementary Appendix.

Discussion

The current trial was designed to determine whether bardoxolone methyl, an activator of the cytoprotective Nrf2 pathway, would reduce the risk of ESRD

among patients with type 2 diabetes mellitus and stage 4 chronic kidney disease
who were receiving guideline-based conventional therapy.

The trial was terminated early because of safety concerns, driven primarily by an increase in cardiovascular events in the bardoxolone methyl group. Bardoxolone methyl did not lower the risk of ESRD or of death from cardiovascular causes, although too few events occurred during the trial to reliably determine the true effect of the drug on the primary composite outcome.

Given the truncated duration of the trial and the number of adjudicated events (46% of the events planned), and assuming no change in any of the original assumptions, we estimated the conditional power of the trial to be less than 40%. Although patients treated with bardoxolone methyl had a significant increase in the estimated GFR, as compared with those who received placebo,

there was a significantly higher incidence of heart failure and of the composite outcome of nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, or death from cardiovascular causes in the bardoxolone methyl group.
There were numerically more deaths from any cause among patients treated with bardoxolone methyl than among those in the placebo group.

Bardoxolone methyl is among the first orally available antioxidant Nrf2 activators. A small previous study showed that bardoxolone methyl

reduced inflammation and oxidative stress13 and
induced a decline in the serum creatinine level.

In the 52-Week Bardoxolone Methyl Treatment: Renal Function in CKD/Type 2 Diabetes (BEAM) trial,15 227 patients with type 2 diabetes mellitus and an estimated GFR of 20 to 45 ml per minute per 1.73 m2

had a significant increase in the estimated GFR (mean change, 8.2 to 11.4 ml per minute per 1.73 m2, depending on the dose group)
that was sustained over the entire trial period.

Muscle spasms and hypomagnesemia were the most common adverse events;

there was no increase in the rate of heart failure or other cardiovascular events.

The current trial was designed to determine whether the change in estimated GFR that we anticipated on the basis of the results of the BEAM trial would translate into a slower progression toward ESRD. Although in the current trial ESRD developed in fewer patients in the bardoxolone methyl group than in the placebo group, the early termination of the trial precludes conclusion of the effect on ESRD events.

The mechanism linking bardoxolone methyl to heart failure is unknown. Since an excess in heart-failure events was unanticipated, echocardiography was not performed routinely before randomization. Although weight declined significantly in the bardoxolone methyl group, we were unable to determine whether there was loss of body fat, intracellular (skeletal muscle) water, or extracellular (interstitial) water.

The fall in serum albumin and hemoglobin levels may reflect hemodilution caused by fluid retention.

Bardoxolone methyl also increased blood pressure.

An increase in preload due to volume expansion and an increase in afterload (as reflected by increased blood pressure),

coupled with an increase in heart rate,
constitute a potentially potent combination of factors that are likely to precipitate heart failure in an at-risk population.

The rise in the level of B-type natriuretic peptide with bardoxolone methyl

is consistent with an increase in left ventricular wall stress owing to one or more of these mediators or to unrecognized factors such as
impaired diastolic filling of the left ventricle.

After recognizing the initial increase in heart-failure events, the independent data and safety monitoring committee tried to identify

clinical characteristics that were associated with patients who were at elevated risk for heart failure
after the initiation of bardoxolone methyl therapy (with the possibility of modifying eligibility criteria or otherwise altering the trial),

but the committee was unable to do so. Other, noncardiovascular adverse events were also observed more frequently among patients exposed to bardoxolone methyl than among those who received placebo. Whether the effects of Nrf2 activation, or one or more counterregulatory responses, rendered this particular population vulnerable, is unknown. Zoja et al.18 found an increase in albuminuria and blood pressure along with weight loss in Zucker diabetic fatty rats treated with an analogue of bardoxolone methyl; these effects were not observed in other studies in Zucker diabetic fatty rats or other rodent models or in 1-year toxicologic studies in monkeys.19-21

Why were these adverse effects identified in the current trial and not in the BEAM trial?

First, the number of patient-months of drug exposure in the current trial was roughly 10 times that in the BEAM trial.
Second, the population in the present trial had more severe chronic kidney disease than did the population in the BEAM trial.

Observational studies have shown significantly higher rates of death and cardiovascular events, including heart failure,

among patients with stage 4 chronic kidney disease than among patients with stage 3 chronic kidney disease.22

Finally, our trial used an amorphous spray-dried dispersion formulation of bardoxolone methyl at a fixed dose rather than at an adjusted dose. We chose the 20-mg dose and the specific formulation used in the BEACON trial

on the basis of four phase 2 studies of chronic kidney disease (three studies used the crystalline formulation, and one used the amorphous formulation),
a crossover pharmacokinetics study involving humans that used both formulations, and
several studies in animals that used both formulations (Meyer C: personal communication),

to provide an activity and safety profile that was similar to that observed with 75 mg of the crystalline formulation, which was one of the dose levels tested in the BEAM trial.

In conclusion, among patients with type 2 diabetes mellitus and stage 4 chronic kidney disease, bardoxolone methyl did not reduce the risk of the primary composite outcome of ESRD or death from cardiovascular causes. Significantly increased risks of heart failure and of the composite cardiovascular outcome (nonfatal myocardial infarction, nonfatal stroke, hospitalization for heart failure, or death from cardiovascular causes) prompted termination of the trial.

Alto, CA 93034, or at gchertow@stanford.edu.

Investigators in the Bardoxolone Methyl Evaluation in Patients with Chronic Kidney Disease and Type 2 Diabetes Mellitus: the Occurrence of Renal Events (BEACON) trial are listed in the Supplementary Appendix, available at NEJM.org.

Table 1. Baseline Characteristics of the Patients in the Intention-to-Treat Population.

Fig 1. Kaplan–Meier Plots of the Time to the First Event of the Primary Outcome and Its Components.

nejmoa1303154_f1 Kaplan–Meier Plot of Cumulative Probabilities of the Primary and Secondary End Points and Death.

Fig 2. Kaplan–Meier Plots of the Time to the First Event of the Discrete Secondary Outcomes

nejmoa1303154_f2 Kaplan–Meier Plot of Cumulative Probabilities of Acute Kidney Injury and Hyperkalemia

Fig 3. Estimated Glomerular Filtration Rate (GFR), Body Weight, and Urinary Albumin-to-Creatinine Ratio

Table 2 Most Commonly Reported Serious Adverse Events in the Intention-to-Treat Population

References

1 Klahr S, Levey AS, Beck GJ, et al. The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. N Engl J Med 1994;330:877-884

2 The ADVANCE Collaborative Group. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med 2008;358:2560-2572

3 Parving HH, Andersen AR, Smidt UM, Svendsen PA. Early aggressive antihypertensive treatment reduces rate of decline in kidney function in diabetic nephropathy. Lancet 1983;1:1175-1179

4 Brenner BM, Cooper ME, de Zeeuw D, et al. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med 2001;345:861-869

5 Lewis EJ, Hunsicker LG, Clarke WR, et al. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med 2001;345:851-860

6 Parving HH, Lehnert H, Brochner-Mortensen J, Gomis R, Andersen S, Arner P. The effect of irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes. N Engl J Med 2001;345:870-878

7 Heerspink HJ, de Zeeuw D. The kidney in type 2 diabetes therapy. Rev Diabet Stud 2011;8:392-402

8 Pfeffer MA, Burdmann EA, Chen CY, et al. A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease. N Engl J Med 2009;361:2019-2032

9 Parving HH, Brenner BM, McMurray JJ, et al. Cardiorenal end points in a trial of aliskiren for type 2 diabetes. N Engl J Med 2012;367:2204-2213

10 Packham DK, Wolfe R, Reutens AT, et al. Sulodexide fails to demonstrate renoprotection in overt type 2 diabetic nephropathy. J Am Soc Nephrol 2012;23:123-130

Combined Angiotensin Inhibition for the Treatment of Diabetic Nephropathy

Linda F. Fried, M.D., M.P.H., Nicholas Emanuele, M.D., Jane H. Zhang, Ph.D., Mary Brophy, M.D., Todd A. Conner, Pharm.D., William Duckworth, M.D., David J. Leehey, M.D., Peter A. McCullough, M.D., M.P.H., Theresa O’Connor, Ph.D., Paul M. Palevsky, M.D., Robert F. Reilly, M.D., Stephen L. Seliger, M.D., Stuart R. Warren, J.D., Pharm.D., Suzanne Watnick, M.D., Peter Peduzzi, Ph.D., and Peter Guarino, M.P.H., Ph.D. for the VA NEPHRON-D Investigators

N Engl J Med 2013; 369:1892-1903November 14, 2013DOI: 10.1056/NEJMoa1303154

Background

Combination therapy with angiotensin-converting–enzyme (ACE) inhibitors and angiotensin-receptor blockers (ARBs) decreases proteinuria; however, its safety and effect on the progression of kidney disease are uncertain.

Methods

We provided losartan (at a dose of 100 mg per day) to patients with type 2 diabetes, a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 300, and an estimated glomerular filtration rate (GFR) of 30.0 to 89.9 ml per minute per 1.73 m2 of body-surface area and then randomly assigned them to receive lisinopril (at a dose of 10 to 40 mg per day) or placebo. The primary end point was the first occurrence of a change in the estimated GFR (a decline of ≥30 ml per minute per 1.73 m2 if the initial estimated GFR was ≥60 ml per minute per 1.73 m2 or a decline of ≥50% if the initial estimated GFR was <60 ml per minute per 1.73 m2), end-stage renal disease (ESRD), or death. The secondary renal end point was the first occurrence of a decline in the estimated GFR or ESRD. Safety outcomes included mortality, hyperkalemia, and acute kidney injury.

Results

The study was stopped early owing to safety concerns. Among 1448 randomly assigned patients with a median follow-up of 2.2 years, there were 152 primary end-point events in the monotherapy group and 132 in the combination-therapy group (hazard ratio with combination therapy, 0.88; 95% confidence interval [CI], 0.70 to 1.12; P=0.30). A trend toward a benefit from combination therapy with respect to the secondary end point (hazard ratio, 0.78; 95% CI, 0.58 to 1.05; P=0.10) decreased with time (P=0.02 for nonproportionality). There was no benefit with respect to mortality (hazard ratio for death, 1.04; 95% CI, 0.73 to 1.49; P=0.75) or cardiovascular events. Combination therapy increased the risk of hyperkalemia (6.3 events per 100 person-years, vs. 2.6 events per 100 person-years with monotherapy; P<0.001) and acute kidney injury (12.2 vs. 6.7 events per 100 person-years, P<0.001).

Conclusions

Combination therapy with an ACE inhibitor and an ARB was associated with an increased risk of adverse events among patients with diabetic nephropathy. (Funded by the Cooperative Studies Program of the Department of Veterans Affairs Office of Research and Development; VA NEPHRON-D ClinicalTrials.gov number, NCT00555217.)

A complete list of investigators in the Veterans Affairs Nephropathy in Diabetes (VA NEPHRON-D) study is provided in the Supplementary Appendix, available at NEJM.org.

Figure 1 Kaplan–Meier Plot of Cumulative Probabilities of the Primary and Secondary End Points and Death.

Figure 2 Kaplan–Meier Plot of Cumulative Probabilities of Acute Kidney Injury and Hyperkalemia

The End of Dual Therapy with Renin–Angiotensin–Aldosterone System Blockade?

Nov 14, 2013 de Zeeuw D. (Editorial)

N Engl J Med 2013; 369:1960-1962

Treatment aimed at multiple risk factors and specific markers such as glucose level, blood pressure, body weight, cholesterol levels, and albuminuria has been the main focus to slow cardiovascular and renal risk among patients with diabetes. Among the agents used, those that interrupt the renin–angiotensin–aldosterone system (RAAS) have shown protection that extends beyond decreasing blood pressure. In part, these additional effects may be explained by a decrease in albuminuria.1 Therefore, angiotensin-converting–enzyme (ACE) inhibitors and angiotensin II–receptor blockers (ARBs) have become first-choice drugs in patients with diabetes. Despite some success, the residual cardiovascular and renal risk among patients with diabetes remains

Diabetes: Mouse Studies Point to Kinase as Treatment Target

Published: Nov 24, 2013
By Kristina Fiore, Staff Writer, MedPage Today

Targeting a pathway that plays a major role in both hepatic glucose production and insulin sensitivity may eventually help treat type 2 diabetes, researchers reported.
In a series of experiments in mice, researchers found that inhibition of the kinase CaMKII — or even some of its downstream components — lowered blood glucose and insulin levels, Ira Tabas, MD, PhD, of Columbia University Medical Center in New York City, and colleagues reported online in Cell Metabolism.
The pathway is activated by glucagon signaling in the liver, and appears to have roles in both insulin resistance as well as hepatic glucose production in the liver.
In an earlier study, Tabas and colleagues showed that inhibiting the CaMKII pathway lowered hepatic glucose production by suppressing p38-mediated FoxO1 nuclear localization.
In the current study, they found CaMKII inhibition suppresses levels of the pseudo-kinase TRB3 to improve Akt-phosphorylation, thereby improving insulin sensitivity.
Thus this single pathway targets “two cardinal features of type 2 diabetes — hyperglycemia and defective insulin signaling,” the researchers wrote.
“When we realized we had one common pathway that was responsible for these two disparate processes that, in essence, comprises all of type 2 diabetes, we though it would be an ideal target for new drug therapy,” Tabas told MedPage Today.
Tabas and colleagues conducted several experiments to evaluate the CaMKII pathway.
In one experiment in obese mice, they found that

no matter how CaMKII was knocked out, it led to lower blood glucose levels and lower fasting plasma insulin levels in response to a glucose challenge.

The improvements also occurred

when they knocked out downstream processes, including p38 and MAPK-activating protein kinase 2 (MK2).

“Thus liver p38 and MK2, like CaMKII, play an important role in the development of hyperglycemia and hyperinsulinemia in obese mice,” they wrote.
In further analyses, the researchers discovered

deleting or inhibiting any of these three elements ultimately improved insulin-induced Akt-phosphorylation in obese mice —
an important part of improving insulin sensitivity.

And unlike the effects on hepatic glucose production, these changes didn’t occur through effects on FoxO1.
Instead, inhibiting the CaMKII pathway suppressed levels of the pseudo-kinase TRB3, which likely occurred because of suppression of ATF4 —

all of which led to an increase in Akt-phosphorylation and insulin sensitivity.

Indeed, when mice were made to overexpress TRB3, the improvement in phosphorylation disappeared, “indicating that

the suppression of TRB3 by CaMKII deficiency is causally important in the improvement in insulin signaling,” they wrote.

As a result, there “appear to be two separate CaMKII pathways,

one involved in CaMKII-p38-FoxO1 dependent hepatic glucose production, and
the other involved in defective insulin-induced p-Akt,” they wrote.

The findings suggest the possibility of a drug that can target both hyperglycemia and insulin resistance in type 2 diabetes, they said.

Association Between a Genetic Variant Related to Glutamic Acid Metabolism and Coronary Heart Disease in Individuals With Type 2 Diabetes

Lu Qi; Qibin Qi; S Prudente; C Mendonca; F Andreozzi; et al.
JAMA. 2013;310(8):821-828. http://dx.doi.org/10.1001/jama.2013.276305.

Importance

Diabetes is associated with an elevated risk of coronary heart disease (CHD). Previous studies have suggested that the genetic factors predisposing to excess cardiovascular risk may be different in diabetic and nondiabetic individuals.

Objective

To identify genetic determinants of CHD that are specific to patients with diabetes.

Design, Setting, and Participants

We studied 5 independent sets of CHD cases and CHD-negative controls from the Nurses’ Health Study (enrolled in 1976 and followed up through 2008), Health Professionals Follow-up Study (enrolled in 1986 and followed up through 2008), Joslin Heart Study (enrolled in 2001-2008), Gargano Heart Study (enrolled in 2001-2008), and Catanzaro Study (enrolled in 2004-2010). Included were a total of 1517 CHD cases and 2671 CHD-negative controls, all with type 2 diabetes. Results in diabetic patients were compared with those in 737 nondiabetic CHD cases and 1637 nondiabetic CHD-negative controls from the Nurses’ Health Study and Health Professionals Follow-up Study cohorts. Exposures included 2 543 016 common genetic variants occurring throughout the genome.

Main Outcomes and Measures

Coronary heart disease—defined as fatal or nonfatal myocardial infarction, coronary artery bypass grafting, percutaneous transluminal coronary angioplasty, or angiographic evidence of significant stenosis of the coronary arteries.

Results

A variant on chromosome 1q25 (rs10911021) was consistently associated with CHD risk among diabetic participants,

with risk allele frequencies of 0.733 in cases vs 0.679 in controls (odds ratio, 1.36 [95% CI, 1.22-1.51]; P = 2 × 10−8).

No association between this variant and CHD was detected among nondiabetic participants, with risk allele frequencies of 0.697 in cases vs 0.696 in controls (odds ratio, 0.99 [95% CI, 0.87-1.13]; P = .89),

consistent with a significant gene × diabetes interaction on CHD risk (P = 2 × 10−4).

Compared with protective allele homozygotes, rs10911021 risk allele

homozygotes were characterized by a 32% decrease in the expression of the neighboring glutamate-ammonia ligase (GLUL) gene in human endothelial cells (P = .0048).
A decreased ratio between plasma levels of γ-glutamyl cycle intermediates pyroglutamic and glutamic acid was also shown in risk allele homozygotes (P = .029).

Conclusion and Relevance

A single-nucleotide polymorphism (rs10911021) was identified that was significantly associated with CHD among persons with diabetes but not in those without diabetes and was functionally related to glutamic acid metabolism, suggesting a mechanistic link.

Adipocyte Heme Oxygenase-1 Induction Attenuates Metabolic Syndrome In Both Male And Female Obese Mice

Angela Burgess1,2, Ming Li2, Luca Vanella1, Dong Hyun Kim1, Rita Rezzani4, et al.

1Department of Physiology and Pharmacology, University of Toledo, Toledo, OH 43614
2Department of Pharmacology, New York Medical College, Valhalla, NY 10595
3Department of Medicine, New York Medical College, Valhalla, NY 10595
4Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy
5Department of Pediatrics and Center for Applied Genomics, Charles University, Prague, Czech Republic
6The Rockefeller University, New York, New York 10065

Hypertension. 2010 December ; 56(6): 1124–1130. http://dx.doi.org/10.1161/HYPERTENSIONAHA.110.151423

Abstract

Increases in visceral fat are associated with

increased inflammation,
dyslipidemia,
insulin resistance,
glucose intolerance and
vascular dysfunction.

We examined the effect of the potent heme oxygenase (HO)-1 inducer, cobalt protoporphyrin (CoPP), on regulation of adiposity and glucose levels in both female and male obese mice. Both lean and obese mice were administered CoPP intraperitoneally, (3mg/kg/once a week) for 6 weeks. Serum levels of

adiponectin,
TNFα,
IL-1β and
IL-6, and
HO-1,
PPARγ,
pAKT, and
pMPK protein expression

were measured in adipocytes and vascular tissue . While female obese mice continued to gain weight at a rate similar to controls, induction of HO-1 slowed the rate of weight gain in male obese mice. HO-1 induction led to lowered blood pressure

levels in obese males and females mice similar to that of lean male and female mice.

HO-1 induction also produced a significant decrease in the plasma levels of IL-6, TNF-α, IL-1β and fasting glucose of obese females compared to untreated female obese mice. HO-1 induction

increased the number and
decreased the size of adipocytes of obese animals.

HO-1 induction increased adiponectin, pAKT, pAMPK, and PPARγ levels in adipocyte of obese animals. Induction of HO-1, in adipocytes was associated with

an increase in adiponectin and
a reduction in inflammatory cytokines.

These findings offer the possibility of treating not only hypertension, but also other detrimental metabolic consequences of obesity

including insulin resistance and dyslipidemia in obese populations
by induction of HO-1 in adipocytes.

Introduction

Moderate to severe obesity is associated with increased risk for cardiovascular complications and insulin resistance in humans1, 2 and animals3, 4. Cardiovascular risk is specifically associated with increased intra-abdominal fat. Women in their reproductive years have a higher BMI than males, which largely reflects increased overall subcutaneous adipose tissue or “gynoid” obesity, this is not associated with increased cardiovascular risk5. However, due to increases in visceral fat with aging, after the age of 60 the fat distribution in females more closely resembles that in males6. Increased androgen levels also often occur after the menopausal transition. These changes in visceral fat content and androgen levels correlate with both central obesity and insulin resistance and are an important determinant of cardiovascular risk7.

Heme oxygenase (HO) catalyzes the breakdown of heme, a potentially harmful pro-oxidant, into its products biliverdin and carbon monoxide, with a concomitant release of iron (reviewed in8). While HO-2 is expressed constitutively, HO-1 is inducible in response to oxidative stress and its induction has been reported to normalize vascular and renal function9–11. Further, induction of HO-1 slows weight gain, decreases levels of TNF-α and IL-6 and increases serum levels of adiponectin in obese rats and obese diabetic mice4, 9, 12.

The association observed between HO-1 and adiponectin has led to the proposal of the existence of a cytoprotective HO-1/adiponectin axis4, 13. Previously, L’Abbate et al,14 have shown that induction of HO-1 is associated with a parallel increase in the serum levels of adiponectin, which has well-documented

insulin-sensitizing,
antiapoptotic,
antioxidative and
anti-inflammatory properties.

Adiponectin is an abundant protein secreted from adipocytes. Once secreted, it mediates its actions by binding to a set of receptors, such as

adipoR1 and adipoR2, and also
through induction of AMPK signaling pathways15, 16.

In addition, increases in adiponectin play a protective role against TNF mediated endothelial activation17.

In this study, we evaluated the effect of CoPP, a potent inducer of HO-1,

on visceral and subcutaneous fat distribution in both female and male obese mice.

We show for the first time a resistance to weight reduction upon administration of CoPP in female obese mice but

a significant decrease in inflammatory cytokines.

Despite continued obesity,

CoPP normalized blood pressure levels,
decreased circulating cytokines, and
increased insulin sensitivity in obese females.

CoPP treatment of obese mice

increased the number and
reduced the size of adipocytes.

CoPP treatment of both male and female obese mice reversed the reduction in adiponectin levels seen in obesity. This study suggests that in spite of continued obesity,

HO-1 induction in female obese mice serves a protective role against obesity associated metabolic consequences via expansion of healthy smaller insulin-sensitive adipocytes.

Results

Effect of induction of HO-1 on body weight, appearance, and fat content of female and male obese mice. Previously, we have shown CoPP treatment results in the prevention of weight gain in several male models of obesity including obese and db/db mice and Zucker fat rats4, 12. We extended our studies to examine the effect of CoPP on weight gain in female obese mice. CoPP-treatment prevented weight gain in male obese mice when compared to age-matched male controls (Figure S1). The revention of body weight gain was accompanied by a

reduction in visceral fat in male obese mice. However, female obese mice administered CoPP did not lose weight but continued to gain weight at the same rate as untreated female obese mice (Figure S1). This was in spite of food intake being comparable between the two

groups. CoPP administration decreased subcutaneous fat content in both obese males and females (p<0.05; p<0.05, respectively). CoPP produced a decrease (p<0.05) in visceral fat in male but not in female obese mice when compared to untreated obese mice (Figure S1D).

We examined adipocyte size by haematoxilin-eosin staining in both lean, obese and CoPP treated obese female mice (Figure 1A, upper panel). CoPP treatment resulted in a decrease in adipocyte size (p<0.05) compared to untreated obese animals (Figure 1A, lower left panel). We then examined the number of adipocytes in lean, obese and CoPP-treated obese female mice. The number of adipocytes (mean±SE) in lean, obese and CoPP-treated obese animals was 40.83±3.50, 18.33±1.80 and 32.00±1.67 respectively indicating that CoPP treatment of obese mice increased the number of adipocytes to levels similar to those in lean animals (Figure 1A, lower right panel). Similar results were seen in male animals.

The induction of HO-1 was associated with a reduction in blood pressure (BP). Systolic blood pressure in obese female mice was 142 ± 6.5 mm Hg compared to obese-CoPP treated, 109 ± 8.1 mm Hg, p<0.05. The value in obese female mice treated with CoPP is similar to the blood pressure seen in lean female mice (110 ± 9.6 mm Hg). The systolic blood pressure in obese male mice was 144± 4.5 mm Hg compared to obese-CoPP treated, 104 ± 3.6 mm Hg, p<0.05.

We further examined whether CoPP affects HO-1 expression in adipocyte using immunohistochemistry and western blot analysis. Immunostaining showed increased levels of HO-1 (green staining), located on the surface of adipocytes, after CoPP treatment (p<0.05), compared with female obese mice, Figure 1B. As seen in Figure 1C, HO-1 and

HO-2 levels in adipocyte isolated from lean, untreated female obese mice or female obese mice treated with CoPP. Densitometry analysis showed that HO-1 was increased

significantly in female obese mice treated with CoPP, compared to non-treated female obese mice, p<0.05, which is in agreement with immunohistochemistry results. This pattern of HO expression in obesity occurs in other tissues, including aortas, kidneys and hearts of male obese mice4, 13.

Effect of CoPP on HO-1 expression and HO activity in female and male obese mice

HO-1 protein levels were increased by CoPP treatments in liver and renal tissues similar to that seen in adipocytes. Western blot analysis showed significant differences (p<0.05) in the ratio of HO-1 to actin in renal of male and female obese and lean mice (Figure S 2A). Obesity decreasd HO-1 levels in both sexes when compared to age matched lean animals.

In addition, HO-1 levels were significantly (p<0.05) lower in obese females compared to obese males (Figure S 2A). This reflects a less active HO system in both male and female

obese animals compared to age matched lean controls. Next, we compared the effect of CoPP on male and female HO-1 gene expression in adipocytes. CoPP increased HO-1

expression in both male and female obese animals compared to untreated obese animals (Figure S 2B, p<0.001 and p<0.001, respectively). Similar results of HO-1 expression were seen in liver tissues (Result not shown).

Effect of CoPP on cytokine levels in female and male obese mice

CoPP administration resulted in a significnt increase in the levels of plasma adiponectin in both female (p<0.001) and male obese (p<0.001) mice (Figure 2A). Untreated female obese animals exhibited a significant (p<0.05) increase in plasma IL-6 levels when compared to age-matched male obese mice (Figure 2B). CoPP decreased plasma IL-6 levels in both female and male obese mice (p<0.05A )p<0.01, respectively) when compared to untreated obese miec. Similar results were observed with plasma TNF-α and IL-1β levels (Figure 2C and 2D). These results indicate that though female obese mice exhibited elevated serum levels of inflammatory cytokines compared to male obese mice, CoPP acts with equal efficacy in both female and male obese animals in reducing inflammation while simultaneously increasing serum adiponectin levels (Figure 2).

Effect of CoPP on blood glucose and LDL levels in female and male obese mice

Fasting glucose levels were determined after the development of insulin resistance. CoPP produced a decrease in glucose levels in both fasting female (p<0.05) and male (p<0.001) obese mice when compared to untreated obese control animals (Figure 3A). CoPP reduced LDL levels in both male (p<0.01) and female (p<0.05) obese mice when compared to untreated obese controls (Figure 3B). Treatment with SnMP, increased LDL levels. In separate experiments two weeks apart, glucose levels and insulin sensitivity were determined after development of insulin resistance (Fig. 4A and B). Blood glucose levels in female obese mice were elevated (p<0.01) 30 min after glucose administration and remained elevated. In CoPP-treated female obese mice produced a decrease in glucose but not in the vehicle-treated female obese mice (p<0.01).

Effect of Obesity on Protein Expression Levels of pAKT, pAMPK, and PPARγ levels in female and male obese mice

Western blot analysis of adipocytes harvested from fat tissues,showed significant differences in basal protein expression levels of pAKT and pAMPK in untreated female obese mice compared to untreated obese male mice. pAMPK levels were higher in obese females compared to obese males (Figure 5A, p< 0.05). This was also the case for pAKT protein levels, where increased levels of pAKT were seen in obese females compared to obese males (Figure 5B, p<0.05). CoPP treatment increased pAMPK and pAKT levels in bothe obese females and obese males. In addition, CoPP administration increased PPARγ levels, in both male (p<0.001) and female (p<0.05) obese mice (Figures 5C).

Discussion

In the current study, we show for the first time that induction of HO-1 regulates adiposity in both male and female animals via an increase in adipocyte HO-1 protein levels. A second novel finding is that induction of HO-1 was associated not only with a decrease in adipocyte cell size but with an increase in adipocyte cell number. Further, induction of HO-1 affects visceral and subcutaneous fat distribution and metabolic function in male obese mice differently than in female obese mice. Despite continued obesity, upregulation of HO-1 induced major improvements in the metabolic profile of female obese mice exhibiting symptoms of Type 2 diabetes including: high plasma levels of proinflammatory cytokines, hyperglycemia, dyslipidemia, and low adiponectin levels. CoPP treatment resulted in increased serum adiponectin levels and decreased blood pressure. Adiponectin is exclusively secreted from adipose tissue, and its expression is higher in subcutaneous rather than invisceral adipose tissue. Increased adiponectin levels reduce adipocyte size and increase adipocyte number12, resulting in smaller, more insulin sensitive adipocytes. Adiponectin has recently attracted much attention because it has insulin-sensitizing properties that enhance fatty acid oxidation, liver insulin action, and glucose uptake and positively affect serum trglyceride levels18–21. Levels of circulating adiponectin are inversely correlated with plasma levels of oxidized LDL in patients with Type 2 diabetes and coronary artery disease, which suggests that low adiponectin levels are associated with an increased oxidative state in the arterial wall22. Thus, increases in adiponectin mediated by upregulation of HO-1 may account for improved insulin sensitivity and reduced levels of LDL and inflammatory cytokines (TNF-α, IL-1β, and IL-6 levels) in both male and female mice.

Females continued to gain weight in spite of the metabolic improvements. One plausible explanation for this anomaly is the direct effects of HO-1 on adiponectin mediating clonal expansion of pre-adipocytes. This supports the concept that expansion of adipogenesis leads to an increased number of adipocytes of smaller cell size; smaller adipocytes are considered to be healthy, insulin sensitive adipocyte cells that are capable of producing adiponectin23. This hypothesis is supported by the increase in the number of smaller adipocytes seen in

CoPP-treated female obese animals without affecting weight gain when compared to female obese animals. Similar results for the presence were seen in males indicating that this effect is not sex specific.

Upregulation of HO-1 was also associated with increased levels of adipocyte pAKT, and pAMPK and PPARγ levels. Previous studies have indicated that insulin resistance and impaired PI3K/pAKT signaling can lead to the of endothelial dysfunction24. In the current study, increased HO-1 expression was associated with increases in both AKT and AMPK phosphorylation; these actions may protect renal arterioles from insulin mediated endothelial damage. By this mechanism, increased levels of HO-1 limit oxidative stress and facilitate activation of an adiponectin-pAMPK-pAKT pathway and increased insulin sensitivity. Induction of adiponectin and activation of the pAMPK-AKT pathway has been shown to provide vascular protection25, 26. A reduction in AMPK and AKT levels may also explain why inhibition of HO activity in CoPP-treated obese mice increased inflammatory cytokine levels while decreasing adiponectin. The action of CoPP in increasing pAKT, pAMPK and PPARγ is associated with improved glucose tolerance and decreased insulin resistant.

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Hyperhomocysteinemia interaction with Protein C and Increased Thrombotic Risk

Posted in Best evidence, Biomarkers & Medical Diagnostics, Chemical Biology and its relations to Metabolic Disease, Coagulation Therapy and Internal Bleeding, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Inferential analysis, Medical Devices R&D and Inventions, Origins of Cardiovascular Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Population Health Management, Genetics & Pharmaceutical, Proteomics, Translational Effectiveness, Translational Research, Translational Science, tagged activated protein C, homocysteine, hypercoagulability, Protein C, T mechanism, thromboembolism on December 3, 2013| Leave a Comment »

Hyperhomocysteinemia interaction with Protein C and Increased Thrombotic Risk

Reporter and Curator: Larry H Bernstein, MD, FCAP

This document explores the relationship between thromboembolic risk related to hyperhomocysteinemia related to the HHcy interaction with and blocking the protective effect of APC.

Previous Venous Thromboembolism Relationships With Plasma Homocysteine Levels

Marco Cattaneo, Franca Franchi, Maddalena L. Zighetti, Ida Martinelli, Daniela Asti, P. Mannuccio Mannucci
Arterioscler Thromb Vasc Biol. 1998;18:1371-1375.
Received January 28, 1998; revision accepted March 16, 1998. From the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Institute of Internal Medicine, IRCCS Ospedale Maggiore, University of Milano, Italy.
Correspondence to Marco Cattaneo, MD, Hemophilia and Thrombosis Center, Via Pace 9, 20122 Milano, Italy. E-mail marco.cattaneo@unimi.it © 1998 American Heart Association, Inc. 1371 Original Contributions

Abstract—

The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro;

this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy).

We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age- and sex-matched healthy controls and

correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML).

Forty- eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study. Because the plasma levels of APC and PC were correlated in healthy controls, the APC/PC ratios were also analyzed.

Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (P < 0.03 and 0.0004, respectively).

Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy.

Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls.

There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls.
The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy.

This study indicates that

APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and
that Hcy does not interfere with the activation of PC in vivo.

Key Words: homocysteine, protein C, thromboembolism, activated protein C, hypercoagulability, T mechanism.

The zymogen protein C is converted to the active protease, activated protein C (APC),

through proteolytic cleavage by thrombin bound to its endothelial membrane receptor thrombomodulin.1

The demonstration that APC is a normal plasma component,2,3whose enzymatic activity can be detected with specific and sensitive methods,4,5indicates that

the protein C anticoagulant pathway is continuously activated in vivo.

Measurement of APC plasma levels might therefore be helpful in determining the in vivo integrity of the protein C anticoagulant pathway. More generally,

APC levels might mirror the in vivo activation of the coagulation system and
serve as a marker of thrombin activity in the circulation.4

The mechanism(s) by which a moderate elevation of plasma levels of homocysteine (Hcy) increases the risk for arterial and venous thrombotic disease is still unclear.6,7 In vitro studies showed that

Hcy inhibits the thrombomodulin- dependent protein C activation to APC and
interferes with the expression of thrombomodulin on human umbilical vein endothelial cells.8–10

These findings may be relevant to unravel the thrombogenic mechanism of Hcy, because

the protein C anticoagulant system is of major physiological importance in the regulation of the hemostatic congenital or acquired disorders

characterized by impaired production or function of APC are associated with a high risk for venous thromboembolism (VTE).11

It must be noted, how ever, that these in vitro findings have been obtained by using very high concentrations of Hcy,

at least 1 order of magnitude higher than the plasma concentrations found in patients with homozygous homocystinuria.12,13

Their clinical relevance is therefore uncertain and awaits confirmation from ex vivo and/or in vivo studies in humans. In this study, we compared the plasma levels of APC with those of the prothrombin fragment F1,2, a marker of thrombin generation,14in healthy subjects and patients with previous episodes of VTE and

tested whether the levels are affected by plasma Hcy concentrations.

Methods

Materials

L-Methionine, tri-n-butylphosphine, 7-fluoro-2,1,3-benzoxadiazole- 4-sulfonamide (ABDF), L-cystine, Tween 20, Tween 80, benzamidine, and HEPES were from Sigma. (4-Amidinophenyl)-methanesulfonylfluoride (APMSF) was from Boehringer, BSA from Calbiochem, and the chromogenic substrate L-homocystine, ovalbumin, S-2366 from Chromogenics. The monoclonal antibody directed against the light chain of protein C (C3-Mab) was a kind gift of Dr H.P. Schwarz (Immuno, Vienna, Austria). All other chemicals were of reagent grade. Subjects We studied 128 patients with previous VTE and 98 healthy controls. All diagnoses of thrombotic episodes, excluding those of superficial veins, had been confirmed by objective methods: compression ultrasonography or venography for deep vein thrombosis; and ventilation/perfusion scintigraphy for pulmonary embolism. The contemporary presence of deep vein thrombosis in patients with superficial vein thrombosis had not been excluded by objective methods. Table 1 shows the characteristics of the patients studied.

They belonged to a cohort of 315 patients who had been screened for thrombophilic states at our Center between December 1993 and July 1995 and were selected on the basis of the following characteristics:
(1) absence of congenital or acquired thrombophilic states except hyperhomocysteinemia (HyperHcy) (see below);
(2) oral anticoagu- lant therapy discontinued at least 1 month before screening;
(3) at least 4 months elapsed since the last thrombotic episode; and
(4) willingness to participate in the study.

The screening for thrombophilia included the following tests:

prothrombin time;
activated partial thromboplastin time;
thrombin time;
plasma levels of fibrinogen,
protein C,
protein S, and
antithrombin;
APC resistance; and
screening for antiphospholipid syndrome15 and
plasma levels of total homocysteine (tHcy)

before and 4 hours after an oral methionine load. Patients with abnormal APC resistance were also screened for factor V Leiden.16

The study was designed and completed before the demonstration that the mutation G20210A of the prothrombin gene is a risk factor for deep vein thrombosis.17 This mutation therefore was looked for retrospectively only in those subjects whose DNA was still available for analysis (all controls and 50 patients): 5 patients (10%) and 2 controls (2.1%) were heterozygous for the mutation. Of the 128 patients enrolled in the study,

48 had hyperhomocysteinemia (VTE-HyperHcy) according to the diagnostic criteria outlined below, and
80 had normal Hcy levels (VTE-NormoHcy).
- The healthy controls, who were age and sex matched with the patients (male/female, 50/45; median age, 40 years [range, 20 to 73 years]), had been chosen from the same geographical area and with the same socioeconomic background as the patients.

Previous episodes of thrombosis had been ruled out by a validated structured questionnaire.18
No subject had abnormal liver or renal function, or overt autoimmune or neoplastic disease.
Informed consent to participate in the study was obtained from all subjects.
The study was approved by the ethics committee of the University of Milano.

Study Protocol

After an overnight fast, blood samples were drawn between 8:30 and 9:30 AM in K3-EDTA for measurement of total Hcy (tHcy), in 0.013 mol/L trisodium citrate for measurement of F1?2 and protein C, and in citrate plus 0.03 mol/L benzamidine (a reversible inhibitor of APC) for measurement of APC. L-Methionine (3.8 g/m2body surface area) was then administered orally in approximately 200 mL of orange juice. Four hours later, a second blood sample was collected in EDTA for tHcy measurement from all subjects and in citrate plus benzamidine for measurement of APC plasma levels from 10 controls. All subjects remained in the fasting state until the second blood sample had been taken. Plasma Hcy Assay Blood samples in K3-EDTA were immediately placed on ice and centrifuged at 2000xG, 4°C, for 15 minutes. The supernatant was stored in aliquots at < 70°C until assay.
The plasma levels of tHcy (free and protein bound) were determined by high-performance liquid chromatography (Waters Millipore 6000A pump, Millipore) and fluorescence detection (Waters 474) by the method of Ubbink et al,19with slight modifications.20 Briefly, 100 uL of plasma was incubated with 10 uL of 10% tri-n-butylphosphine in dimethylfor- mamide at 4°C for 30 minutes to reduce homocystine and mixed disulfide and deconjugate Hcy from plasma proteins. Then, 100 uL of 10% trichloroacetic acid was added, and the mixture was centrifuged in an Eppendorf microcentrifuge at 13 000 rpm for 10 minutes.
After centrifugation, the mixture was incubated with 1 mg/mL ABDF in borate buffer to derivatize the thiols. The mobile phase, pumped at 1 mL/min, consisted of 0.1 mol/L potassium dihydrogenophosphate, 0.06 mmol/L EDTA, and 12% acetonitrile (pH = 2.1).

Criteria for Diagnosis of HyperHcy HyperHcy was diagnosed when

fasting plasma levels of tHcy or its postmethionine load absolute increments above fasting levels exceeded the 95th percentiles of distribution of values obtained in 388 healthy controls.

Measurement of Plasma APC Plasma APC levels were measured with < enzyme capture assay, essentially as described by Gruber and Griffin.4 Blood samples were

TABLE 1.
Patients With Previous VTE-NormoHcy

Demographic Characteristics of Patients With Previous VTE-HyperHcy
VTE-HyperHcyVTE-NormoHcy 4880

No. Males/females 23/25
Median age, y (range)   36 (19–69)
Median age at the first thrombotic episode, y (range)   32 (17–62)
Time elapsed since last episode, mo (range)   14 (4–70)
Time elapsed since discontinuation of oral anticoagulant therapy, mo (range) 11 (1–64)Type of first thrombotic episode
Deep vein thrombosis   31/49
Pulmonary embolism 36 (14–62)
Superficial vein thrombosis   31 (13–60)
Venous thrombosis of other sites   14 (4–90)

With 1 or more episodes 11 (1–70)
2233 26 (54.2%)
With circumstantial risk factors* at first episode 44 (55%)

*The following circumstantial risk factors were considered: surgery (26), trauma (50), immobilization (47), pregnancy/puerperium (16,21), and oral contraceptives (22).

1372

Activated Protein C, Thrombosis, and Homocysteine

centrifuged within 60 minutes from collection at 1200xG, 4°C, for 30 minutes to obtain platelet-poor plasma, which was frozen in aliquots at < 70°C. A plasma pool from 30 healthy individuals (15 men, 15 women) was obtained in the same way and used to prepare the standards.
(removed)… The chromogenic substrate for APC S-2366 (0.46 mmol/L in Tris-buffered saline, pH 7.4) was then added to the wells. After incubation of the sealed plates at 4°C in wet chambers for 3 weeks, hydrolysis of the substrate was monitored at a dual wavelength setting of 405/655 nm.
The concentration of APC in the unknown samples was calculated from the absorbance of each sample with the standard curve as a reference. Results were expressed as percentage of pooled normal plasma. Measurement of Plasma F1?2 F1?2 was assayed by a commercial ELISA (Behringwerke), as previously described.21

Statistical Analysis

The two-tailed t test was used to compare VTE patients and healthy controls. ANOVA was used to compare VTE-HyperHcy, VTE controls, and healthy controls, followed by the Dunnett’s test for internal contrasts. The Pearson r value was calculated for correla- tions between the variables studied.

Results

The results obtained in all VTE patients and controls are presented, including those with the heterozygous G20210A mutation of the prothrombin gene. A subanalysis of the results obtained in the 40 patients and 98 controls, in whom the mutation was looked for, revealed that

exclusion of the subjects heterozygous for the mutation did not significantly affect the results.

Plasma tHcy Levels

The mean (SD) fasting levels of plasma tHcy were significantly higher in VTE-HyperHcy (28.8?19.5 ?mol/L) than in VTE-NormoHcy (12.0+5.2, P<0.001) and healthy con- trols (11.0+5.3, P<0.001). The mean postmethionine load increments of tHcy above fasting levels were also higher in VTE-HyperHcy (32.9+13.5 umol/L) than in VTE- NormoHcy (19.8+7.5, P<0.001) and healthy controls (16.1+7.6, P<0.001). Differences between VTE-NormoHcy and healthy controls were not statistically significant. Six healthy controls (6.3%) had HyperHcy, according to the diagnostic criteria previously outlined. Plasma Levels of APC Healthy Controls The mean plasma level of APC in healthy controls was 116(20%). There was a statistically significant correlation between the plasma levels of APC and those of protein C (r?0.48, P?0.001) (Figure 1). Therefore, because APC levels are influenced by the concentration of their zymogen, both the absolute APC levels and the activated protein C/protein C (APC/PC) ratios were used for subsequent analysis. The mean value of the APC/PC ratio in healthy controls was 1.01?0.2.

There was no correlation between the plasma levels of APC (not shown) or the APC/PC ratios and the fasting plasma levels of tHcy (Figure 2) or its postmethionine load increments above fasting levels (not shown). The mean APC plasma levels and APC/PC ratios were similar in healthy controls whose tHcy plasma levels fell within the first (115 and 1.0), second (118 and 0.96), or third (115 and 1.01) tertiles of distribution. The mean fasting plasma levels of APC and the APC/PC ratios of 10 healthy controls

– did not significantly differ from those measured in the same subjects 4 hours after an oral methionine load,
– which increased the concentration of tHcy by more than 2-fold (Table 2).

VTE Patients

The mean plasma levels of APC and APC/PC ratios were higher in VTE patients than in healthy controls (124?32 versus 116?20, P?0.03 and 1.12?0.32 versus 0.99?0.19, P?0.0004). This difference was mostly due to VTE- HyperHcy patients whose plasma APC levels and APC/PC ratios were significantly higher than those of healthy controls (Table 3). In contrast, differences between VTE-NormoHcy and healthy controls and between VTE-HyperHcy and VTE- NormoHcy did not reach statistical significance (Table 3). Results did not change substantially when we excluded patients with thrombosis of the superficial veins (APC levels, 124+26 in VTE-HyperHcy and 121?31 in VTE-NormoHcy; APC/PC ratio, 1.17?0.25 in VTE-HyperHcy and 1.09?0.3 Figure 1. Correlation between the plasma levels of protein C and APC in 98 healthy volunteers. Values are expressed as per- centage of the concentrations measured in pooled normal plasma from 30 healthy blood donors. Figure 2. Correlation between the fasting plasma levels of tHcy and APC/PC ratios of 98 healthy volunteers. Cattaneo et al September 1998 1373 in VTE-NormoHcy) or women taking oral contraceptives (APC levels, 115?19 in controls, 130?29 in VTE- HyperHcy, and 121+33 in VTE-NormoHcy; APC/PC ratio, 0.98?0.23 in controls, 1.13?0.4 in VTE-HyperHcy, and 1.08?0.3 in VTE-NormoHcy). The prevalence of high APC/PC ratios was significantly higher in VTE patients than in controls, independent of the tHcy levels in their plasma (Table 4),

-whereas that of high plasma APC levels was significantly increased in VTE- HyperHcy patients only (Table 4).

Plasma Levels of F1?2

The mean plasma level of F1?2 in VTE patients (1.6?0.5 nmol/L) did not significantly differ from that measured in healthy controls (1.5?0.6 nmol/L). There was no statistically significant difference between plasma levels of F1?2 in VTE-HyperHcy (1.6?0.6 nmol/L), VTE-NormoHcy (1.6?0.6 nmol/L), and healthy controls. The mean F1?2 plasma levels were similar in healthy controls whose plasma levels of tHcy fell within the first, second, or third tertiles of distribution (not shown). F1?2 levels and APC/PC ratios were significantly correlated in controls (r?0.28, P?0.005) but not in VTE-HyperHcy (r? ?0.03, P?0.05) or VTE- NormoHcy (r?0.08, P?0.05).

Discussion

This study shows that

– patients with previous episodes of VTE have higher circulating plasma levels of APC than healthy controls, particularly if they have HyperHcy.

The patients studied had none of the known congenital or acquired thrombophilic states, in which

– the circulating levels of markers of activation of the coagulation system may be increased.21–24Even
– though the recently described G20210A mutation of the prothrombin gene17could be looked for retrospectively in only approximately one third of the pa- tients, also those patients in whom the prothrombin mutation was ruled out had high APC levels,
– excluding that they were mainly due to the presence of the mutation.

APC is generated from its plasma precursor, protein C, on activation by thrombin-thrombomodulin complex on the endothelial cell surface, probably acting in concert with the endothelial cell protein C receptor.1Subcoagulant amounts of thrombin in the circulation may increase the plasma levels of endogenous APC, which can therefore be considered markers of a hypercoagulable state.4Accordingly, the high APC plasma levels that we measured in patients with previous episodes of VTE may be interpreted as an index of ongoing thrombin formation,
despite the fact that at least 4 months (and a median of 14 months) elapsed since their last thrombotic episode. However,

– the plasma concentrations of F1?2, a marker of thrombin generation, were not increased signifi cantly in the same VTE patients and were not correlated with APC levels or APC/PC ratios.

In contrast to VTE patients, a statistically significant correlation between APC and F1?2 plasma levels was found in healthy controls. On the basis of these data, we hypothesize that

– the increased plasma levels of APC found in patients with previous episodes of VTE are not caused by heightened thrombin generation but by alternative mechanisms. Although we did not measure markers of activation of the fibrinolytic system,

– the possibility that high plasma levels of plasmin could be responsible for protein C activation25in these patients should be considered.

The greatest increase of APC plasma levels in VTE patients was observed in subjects with fasting and/or postmethionine-loading HyperHcy. VTE patients with nor mal plasma levels of tHcy had lower concentrations of APC than patients with HyperHcy, but this

– difference could be due to chance alone, because it was not statistically significant. These results contrast with the alleged inhibitory effect of Hcy on protein C activation that was shown in in vitro studies.8–10

Our data obtained in healthy individuals

– support the view that Hcy does not affect protein C activation in vivo, because the
– mean plasma levels of APC of subjects in the highest tertile of distribution of tHcy levels were not different from those of subjects in the lowest tertile. Moreover,
– the rapid increase in plasma tHcy brought about by an oral methionine load did not affect the concentration of circulating APC

TABLE 2. Healthy Controls Before and 4 Hours After Methionine Loading (PML) Plasma Levels tHcy, APC, and APC/PC Ratios in 10 tHcy, ?mol/LAPC, %APC/PC Ratio Baseline 4 h PML* P† 10.5?3.8 29.5?7.6 0.0001 118?43 113?32 0.57 0.98?0.2 0.95?0.1 0.7 Data are mean?SD. *Methionine was given orally at a dose of 3.8 g/m2body surface area. †t test for paired samples. TABLE 4. APC/PC Ratios in Healthy Controls, Patients With Previous VTE-HyperHcy, and Patients With Previous VTE-NormoHcy Prevalences of High Plasma Levels of APC and Subjectsn With High APC LevelsWith High APC/PC Ratio n (%)OR (95% CI) n (%)OR (95% CI) Healthy controls 98 10 (10.2) 1.0 (reference) 10 (10.2) 1.0 (reference) VTE-HyperHcy48 12 (25.0) 2.9 (1.1–8.3) VTE-NormoHcy80 16 (20.0) 2.2 (0.9–5.7) 16 (33.3) 4.4 (1.7–11.4) 22 (27.5) 3.3 (1.4–8.1) CI indicates confidence interval. The cutoff points, which corresponded to the 90th percentiles of distribution among healthy controls, were 143.1% for APC levels and 1.22 for APC/PC ratios.

TABLE 3. Controls, Patients With Previous VTE-HyperHcy, and Patients With Previous VTE-NormoHcy

Plasma Levels of APC and APC/PC Ratios in Healthy Subjects nAPC,* %APC/PC Ratio† Healthy controls VTE-HyperHcy VTE-NormoHcy P (ANOVA) 98 48 80 116?20 128?29 121?33 0.03 0.99?0.19 1.15?0.33 1.10?0.31 0.002 Data are mean?SD. *VTE-HyperHcy versus VTE-NormoHcy (Dunnett’s test), P?NS; VTE- HyperHcy versus healthy controls, P?0.01; VTE-NormoHcy versus healthy controls, P?NS. †VTE-HyperHcy versus VTE-NormoHcy (Dunnett’s test), P?NS; VTE- yperHcy versus healthy controls, P?0.001; VTE-NormoHcy versus healthy controls, P?0.01. 1374

Activated Protein C, Thrombosis, and Homocysteine

Therefore, the results of our study suggest that Hcy does not negatively influence the plasma APC levels and argue against the hypothesis that

– it inhibits the activation of protein C in vivo by interfering with the activity of thrombomodulin.

Recently, Lentz et al,26in an experimental study of mon- keys with diet-induced moderate HyperHcy, showed that

– the thrombin-stimulated endothelium of aortas from hyperhomocysteinemic animals activated protein C in vitro less effectively than that of control animals.

This study, which supports the hypothesis that Hcy interferes with protein C activation, is in apparent contradiction with our results. At least two possible explanations for their different results can be proposed.

First, Hcy would not affect protein C activation that is ongoing in vivo under physiological conditions, whereas it would interfere with its activation at sites at which athero- genic or thrombogenic stimuli injured the endothelium and increased the local concentration of thrombin.
Second, due to the different relative densities of endothelial cell protein C receptor and thrombomodulin on the endothelium of large vessels and capillaries,1the regulation of protein C activation may differ in the two vascular districts. Although Lentz et al26 measured protein C activation by the endothelium of the aorta, we measured circulating APC, which mostly reflects protein C activation occurring in the microcirculation.

On the basis of the considerations above, we speculate that
– Hcy does not interfere with protein C activation ongoing in the micro- circulation under physiological conditions, whereas
– it could inhibit protein C activation on large, injured vessels.

In conclusion, our study shows that APC plasma levels are high in patients with previous episodes of VTE in whom the plasma levels of F1?2 are normal. Therefore, APC plasma levels represent a sensitive marker of activation of the hemostatic system. In addition, the study showed that high Hcy levels are not associated with heightened thrombin generation and do not interfere with the activation of protein C under physiological conditions in vivo. Further studies are needed to unravel the mechanism(s) by which HyperHcy increases the risks for atherosclerosis and thrombosis.

References

1. Esmon CT, Ding W, Yasuhiro K, Gu J-M, Ferrel G, Regan LM, Stearns- Kurosawa DJ, Kurosawa S, Mather T, Laszik Z, Esmon NL. The protein C pathway: new insights. Thromb Haemost. 1997;78:70–74.
2. Bauer KA, Kass BL, Beeler DL, Rosenberg RD. Detection of protein C activation in humans. J Clin Invest. 1984;74:2033–2041.
3. Heeb MJ, Mosher D, Griffin JH. Inhibition and complexation of activated protein C by two major inhibitors in plasma. Blood. 1989;73:446–454.
4. Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood. 1992;79:2340–2348.
5. Espan ˜a F, Zuazu I, Vicente V, Estelle ´s A, Marco P, Aznar J. Quantifi- cation of circulating activated protein C in human plasma by immuno- assays: enzyme levels are proportional to total protein C levels. Thromb Haemost. 1996;75:56–61.
6. Cattaneo M. Hyperhomocysteinemia: a risk factor for arterial and venous thrombotic disease. Int J Clin Lab Res. 1997;27:139–144.
7. Harpel PC, Zhang X, Borth W. Homocysteine and hemostasis: patho- genetic mechanisms predisposing to thrombosis. J Nutr. 1996;126: 1285S–1289S.
8. Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood. 1990;75:895–901.
9. Lentz SR, Sadler JE. Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest. 1991;88:1906–1914.
10. Hayashi T, Honda G, Suzuki K. An atherogenic stimulus homocysteine inhibits cofactor activity of thrombomodulin and enhances thrombo- modulin expression in human umbilical vein endothelial cells. Blood. 1992;79:2930–2936.
saee original manuscript for further referencesz and for figures (not shown)

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Risk of Bias in Translational Science

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Risk of Bias in Translational Science

Author: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

Assessment of risk of bias in translational science

Andre Barkhordarian¹, Peter Pellionisz², Mona Dousti¹, Vivian Lam¹,Lauren Gleason¹, Mahsa Dousti¹, Josemar Moura³ and Francesco Chiappelli¹⁴^*

¹Oral Biology & Medicine, School of Dentistry, UCLA, Evidence-Based Decisions Practice-Based Research Network, Los Angeles, USA

²Pre-medical program, UCLA, Los Angeles, CA

³School of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

⁴Evidence-Based Decisions Practice-Based Research Network, UCLA School of Dentistry, Los Angeles, CA

Journal of Translational Medicine 2013, 11:184 http://dx.doi.org/10.1186/1479-5876-11-184
http://www.translational-medicine.com/content/11/1/184

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
http://creativecommons.org/licenses/by/2.0

Abstract

Risk of bias in translational medicine may take one of three forms:

a systematic error of methodology as it pertains to measurement or sampling (e.g., selection bias),
a systematic defect of design that leads to estimates of experimental and control groups, and of effect sizes that substantially deviate from true values (e.g., information bias), and
a systematic distortion of the analytical process, which results in a misrepresentation of the data with consequential errors of inference (e.g., inferential bias).

Risk of bias can seriously adulterate the internal and the external validity of a clinical study, and, unless it is identified and systematically evaluated, can seriously hamper the process of comparative effectiveness and efficacy research and analysis for practice. The Cochrane Group and the Agency for Healthcare Research and Quality have independently developed instruments for assessing the meta-construct of risk of bias. The present article begins to discuss this dialectic.

Background

As recently discussed in this journal [1], translational medicine is a rapidly evolving field. In its most recent conceptualization, it consists of two primary domains:

translational research proper and

translational effectiveness.

This distinction arises from a cogent articulation of the fundamental construct of translational medicine in particular, and of translational health care in general.

The Institute of Medicine’s Clinical Research Roundtable conceptualized the field as being composed by two fundamental “blocks”:

one translational “block” (T1) was defined as “…the transfer of new understandings of disease mechanisms gained in the laboratory into the development of new methods for diagnosis, therapy, and prevention and their first testing in humans…”, and

the second translational “block” (T2) was described as “…the translation of results from clinical studies into everyday clinical practice and health decision making…” [2].

These are clearly two distinct facets of one meta-construct, as outlined in Figure 1. As signaled by others, “…Referring to T1 and T2 by the same name—translational research—has become a source of some confusion. The 2 spheres are alike in name only. Their goals, settings, study designs, and investigators differ…” [3].

Figure 1. Schematic representation of the meta-construct of translational health carein general, and translational medicine in particular, which consists of two fundamental constructs: the T1 “block” (as per Institute of Medicine’s Clinical Research Roundtable nomenclature), which represents the transfer of new understandings of disease mechanisms gained in the laboratory into the development of new methods for diagnosis, therapy, and prevention as well as their first testing in humans, and the T2 “block”, which pertains to translation of results from clinical studies into everyday clinical practice and health decision making [[3]]. The two “blocks” are inextricably intertwined because they jointly strive toward patient-centered research outcomes (PCOR) through the process of comparative effectiveness and efficacy research/review and analysis for clinical practice (CEERAP). The domain of each construct is distinct, since the “block” T1 is set in the context of a laboratory infrastructure within a nurturing academic institution, whereas the setting of “block” T2 is typically community-based (e.g., patient-centered medical/dental home/neighborhoods [4]; “communities of practice” [5]).

For the last five years at least, the Federal responsibilities for “block” T1 and T2 have been clearly delineated. The National Institutes of Health (NIH) predominantly concerns itself with translational research proper – the bench-to-bedside enterprise (T1); the Agency for Healthcare Research Quality (AHRQ) focuses on the result-translation enterprise (T2). Specifically: “…the ultimate goal [of AHRQ] is research translation—that is, making sure that findings from AHRQ research are widely disseminated and ready to be used in everyday health care decision-making…” [6]. The terminology of translational effectiveness has emerged as a means of distinguishing the T2 block from T1.

Therefore, the bench-to-bedside enterprise pertains to translational research, and the result-translation enterprise describes translational effectiveness. The meta-construct of translational health care (viz., translational medicine) thus consists of these two fundamental constructs:

translational research and

translational effectiveness,

which have distinct purposes, protocols and products, while both converging on the same goal of new and improved means of

individualized patient-centered diagnostic and prognostic care.

It is important to note that the U.S. Patient Protection and Affordable Care Act (PPACA, 23 March 2010) has created an environment that facilitates the pursuit of translational health care because it emphasizes patient-centered outcomes research (PCOR). That is to say, it fosters the transaction between translational research (i.e., “block” T1)(TR) and translational effectiveness (i.e., “block” T2)(TE), and favors the establishment of communities of practice-research interaction. The latter, now recognized as practice-based research networks, incorporate three or more clinical practices in the community into

a community of practices network coordinated by an academic center of research.

Practice-based research networks may be a third “block” (T3)(PBTN) in translational health care and they could be conceptualized as a stepping-stone, a go-between bench-to-bedside translational research and result-translation translational effectiveness [7]. Alternatively, practice-based research networks represent the practical entities where the transaction between

translational research and translational effectiveness can most optimally be undertaken.

It is within the context of the practice-based research network that the process of bench-to-bedside can best seamlessly proceed, and it is within the framework of the practice-based research network that

the best evidence of results can be most efficiently translated into practice and
be utilized in evidence-based clinical decision-making, viz. translational effectiveness.

Translational effectiveness

As noted, translational effectiveness represents the translation of the best available evidence in the clinical practice to ensure its utilization in clinical decisions. Translational effectiveness fosters evidence-based revisions of clinical practice guidelines. It also encourages

effectiveness-focused,
patient-centered and
evidence-based clinical decision-making.

Translational effectiveness rests not only on the expertise of the clinical staff and the empowerment of patients, caregivers and stakeholders, but also, and

most importantly on the best available evidence [8].

The pursuit of the best available evidence is the foundation of

translational effectiveness and more generally of
translational medicine in evidence-based health care.

The best available evidence is obtained through a systematic process driven by

a research question/hypothesis that is articulated about clearly stated criteria that pertain to the
patient (P), the interventions (I) under consideration (C), for the sought clinical outcome (O), within a given timeline (T) and clinical setting (S).

PICOTS is tested on the appropriate bibliometric sample, with tools of measurements designed to establish the level (e.g., CONSORT) and the quality of the evidence. Statistical and meta-analytical inferences, often enhanced by analyses of clinical relevance [9], converge into the formulation of the consensus of the best available evidence. Its dissemination to all stakeholders is key to increase their health literacy in order to ensure their full participation

in the utilization of the best available evidence in clinical decisions, viz., translational effectiveness.

To be clear, translational effectiveness – and, in the perspective discussed above, translational health care – is anchored on obtaining the best available evidence,

which emerges from highest quality research.
which is obtained when errors are minimized.

In an early conceptualization [10], errors in research were presented as

those situations that threaten the internal and the external validity of a research study –

that is, conditions that impede either the study’s reproducibility, or its generalization. In point of fact, threats to internal and external validity [10] represent specific aspects of systematic errors (i.e., bias) in the

research design,
methodology and
data analysis.

Thence emerged a branch of science that seeks to

understand,
control and
reduce risk of bias in research.

Risk of bias and the best available evidence

It follows that the best available evidence comes from research with the fewest threats to internal and to external validity – that is to say, the fewest systematic errors: the lowest risk of bias. Quality of research, as defined in the field of research synthesis [11], has become synonymous with

low bias and contained risk of bias [12–15].

Several years ago, the Cochrane group embarked on a new strategy for assessing the quality of research studies by examining potential sources of bias. Certain original areas of potential bias in research were identified, which pertain to

(a) the sampling and the sample allocation process, to measurement, and to other related sources of errors (reliability of testing),

(b) design issues, including blinding, selection and drop-out, and design-specific caveats, and

A Risk of Bias tool was created (Cochrane Risk of Bias), which covered six specific domains:

1. selection bias,

2. performance bias,

3. detection bias,

4. attrition bias,

5. reporting bias, and

6. other research protocol-related biases.

Assessments were made within each domain by one or more items specific for certain aspects of the domain. Each items was scored in two distinct steps:

1. the support for judgment was intended to provide a succinct free-text description of the domain being queried;

2. each item was scored high, low, or unclear risk of material bias (defined here as “…bias of sufficient magnitude to have a notable effect on the results or conclusions…” [16]).

It was advocated that assessments across items in the tool should be critically summarized for each outcome within each report. These critical summaries were to inform the investigator so that the primary meta-analysis could be performed either

only on studies at low risk of bias, or for
the studies stratified according to risk of bias [16].

This is a form of acceptable sampling analysis designed to yield increased homogeneity of meta-analytical outcomes [17]. Alternatively, the homogeneity of the meta-analysis can be further enhanced by means of the more direct quality-effects meta-analysis inferential model [18].

Clearly, one among the major drawbacks of the Cochrane Risk of Bias tool is

the subjective nature of its assessment protocol.

In an effort to correct for this inherent weakness of the instrument, the Cochrane group produced

detailed criteria for making judgments about the risk of bias from each individual item[16], and
that judgments be made independently by at least two people, with any discrepancies resolved by discussion [16].

This approach to increase the reliability of measurement in research synthesis protocols

is akin to that described by us [19,20] and by AHRQ [21].

In an effort to aid clinicians and patients in making effective health care related decisions, AHRQ developed an alternative Risk of Bias instrument for enabling systematical evaluation of evidence reporting [22]. The AHRQ Risk of Bias instrument was created to monitor four primary domains:

1. risk of bias: design, methodology, analysis scoring – low, medium, high

2. consistency: extent of similarity in effect sizes across studies within a bibliome scoring – consistent, inconsistent, unknown

3. directness: unidirectional link between the interventions of interest and the sought outcome, as opposed to multiple links in a casual chain scoring – direct, indirect

4. precision: extent of certainty for estimate of effect with respect to the outcome scoring – precise, imprecise In addition, four secondary domains were identified:

a. Dose response association: pattern of a larger effect with greater exposure (Present/Not Present/Not Applicable or Not Tested)

a. Confounders: consideration of confounding variables (Present/Absent)

a. Strength of association: likelihood that the observed effect is large enough that it cannot have occurred solely as a result of bias from potential confounding factors (Strong/Weak)

a. Publication bias

The AHRQ Risk of Bias instrument is also designed to yield an overall grade of the estimated risk of bias in quality reporting:

•Strength of Evidence Grades (scored as high – moderate – low – insufficient)

This global assessment, in addition to incorporating the assessments above, also rates:

–major benefit

–major harm

–jointly benefits and harms

–outcomes most relevant to patients, clinicians, and stakeholders

The AHRQ Risk of Bias instrument suffers from the same two major limitations as the Cochrane tool:

1. lack of formal psychometric validation as most other tools in the field [21], and

2. providing a subjective and not quantifiable assessment.

To begin the process of engaging in a systematic dialectic of the two instruments in terms of their respective construct and content validity, it is necessary

to validate each for reliability and validity either by means of the classic psychometric theory or generalizability (G) theory, which allows
the simultaneous estimation of multiple sources of measurement error variance (i.e., facets)
while generalizing the main findings across the different study facets.

G theory is particularly useful in clinical care analysis of this type, because it permits the assessment of the reliability of clinical assessment protocols.

the reliability and minimal detectable changes across varied combinations of these facets are then simply calculated [23], but
it is recommended that G theory determination follow classic theory psychometric assessment.

Therefore, we have commenced a process of revision the AHRQ Risk of Bias instrument by rendering questions in primary domains quantifiable (scaled 1–4),

which established the intra-rater reliability (r = 0.94, p < 0.05), and
the criterion validity (r = 0.96, p < 0.05) for this instrument (Figure 2).

Figure 2. Proportion of shared variance in criterion validity (A) and inter-rater reliability (B) in the AHRQ Risk of Bias instrument revised as described.
Two raters were trained and standardized [20] with the revised AHRQ Risk of Bias and with the R-Wong instrument, which has been previously validated[24]. Each rater independently produced ratings on a sample of research reports with both instruments on two separate occasions, 1–2 months apart. Pearson correlation coefficient was used to compute the respective associations. The figure shows Venn diagrams to illustrate the intersection between each two sets data used in the correlations. The overlap between the sets in each panel represents the proportion of shared variance for that correlation. The percent of unexplained variance is given in the insert of each panel.

A similar revision of the Cochrane Risk of Bias tool may also yield promising validation data. G theory validation of both tools will follow. Together, these results will enable a critical and systematic dialectical comparison of the Cochrane and the AHRQ Risk of Bias measures.

Discussion

The critical evaluation of the best available evidence is critical to patient-centered care, because biased research findings are fundamentally invalid and potentially harmful to the patient. Depending upon the tool of measurement, the validity of an instrument in a study is obtained by means of criterion validity through correlation coefficients. Criterion validity refers to the extent to which one measures or predicts the value of another measure or quality based on a previously well-established criterion. There are other domains of validity such as: construct validity and content validity that are rather more descriptive than quantitative. Reliability however is used to describe the consistency of a measure, the extent to which a measurement is repeatable. It is commonly assessed quantitatively by correlation coefficients. Inter-rater reliability is rendered as a Pearson correlation coefficient between two independent readers, and establishes equivalence of ratings produced by independent observers or readers. Intra-rater reliability is determined by repeated measurement performed by the same subject (rater/reader) at two different points in time to assess the correlation or strength of association of the two sets of scores.

To establish the reliability of research quality assessment tools it is necessary, as we previously noted [20]:

•a) to train multiple readers in sharing a common view for the cognitive interpretation of each item. Readers must possess declarative knowledge a factual form of information known to be static in nature a certain depth of knowledge and understanding of the facts about which they are reviewing the literature. They must also have procedural knowledge known as imperative knowledge that can be directly applied to a task in this case a clear understanding of the fundamental concepts of research methodology, design, analysis and inference.

•b) to train the readers to read and evaluate the quality of a set of papers independently and blindly. They must also be trained to self-monitor and self-assess their skills for the purpose of insuring quality control.

•c) to refine the process until the inter-rater correlation coefficient and Cohen coefficient of agreement are about 0.9 (over 81% shared variance). This will establishes that the degree of attained agreement among well-trained readers is beyond chance.

•d) to obtain independent and blind reading assessments from readers on reports under study.

•e) to compute means and standard deviation of scores for each question across the reports, repeat process if the coefficient of variations are greater than 5% (i.e., less than 5% error among the readers across each questions).

The quantification provided by instruments validated in such a manner to assess the quality and the relative lack of bias in the research evidence allows for the analysis of the scores by means of the acceptable sampling protocol. Acceptance sampling is a statistical procedure that uses statistical sampling to determine whether a given lot, in this case evidence gathered from an identified set of published reports, should be accepted or rejected [12,25]. Acceptable sampling of the best available evidence can be obtained by:

•convention: accept the top 10 percentile of papers based on the score of the quality of the evidence (e.g., low Risk of Bias);

•confidence interval (CI⁹⁵): accept the papers whose scores fall at of beyond the upper confidence limit at 95%, obtained with mean and variance of the scores of the entire bibliome;

•statistical analysis: accept the papers that sustain sequential repeated Friedman analysis.

To be clear, the Friedman test is a non-parametric equivalent of the analysis of variance for factorial designs. The process requires the 4-E process outlined below:

•establishing a significant Friedman outcome, which indicates significant differences in scores among the individual reports being tested for quality;

•examining marginal means and standard deviations to identify inconsistencies, and to identify the uniformly strong reports across all the domains tested by the quality instrument

•excluding those reports that show quality weakness or bias

•executing the Friedman analysis again, and repeating the 4-E process as many times as necessary, in a statistical process akin to hierarchical regression, to eliminate the evidence reports that exhibit egregious weakness, based on the analysis of the marginal values, and to retain only the group of report that harbor homogeneously strong evidence.

Taken together, and considering the domain and the structure of both tools, expectations are that these analyses will confirm that these instruments are two related entities, each measuring distinct aspects of bias. We anticipate that future research will establish that both tools assess complementary sub-constructs of one and the same archetype meta-construct of research quality.

References

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JAMA 2003, 289:1278-1287. PubMed Abstract | Publisher Full Text
Woolf SH: The meaning of translational research and why it matters.

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Chiappelli F: From translational research to translational effectiveness: the “patient-centered dental home” model.

Dental Hypotheses 2011, 2:105-112. Publisher Full Text
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Archive for the ‘Inferential analysis’ Category

Ecdysteroid-Dioxolanes-as-MDR-Modulators-in Cancer

Synthesis and Structure-Activity Relationships of Novel Ecdysteroid Dioxolanes as MDR Modulators in Cancer

1. Introduction

2. Results and Discussion

2.1. Semi-Synthesis

Figure 1. Semi-synthetic transformations of 20E and structures of the products obtained.

Table 1. 1H- and 13C-NMR shifts of compounds 4, 6, 22, 30, 33, 9 and 10; in ppm, in methanol-d4. (go to source)

Table 2. 1H- and 13C-NMR shifts of compounds 16–20 and 26–27; in ppm, in methanol-d4. (go to source)

Table 3. 1H- and 13C-NMR shifts of compounds 11–14; 31 and 32; in ppm, in methanol-d4. (go to source)

Figure 2. Stereostructure of 22.

2.2. Anti-Proliferative Effect of Ecdysteroid Derivatives on PAR and MDR Mouse Lymphoma Cells

Table 4. IC50 values of the ecdysteroid derivatives and fluorescence activity ratio (FAR)

2.3. Inhibition of the ABCB1 Pump of MDR Mouse Lymphoma Cells (Rhodamine 123 Accumulation Assay)

Figure 3. Fraction affected (Fa) vs. combination index (CI) value plot for compounds 5 and 15, in comparison with the original lead compound 1.

Table 5.

Figure 4. SAR summary for compounds 1–33.

3. Experimental

3.1 General Information

3.2. Semi-Synthesis and Purification of Monosubstituted Ecdysteroid Dioxolane Derivatives 2–16

3.3. Semi-Synthesis and Purification of Disubstituted Ecdysteroid Derivatives 17–25 in One-Step

3.4. Semi-Synthesis and Purification of Disubstituted Ecdysteroid Derivatives 26–33 in Two-Steps

3.5. Further Experimental Data for the New Compounds

3.7. Cell Lines

3.8. Anti-proliferative Assay

3.9. Inhibition of ABCB1 Pump of MDR Mouse Lymphoma Cells (Rhodamine 123 Accumulation Assay)

3.10. Combination Assays

4. Conclusions

Acknowledgments

References

Archival Supplement

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Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker

Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker

Anemia as an Independent Predictor of Elevated N-terminal proBNP

The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Perspective

Introduction

FIRST ARTICLE

Anemia as an Independent Predictor of Elevated N-terminal proBNP

Table 1. Patient Characteristics

ABSTRACT

Introduction

Objective

Methods

Results

Conclusion

INTRODUCTION

METHODS

Patient population

Baseline patient data

Statistical analysis

RESULTS

DISCUSSION

CONCLUSION

REFERENCES

Table Legends

Table I. Clinical characteristics of the study population

Table II. Comparison of NT- proBNP means under WHO criteria at different GFR

Table I

Table II

FIGURE LEGENDS

Supplementary Material

Table based on LatentGOLD Statistical Innovations, Inc., Belmont, MA., 2000: Jeroen Vermunt & Jay Magidson)

LogNTpr < 1.5 0.0843 0.2457 0.0006 0.0084 1.6-2.5 0.6179 0.6458 0.0709 0.2809 2.5-3.5 0.2848 0.1067 0.5319 0.5883 > 3.5 0.0130 0.0018 0.3966 0.1224

MDRD > 90 0.1341 0.7919 0.0063 0.6106 61-90 0.6019 0.2040 0.1633 0.3713 41-60 0.2099 0.0041 0.3317 0.0175 < 41 0.0542 0.0001 0.4987 0.0006

age under 51 0.0668 0.5646 0.0568 0.0954 51-70 0.3462 0.3602 0.3271 0.3880 over 70 0.5870 0.0752 0.6161 0.5166

WHO No anemia 0.7518 0.6556 0.2041 0.0998 Anemia 0.2482 0.3444 0.7959 0.9002

LogNTpro < 1.5 0.2492 0.7379 0.0013 0.0116 1.6-2.5 0.4163 0.4243 0.0427 0.1167 2.6-3.5 0.2296 0.0887 0.3723 0.3095 > 3.5 0.0328 0.0023 0.7982 0.1666

MDRD > 90 0.1001 0.5998 0.0043 0.2958 61-90 0.5198 0.1716 0.1136 0.1950 41-60 0.3860 0.0055 0.5847 0.0238 < 41 0.1205 0.0002 0.8785 0.0008

age < 51 0.0720 0.7458 0.0910 0.0912 51-70 0.3036 0.3084 0.2013 0.1867 over 70 0.3773 0.0409 0.3633 0.2186

WHO No anemia 0.4589 0.3957 0.1076 0.0378 Anemia 0.1342 0.1844 0.3742 0.3073

SECOND ARTICLE

The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

OBJECTIVES

BACKGROUND

METHODS

Synthesis and Structure-Activity Relationships of
Novel Ecdysteroid Dioxolanes as MDR Modulators in Cancer

Figure 1. Semi-synthetic transformations of 20E
and structures of the products obtained.

Table 1. 1H- and 13C-NMR shifts of compounds 4, 6, 22, 30, 33, 9 and 10; in ppm, in methanol-d4.
(go to source)

Table 2. 1H- and 13C-NMR shifts of compounds 16–20 and 26–27; in ppm, in methanol-d4.
(go to source)

Table 3. 1H- and 13C-NMR shifts of compounds 11–14; 31 and 32; in ppm, in methanol-d4.
(go to source)

2.2. Anti-Proliferative Effect of Ecdysteroid Derivatives on
PAR and MDR Mouse Lymphoma Cells

2.3. Inhibition of the ABCB1 Pump of MDR Mouse Lymphoma Cells
(Rhodamine 123 Accumulation Assay)

LogNTpr
< 1.5 0.0843 0.2457 0.0006 0.0084
1.6-2.5 0.6179 0.6458 0.0709 0.2809
2.5-3.5 0.2848 0.1067 0.5319 0.5883
> 3.5 0.0130 0.0018 0.3966 0.1224

MDRD
> 90 0.1341 0.7919 0.0063 0.6106
61-90 0.6019 0.2040 0.1633 0.3713
41-60 0.2099 0.0041 0.3317 0.0175
< 41 0.0542 0.0001 0.4987 0.0006

age
under 51 0.0668 0.5646 0.0568 0.0954
51-70 0.3462 0.3602 0.3271 0.3880
over 70 0.5870 0.0752 0.6161 0.5166

WHO
No anemia 0.7518 0.6556 0.2041 0.0998
Anemia 0.2482 0.3444 0.7959 0.9002

LogNTpro
< 1.5 0.2492 0.7379 0.0013 0.0116
1.6-2.5 0.4163 0.4243 0.0427 0.1167
2.6-3.5 0.2296 0.0887 0.3723 0.3095
> 3.5 0.0328 0.0023 0.7982 0.1666

MDRD
> 90 0.1001 0.5998 0.0043 0.2958
61-90 0.5198 0.1716 0.1136 0.1950
41-60 0.3860 0.0055 0.5847 0.0238
< 41 0.1205 0.0002 0.8785 0.0008

age
< 51 0.0720 0.7458 0.0910 0.0912
51-70 0.3036 0.3084 0.2013 0.1867
over 70 0.3773 0.0409 0.3633 0.2186

WHO
No anemia 0.4589 0.3957 0.1076 0.0378
Anemia 0.1342 0.1844 0.3742 0.3073

Table 4. Changes from Baseline to Final New York Heart Association (NYHA) Class
Initial minus ﬁnal