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Genomics in Medicine – Tomorrow’s Promise

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, Health Law & Patient Safety, International Global Work in Pharmaceutical, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , on March 3, 2013| 3 Comments »

Genomics in Medicine – Tomorrow’s Promise

Reporter: Larry H Bernstein, MD, FCAP

Genomics in Medicine: Today’s Issues, Tomorrow’s Promise

KM Beima-Sofie, EH Dorfman, JM Kocarnik, MY Laurino
Feb 13, 2013 Medscape Genomic Medicine

What do you think about these issues before reading this piece?

The Broader Implications of Genetic Sciences
The 62nd annual meeting of the American Society of Human Genetics (ASHG), which was held in San Francisco, California, in November 2012, featured a diverse array of research in basic, clinical, and population science contributed by human geneticists across the globe.
Genetic Sequencing Moves Beyond the Laboratory
Several presentations at the meeting focused on the lessons learned from the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project. The goal of the project was to
  • develop and validate a cost-effective and high-throughput sequencing technology
  • capable of analyzing the DNA sequence in the exome, which
  • consists of all protein-coding regions in the human genome.
At previous ASHG meetings, presentations and discussions largely focused on
  • the development of sequencing technology and on applications of this technology for research.
Now that sequencing is an increasing reality, this year’s conference featured presentations on
  • what to do with the resulting information, in both research and clinical settings.
Issues discussed include the challenges of
  • interpreting sequence data,
  • determining which results should be returned to various parties, and
  • the potential impacts of different testing techniques.
Results from the NHLBI Exome Sequencing Project and other projects are fueling the discussion on
legal issues surrounding gene patenting, a hotly debated topic that is currently under consideration by the US Supreme Court. During a plenary session on gene discovery and patent law,
Of particular focus was the lawsuit brought by the American Civil Liberties Union against Myriad Genetics,
  • contesting the company’s patent of the BRCA1 and BRCA2 genes for hereditary breast and ovarian cancer.
At present, Myriad has exclusive rights to offer clinical genetic testing for these genes; one of the main arguments of the lawsuit is
  • that gene patents hinder the pursuit of confirmatory tests and limit the testing options available to women.
DNAPrint Genomics

DNAPrint Genomics (Photo credit: Wikipedia)

English: Exome sequencing workflow: Part 2. Ta...

English: Exome sequencing workflow: Part 2. Target exons are enriched, eluted and then amplified by ligation-mediated PCR. Amplified target DNA is then ready for high-throughput sequencing. (Photo credit: Wikipedia)

Cost per Megabase of DNA Sequence (Why biologi...

Cost per Megabase of DNA Sequence (Why biologists panic about compute) (Photo credit: dullhunk)

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Virtual Biopsy – is it possible?

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Chemical Biology and its relations to Metabolic Disease, Ecosystems & Industrial Concentration in the Medical Device Sector, Health Economics and Outcomes Research, Imaging-based Cancer Patient Management, Medical Devices R&D and Inventions, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Personalized and Precision Medicine & Genomic Research, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Statistical Methods for Research Evaluation, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , on March 3, 2013| 7 Comments »

Virtual Biopsy – is it possible?

Author and Curator: Dror Nir, PhD

Article 11.5.3 Virtual Biopsy is it possible

In a remark made to my last post: New envelopment in measuring mechanical properties of tissue, Dr. Aviva Lev-Ari, PhD, RN, Director and Founder of our Open Access Online Scientific Journal:  Leaders of Pharmaceutical Business Intelligence, asked whether OCT can be used for the purpose of performing biopsy. My answer to her question was “YES”. I thought that it will be worthwhile explaining why I am so “optimistic” about this:

A conventional biopsy is a process where a tissue sample is being cut out of the body and after being subjected to all kind of chemical processes a thin-film of tissue is trimmed and read under the microscope by a trained pathologist. Can imaging provide histological assessment of “thin-film” of tissue without cutting it out of the body? The answer would be positive if the imaging will result with high resolution reconstruction of a tissue sample identical in quality to a “live-sample” that is put under the microscope.

I was happy to find support to my optimism regarding the feasibility of constructing such device in the following article: Virtual skin biopsy by optical coherence tomography: the first quantitative imaging biomarker for scleroderma published on February 20th 2013 in Ann Rheum Dis doi:10.1136/annrheumdis-2012-202682

 This article reports an original, first study to perform histological comparison and explore Optical coherence tomography (“OCT”) as a potential imaging technique for the clinical assessment of patients presenting with systemic sclerosis (“SSc”). In their study the investigators used a device emitting low-intensity infrared laser beam, capable of producing high-contrast images of skin up to 2 mm deep with resolutions of 4–10 μm.

[START ORIGINAL PAPER]

ABSTRACT

Background

Skin involvement is of major prognostic value in systemic sclerosis (SSc) and often the primary outcome in clinical trials. Nevertheless, an objective, validated biomarker of skin fibrosis is lacking. Optical coherence tomography (OCT) is an imaging technology providing high-contrast images with 4 μm resolution, comparable with microscopy (‘virtual biopsy’). The present study evaluated OCT to detect and quantify skin fibrosis in SSc.

Methods

We performed 458 OCT scans of hands and forearms on 21 SSc patients and 22 healthy controls. We compared the findings with histology from three skin biopsies and by correlation with clinical assessment of the skin. We calculated the optical density (OD) of the OCT images employing Matlab software and performed statistical analysis of the results, including intraobserver/ interobserver reliability, employing SPSS software.

 Results

Comparison of OCT images with skin histology indicated a progressive loss of visualisation of the dermal–epidermal junction associated with dermal fibrosis. Furthermore, SSc affected skin showed a consistent decrease of OD in the papillary dermis, progressively worse in patients with worse modified Rodnan skin score (p<0.0001). Additionally, clinically unaffected skin was also distinguishable from healthy skin for its specific pattern of OD decrease in the reticular dermis (p<0.001). The technique showed an excellent intraobserver and interobserver reliability (intraclass correlation coefficient >0.8).

Conclusions

OCT of the skin could offer a feasible and reliable quantitative outcome measure in SSc. Studies determining OCT sensitivity to change over time and its role in defining skin vasculopathy may pave the way to defining OCT as a valuable imaging biomarker in SSc.

Virtual skin biopsy by OCT

The OCT images acquisition allowed the reconstruction of a virtual skin biopsy measuring 4×0.4×2 mm. The main structure of the healthy skin was easily recognisable by OCT (figure 1).

Virtual biopsy of forearm skin by optical coherence tomography. Representative 3D reconstruction from the tomography of healthy and systemic sclerosis (SSc) (site modified Rodnan skin score=3) skin scans. The keratin of the skin appears as a white line on the surface (k). The epidermis (ED) is quite visible in the healthy skin by the contrast with the increased optical density of the papillary dermis (PD). The dermal– epidermal junction (DEJ) is quite visible in the healthy skin between the ED and PD. On the contrary, neither clear distinction of ED and PD or DEJ is appreciable in the SSc skin. The vessels (*) are numerous and very well recognisable in healthy skin, whereas they appear less numerous and less distinct in the OCT image of SSc skin. Total depth of 3D reconstruction=1.2 mm. Scale bars are calculated by ImageJ.

Virtual biopsy of forearm skin by optical coherence tomography. Representative 3D reconstruction from the tomography of healthy and systemic sclerosis (SSc) (site modified Rodnan skin score=3) skin scans. The keratin of the skin appears as a white line on the surface (k). The epidermis (ED) is quite visible in the healthy skin by the contrast with the increased optical density of the papillary dermis (PD). The dermal– epidermal junction (DEJ) is quite visible in the healthy skin between the ED and PD. On the contrary, neither clear distinction of ED and PD or DEJ is appreciable in the SSc skin. The vessels (*) are numerous and very well recognisable in healthy skin, whereas they appear less numerous and less distinct in the OCT image of SSc skin. Total depth of 3D reconstruction=1.2 mm. Scale bars are calculated by ImageJ.

Some quantitative results  – in images:

Validation of optical coherence tomography (OCT) images by histology. (A and B) H&E staining (A) and corresponding OCT scan (B) from a healthy control (HC). The green line is the mean A-scan of the entire OCT image (100 scans) overlaid by matching the scale bars of OCT and histology. The green arrow indicates the nadir of the valley in the mean A-scan, which corresponds to the dermal–epidermal junction clearly visible on both images. The green arrowhead indicates the second peak of the mean OCT A-Scan which corresponds by the overlay to the most superficial region of the papillary dermis. (C and D) H&E staining (C) and corresponding OCT scan (D) from a systemic sclerosis (SSc) patient (site modified Rodnan skin score =3). The red line is the mean A-scan of the OCT image, overlaid by matching the scale bars in the two panels. The red arrow indicates the nadir in the valley of the mean A-scan, which in this case does not correspond to the dermal–epidermal junction. The red arrowhead corresponds to the second peak in mean A-Scan. (E) Overlay of HC and SSc. Scale bar=240 μm.

Validation of optical coherence tomography (OCT) images by histology. (A and B) H&E staining (A) and corresponding OCT scan (B) from a healthy control (HC). The green line is the mean A-scan of the entire OCT image (100 scans) overlaid by matching the scale bars of OCT and histology. The green arrow indicates the nadir of the valley in the mean A-scan, which corresponds to the dermal–epidermal junction clearly visible on both images. The green arrowhead indicates the second peak of the mean OCT A-Scan which corresponds by the overlay to the most superficial region of the papillary dermis. (C and D) H&E staining (C) and corresponding OCT scan (D) from a systemic sclerosis (SSc) patient (site modified Rodnan skin score =3). The red line is the mean A-scan of the OCT image, overlaid by matching the scale bars in the two panels. The red arrow indicates the nadir in the valley of the mean A-scan, which in this case does not correspond to the dermal–epidermal junction. The red arrowhead corresponds to the second peak in mean A-Scan. (E) Overlay of HC and SSc. Scale bar=240 μm.

Optical coherence tomography (OCT) of affected and not affected skin in plaque morphea. (A) OCT of not affected skin. Vertical scale represents depth in micrometre from the surface. The dermal–epidermal junction (DEJ) level is indicated by the white dotted line. Mean A-scan curve is overlaid and displayed in green. (B) OCT of affected skin in morphea patient. Mean A-scan curve is overlaid and displayed in red. Note the poorly visible DEJ and the valley of the curve below the DEJ (arrowhead). (C) Overlay of mean A-scan curves from the analysis of affected and unaffected skin in a morphea patient. Note that in the curves overlay graph both the difference depth of the first valley is clearly appreciable (arrowheads). Similarly the second mean A-scan peak (arrow) is subtle in the affected skin, similar to scleroderma affected skin.

Optical coherence tomography (OCT) of affected and not affected skin in plaque morphea. (A) OCT of not affected skin. Vertical scale represents depth in micrometre from the surface. The dermal–epidermal junction (DEJ) level is indicated by the white dotted line. Mean A-scan curve is overlaid and displayed in green. (B) OCT of affected skin in morphea patient. Mean A-scan curve is overlaid and displayed in red. Note the poorly visible DEJ and the valley of the curve below the DEJ (arrowhead). (C) Overlay of mean A-scan curves from the analysis of affected and unaffected skin in a morphea patient. Note that in the curves overlay graph both the difference depth of the first valley is clearly appreciable (arrowheads). Similarly the second mean A-scan peak (arrow) is subtle in the affected skin, similar to scleroderma affected skin.

DISCUSSION

The current gold standard for semiquantitative assessment of skin fibrosis, the mRSS, suffers from several shortcomings ranging from the subjectivity of skin palpation assessments and the high level of skill required from the clinical investigator. Even more importantly, a meta-analysis of three independent studies determined an overall within patient interobserver SD of five units independently of the mean skin score,[6 21] which represents an SE ranging from 20% to 26%. A primary outcome measure with 25% of SE entails the recruitment of a large number of patients to attain statistical validity in minimally significant changes, a task often difficult to accomplish given the comparatively low incidence of SSc.

A robust imaging biomarker for the assessment of skin fibrosis in SSc has not previously been reported. Herein we report the first study aimed to validate OCT for the quantitative assessment of skin involvement in SSc.

To date, the limited data on surrogate outcome measures for skin involvement are largely composed of histopathological or molecular changes in affected skin.[22 23] Despite conceptually very valuable, these studies, involving skin biopsies, are invasive and limited because of a site bias, referring to only one precise body area. Moreover, they are difficult to repeat in longitudinal manner and showed no sensitivity to change over time.[24] In this study, we evaluated OCT skin scanning as a reliable and quanti­tative tool that could be used as a surrogate marker of skin fibrosis. The technique requires minimal operator training, less than 10s per site examined, and offers the great advantage of saving image files for further or centralised operator independ­ent analysis. This latter is a particularly useful tool limiting the ‘hands on’ time in the clinic office and allowing a centralised, blinded assessment of results in clinical trials.

We observed an excellent correlation of OCT mean A-Scan curves and mRSS score at the site of analysis. More importantly, the corroboration of our OCT findings with pathological changes at the DEJ provides a robust construct validity for the technique. Of interest, we found that the changes of the OD of the dermis in SSc are similar to the ones observed in a case of plaque morphea, corroborating even further the potential value of OCT in measuring skin fibrosis.

Additional Comment

HFUS (High Frequensy Ultrasound) has been recently suggested to offer a quantitative assessment of skin thickness in SSc by several studies.8–10 In contrast with ultrasound, OCT does not require any use of gels, is able to give a higher resolution images and the analysis algo­rithm is automatic, not involving any operator interpretation. Nevertheless, since the penetration of OCT is limited to the first millimetre of skin, OCT and HFUS may be explored as comple­mentary imaging biomarkers in SSc.

REFERENCES

1     Jimenez SA, Derk CT. Following the molecular pathways toward an understanding
of the pathogenesis of systemic sclerosis. Ann Intern Med 2004;140:37–50.

2     Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem fibrotic disorder.
J Clin Invest 2007;117:557–67.

3     Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med
2009;360:1989–2003.

4     Clements PJ, Hurwitz EL, Wong WK, et al. Skin thickness score as a predictor and
correlate of outcome in systemic sclerosis: high-dose versus low-dose penicillamine trial. Arthritis Rheum 2000;43:2445–54.

5     Steen VD, Medsger TA Jr. Improvement in skin thickening in systemic sclerosis
associated with improved survival. Arthritis Rheum 2001;44:2828–35.

6     Pope JE, Baron M, Bellamy N, et al. Variability of skin scores and clinical
measurements in scleroderma. J Rheumatol 1995;22:1271–6.

Clements PJ, Lachenbruch PA, Seibold JR, et al. Skin thickness score in systemic

sclerosis: an assessment of interobserver variability in 3 independent studies. J Rheumatol 1993;20:1892–6.

   8     Akesson A, Hesselstrand R, Scheja A, et al. Longitudinal development of skin
involvement and reliability of high frequency ultrasound in systemic sclerosis. Ann Rheum Dis 2004;63:791–6.

   9     Moore TL, Lunt M, McManus B, et al. L. Seventeen-point dermal ultrasound scoring
system—a reliable measure of skin thickness in patients with systemic sclerosis. Rheumatology (Oxford) 2003;42:1559–63.

10     Kaloudi O, Bandinelli F, Filippucci E, et al. High frequency ultrasound

measurement of digital dermal thickness in systemic sclerosis. Ann Rheum Dis 2010;69:1140–3.

11     Aden N, Shiwen X, Aden D, et al. Proteomic analysis of scleroderma lesional skin
reveals activated wound healing phenotype of epidermal cell layer. Rheumatology (Oxford) 2008;47:1754–60.

12     Aden N, Nuttall A, Shiwen X, et al. Epithelial Cells Promote Fibroblast Activation via
IL-1alpha in Systemic Sclerosis. J Invest Dermatol 2010;130:2191–200.

13     Gambichler T, Jaedicke V, Terras S. Optical coherence tomography in dermatology:
technical and clinical aspects. Arch Dermatol Res 2011;303:457–73.

14     Marschall S, Sander B, Mogensen M, et al. Optical coherence tomography-current
technology and applications in clinical and biomedical research. Anal Bioanal Chem 2011;400:2699–720.

15     Coleman AJ, Richardson TJ, Orchard G, et al. Histological correlates of optical
coherence tomography in non-melanoma skin cancer. Skin Res Technol 2013;19: e10–9.

16     Preliminary criteria for the classification of systemic sclerosis (scleroderma).
Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum 1980;23:581–90.

17     Collins TJ. ImageJ for microscopy. Biotechniques 2007;43:25–30.

18     Clendenon JL, Phillips CL, Sandoval RM, et al. Voxx: a PC-based, near real-time

volume rendering system for biological microscopy. Am J Physiol Cell Physiol 2002;282:C213–18.

19     Bland JM, Altman DG. Statistical methods for assessing agreement between two
methods of clinical measurement. Lancet 1986;1:307–10.

20     LeRoy EC, Black C, Fleischmajer R, et al. Scleroderma (systemic sclerosis):
classification, subsets and pathogenesis. J Rheumatol 1988;15:202–5.

21     Merkel PA, Silliman NP, Clements PJ, et al. Patterns and predictors of change in
outcome measures in clinical trials in scleroderma: an individual patient meta-analysis of 629 subjects with diffuse cutaneous systemic sclerosis. Arthritis Rheum 2012;64:3420–9.

22     Farina G, Lafyatis D, Lemaire R, et al. A four-gene biomarker predicts skin disease
in patients with diffuse cutaneous systemic sclerosis. Arthritis Rheum 2010;62:580–8.

23     Milano A, Pendergrass SA, Sargent JL, et al. Molecular subsets in the gene
expression signatures of scleroderma skin. PLoS One 2008;3:e2696.

  24   Pendergrass SA, Lemaire R, Francis IP, et al. Intrinsic gene expression subsets of

diffuse cutaneou

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Targeted Nucleases

Posted in Chemical Genetics, Computational Biology/Systems and Bioinformatics, Genome Biology, Infectious Disease & New Antibiotic Targets, International Global Work in Pharmaceutical, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , on March 2, 2013| Leave a Comment »

Targeted Nucleases

Curator: Larry H Bernstein, MD, FCAP

A REVIEW of 3 published works

Targeted nucleases: spreading the joy
Nature Methods 10, 179 (2013)   http://dx.doi.org/10.1038/nmeth.2402

Published online 27 February 2013
New RNA-guided endonucleases (RGENs) are directed to their target sites
  • by a complementary RNA molecule.
In contrast to previous tools,
  • zinc-finger nucleases (ZFNs) and
  • transcription activator–like effector nucleases (TALENs),
the RGEN nuclease component itself does not require re-engineering to
  • target a new sequence.
The ability to manipulate DNA has led to a new genetics.
Professor of Genetics at Washington University’s School of Medicine
Backgrounders – introduction to issues of current interest

Restriction Endonucleases

In 1980, geneticists used the relatively new technique of gene splicing, which we will describe in this chapter, to introduce
  • the human gene that encodes interferon into a bacterial cell’s genome.
Interferon is a rare blood protein that increases human resistance to viral infection, and medical scientists have been interested in its possible usefulness in cancer therapy. Purification of the large amounts of interferon required for clinical testing would have been prohibitively expensive at the time.   Introducing the gene responsible for its production into a bacterial cell made that possible. The cell that had
  • acquired the human interferon gene proceeded to produce interferon at a rapid rate, and to grow and divide.

The  millions of interferon-producing bacteria growing in the culture were all descendants of the cell that had originally received the human interferon gene.

The Advent of Genetic Engineering
The human insulin gene has also been cloned in bacteria, and now insulin can be manufactured at little expense. Furthermore, cloning and related molecular techniques are needed to provide basic information about how genes are put together and regulated.
The essence of genetic engineering is
  • the ability to cut DNA into recognizable pieces and rearrange those pieces in different ways.
In the interferon experiment,
  • a piece of DNA carrying the interferon gene was
  • inserted into a plasmid,which
    • carried the gene into a bacterial cell.
Most other genetic engineering approaches bring the gene of interest into the target cell by first incorporating it into a plasmid or an infective virus.
This cutting is performed
  • by enzymes that recognize and
  • cleave specific sequences of nucleotides in DNA.
Discovery of Restriction Endonucleases
Scientific discoveries often have their origins in seemingly unimportant observations that receive little attention by researchers before their general significance is appreciated. In the case of genetic engineering, the original observation was that bacteria use enzymes to defend themselves against viruses.
Most organisms eventually evolve means of defending themselves from predators and parasites, and bacteria are no exception. Among the natural enemies of bacteria are bacteriophages, viruses that infect bacteria and multiply within them. At some point, they cause the bacterial cells to burst, releasing thousands more viruses.
Some types of bacteria have acquired powerful weapons against these viruses: they contain enzymes called restriction endonucleases
  • that fragment the viral DNA as soon as it enters the bacterial cell.
Many restriction endonucleases recognize
  1. specific nucleotide sequences in a DNA strand,
  2. bind to the DNA at those sequences, and
  3. cleave the DNA at a particular place within the recognition sequence.
Why don’t restriction endonucleases cleave the bacterial cells’ own DNA as well as that of the viruses?
  • bacteria modify their own DNA, using other enzymes known as methylases to add methyl (CH3) groups
  • to some of the nucleotides in the bacterial DNA.
When nucleotides within a restriction endonuclease’s recognition sequence have been methylated,
  • the endonuclease cannot bind to that sequence.
  • the bacterial DNA is protected from being degraded at that site.
  • but viral DNA has not been methylated, and therefore
    • is not protected from enzymatic cleavage.
How Restriction Endonucleases Cut DNA
The sequences recognized by restriction endonucleases are
  • typically four to six nucleotides long, and
  • they are often palindromes.
    • the nucleotides at one end of the recognition sequence are complementary to those at the other end, so that
    • the two strands of the DNA duplex have the same nucleotide sequence running in opposite directions for the length of the recognition sequence.

Two important consequences arise from this arrangement of nucleotides to be discussed.

Biochemistry. 5th edition.
Berg JM, Tymoczko JL, Stryer L.
Section 9.3  Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions
Bacteria and archaea have evolved mechanisms to protect themselves from viral infections so that viruses inject their DNA genomes into cells and the viral DNA hijacks the cell’s machinery A major protective strategy for the host is to use restriction endonucleases (restriction enzymes) to degrade the viral DNA. These  particular base sequences the enzymes recognize are called recognition sequences or recognition sites.
  • theycleave that DNA at defined positions.
  • The most well studied class are the so-called type II restriction enzymes.
Restriction endonucleases must show tremendous specificity at two levels.
  • First, they must cleave only DNA molecules that contain recognition sites (hereafter referred to as cognate DNA) without cleaving DNA molecules that lack these sites.
    •  endonucleases must cleave cognate DNA molecules much more than 5000 times as efficiently as they cleave nonspecific sites.
  • Second, restriction enzymes must not degrade the host DNA.

How do these enzymes manage to degrade viral DNA while sparing their own?

The restriction endonuclease EcoRV (from E. coli) cleaves double-stranded viral DNA molecules that contain the sequence 5′-GATATC-3′ but leaves intact host DNA containing hundreds of such sequences. The host DNA is protected by other enzymes called methylases, which methylate adenine bases within host recognition sequences (Figure 9.32). For each restriction endonuclease, the host cell produces a corresponding methylase that marks the host DNA and prevents its degradation.
  • These pairs of enzymes are referred to as restriction-modification systems.
Hydrolysis of a Phosphodiester Bond.
All restriction enzymes catalyze the hydrolysis of DNA phosphodiester bonds, leaving a phosphoryl group attached to the 5′ end. The bond that is cleaved is shown in red.
Mechanism Type 1 (covalent intermediate)
Mechanism Type 2 (direct hydrolysis)
Each postulates a different nucleophile to carry out the attack on the phosphorus. In either case, each reaction takes place by an in-line displacement path:
  • The incoming nucleophile attacks the phosphorus atom, and
  • a pentacoordinate transition state is formed.

This species has a trigonal bipyramidal geometry centered at the phosphorus atom, with

  • the incoming nucleophile at one apex of the two pyramids and the group that is displaced (the leaving group, L) at the other apex.
  • The two mechanisms differ in the number of times the displacement occurs in the course of the reaction.
The incoming nucleophile attacks the phosphorus atom, and
  • a pentacoordinate transition state is formed.
The analysis revealed that the stereochemical configuration at the phosphorus atom was inverted only once with cleavage. This result is consistent with a direct attack of water at phosphorus and
  • rules out the formation of any covalently bound intermediate (Figure 9.35).
Stereochemistry of Cleaved DNA.
Cleavage of DNA by EcoRV endonuclease results in overall inversion of the stereochemical configuration at the phosphorus atom.
9.3.2 Restriction Enzymes Require Magnesium for Catalytic Activity
Restriction endonucleases as well as many other enzymes that act on phosphate-containing substrates require Mg2+ or some other similar divalent cation for activity. What is the function of this metal?
It has been possible to examine the interactions of the magnesium ion when it is bound to the enzyme. Crystals have been produced of EcoRV endonuclease
  • bound to oligonucleotides that contain the appropriate recognition sequences.
These crystals are grown in the absence of magnesium to prevent cleavage; then,
  • when produced, the crystals are soaked in solutions containing the metal.
  • No cleavage takes place, allowing the location of the magnesium ion binding sites to be determined (Figure 9.36).
The magnesium ion was found to be bound to six ligands:
  1. three are water molecules,
  2. two are carboxylates of the enzyme’s aspartate residues, and
  3. one is an oxygen atom of the phosphoryl group at the site of cleavage.
The magnesium ion holds a water molecule in a position from which the water molecule can attack the phosphoryl group and,
  • in conjunction with the aspartate residues,
  • helps polarize the water molecule toward deprotonation.
Cleavage does not take place within these crystals. But a second magnesium ion must be present in an adjacent site for EcoRV endonuclease to cleave its substrate.
Magnesium Ion Binding Site in ECORV Endonuclease. The magnesium ion helps to activate a water molecule and positions it so that it can attack the phosphate.
9.3.3 The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity
Specificityis the defining feature of restriction enzymes. The recognition sequences for most restriction endonucleases are inverted repeats.
This arrangement gives the three-dimensional structure of the recognition site
  • a twofold rotational symmetry (Figure 9.37).
The restriction enzymes display a corresponding symmetry to facilitate recognition:
they are dimers whose two subunits are related by twofold rotational symmetry.
  • The matching symmetry of the recognition sequence and the enzyme has been confirmed
  • by the determination of the structure of the complex between EcoRV endonuclease and DNA fragments containing its recognition sequence (Figure 9.38).

The enzyme surrounds the DNA in a tight embrace.

Structure of the Recognition Site of ECORV Endonuclease.
(A) The sequence of the recognition site, which is symmetric around the axis of rotation designated in green.
(B) The inverted repeat within the recognition sequence of EcoRV
 Structure of the ECORV – Cognate DNA Complex.
This view of the structure of EcoRV endonuclease bound to a cognate DNA fragment is down the helical axis of the DNA. The two protein subunits are in yellow and blue, and the DNA backbone is in red.
A unique set of interactions occurs between the enzyme and a cognate DNA sequence. Within the 5′-GATATC-3′ sequence,
the G and A bases at the 5′ end of each strand and their Watson-Crick partners directly contact the enzyme
  • by hydrogen bonding with residues that are located in two loops,
  • one projecting from the surface of each enzyme subunit (Figure 9.39).
The most striking feature of this complex is the distortion of the DNA, which is substantially kinked in the center (Figure 9.40). The central two TA base pairs in the recognition sequence play a key role in producing the kink. They do not make contact with the enzyme but appear to be required because of their ease of distortion. 5′-TA-3′ sequences are known to be among the most easily deformed base pairs.
The distortion of the DNA at this site has severe effects on the specificity of enzyme action.
Hydrogen Bonding Interactions between ECORV Endonuclease and Its DNA Substrate.
One of the DNA-binding loops (in green) of EcoRV endonuclease is shown interacting with the base pairs of its cognate DNA binding site. Key amino acid residues are shown.
Distortion of the Recognition Site.
The DNA is represented as a ball-and-stick model. The path of the DNA helical axis, shown in red, is substantially distorted on binding to the enzyme. For the B form of DNA, the axis is straight (not shown).
Specificity is often determined by an enzyme’s binding affinity for substrates. In regard to EcoRV endonuclease, however, binding studies performed in the absence of magnesium have demonstrated that
  • the enzyme binds to all sequences, both cognate and noncognate, with approximately equal affinity.
  • the structures of complexes formed with noncognate DNA fragments are strikingly different from those formed with cognate DNA:
    • the noncognate DNA conformation is not substantially distorted (Figure 9.41).

This lack of distortion has important consequences with regard to catalysis. No phosphate is positioned sufficiently close to the active-site aspartate residues to complete a magnesium ion binding site (see Figure 9.36). Hence, the nonspecific complexes do not bind the magnesium ion and

  • the complete catalytic apparatus is never assembled.
The distortion of the substrate and the subsequent binding of the magnesium ion account for
  • the catalytic specificity of more than 1,000,000-fold that is observed for EcoRV endonuclease
Nonspecific and Cognate DNA within ECORV Endonuclease.
A comparison of the positions of the nonspecific (orange) and the cognate DNA (red) within EcoRV reveals that,
  • in the nonspecific complex, the DNA backbone is too far from the enzyme
We can now see the role of binding energy in this strategy for attaining catalytic specificity.
In binding to the enzyme, the DNA is distorted in such a way that
  • additional contacts are made between the enzyme and the substrate, increasing the binding energy.
However, this increase is canceled by the energetic cost of distorting the DNA from its relaxed conformation (Figure 9.42). Thus, for EcoRV endonuclease,
there is little difference in binding affinity for cognate and nonspecific DNA fragments.
  • However, the distortion in the cognate complex dramatically affects catalysis by completing the magnesium ion binding site.
  • This example illustrates how enzymes can utilize available binding energy to deform substrates and poise them for chemical transformation.
  • Interactions that take place within the distorted substrate complex
    • stabilize the transition state leading to DNA hydrolysis.
Greater Binding Energy of EcoRV Endonuclease Bound to Cognate Versus Noncognate Dna.
The additional interactions between EcoRV endonuclease and cognate DNA increase the binding energy, which can be used to drive DNA distortions.
The distortion in the DNA explains how methylation blocks catalysis and protects host-cell DNA. When a methyl group is added to the amino group of the adenine nucleotide at the 5′ end of the recognition sequence,
  • the methyl group’s presence precludes the formation of a hydrogen bond between the amino group and the side-chain carbonyl group of asparagine 185 (Figure 9.43).
  • This asparagine residue is closely linked to the other amino acids that form specific contacts with the DNA.
  • The absence of the hydrogen bond disrupts other interactions between the enzyme and the DNA substrate, and
    • the distortion necessary for cleavage will not take place.
Methylation of Adenine.
The methylation of adenine blocks the formation of hydrogen bonds
  • between EcoRV endonuclease and cognate DNA molecules and
  • prevents their hydrolysis.
 9.3.4 Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer
 Type II restriction enzymes are prevalent in Archaea and Eubacteria. What can we tell of the evolutionary history of these enzymes?
Comparison of the amino acid sequences of a variety of type II restriction endonucleases did not reveal significant sequence similarity between most pairs of enzymes. However, a careful examination of three-dimensional structures, taking into account the location of the active sites, revealed
  • the presence of a core structure conserved in the different enzymes.
  • This structure includes β strands that contain the aspartate (or, in some cases, glutamate) residues forming the magnesium ion binding sites (Figure 9.44).
 A Conserved Structural Core in Type II Restriction Enzymes.
Four conserved structural elements, including the active-site region (in blue), are highlighted in color in these models of a single monomer from each dimeric enzyme.
These observations indicate that many type II restriction enzymes are indeed evolutionary related. Analyses of the sequences in greater detail suggest that bacteria may have obtained genes encoding these enzymes 
  • from other species by horizontal gene transfer, the passing between species of pieces of DNA (such as plasmids) that provide
  • a selective advantage in a particular environment.
For example, EcoRI (from E. coli) and RsrI (from Rhodobacter sphaeroides) are 50% identical in sequence over 266 amino acids, clearly
  • indicative of a close evolutionary relationship.
  • these species of bacteria are not closely related,
  • as is known from sequence comparisons of other genes and other evidence.
Thus, it appears that these species obtained the gene for this restriction endonuclease from a common source
  • more recently than the time of their evolutionary divergence.
  • the gene encoding EcoRI endonuclease uses particular codons to specify given amino acids that are
  • strikingly different from the codons used by most E. coli genes, which
    • suggests that the gene did not originate in E. coli.
  • Horizontal gene transfer may be a relatively common event.
    • genes that inactivate antibiotics are often transferred, leading to the transmission of antibiotic resistance from one species to another.
For restriction-modification systems,
  • protection against viral infections may have favored horizontal gene transfer.
Biochemistry. 5th edition.
Berg JM, Tymoczko JL, Stryer L.
New York: W H Freeman; 2002.
  • Cleavage Is by In-Line Displacement of 3′ Oxygen from Phosphorus by Magnesium-Activated Water
  • Restriction Enzymes Require Magnesium for Catalytic Activity
  • The Complete Catalytic Apparatus Is Assembled Only Within Complexes of Cognate DNA Molecules, Ensuring Specificity
  • Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer

By Richard Wheeler (Zephyris) 2007. Image of E...

By Richard Wheeler (Zephyris) 2007. Image of EcoRV homodimer in complex with a DNA substrate. From . (Photo credit: Wikipedia)

HindIII restriction endonuclease in complex wi...

HindIII restriction endonuclease in complex with cognate DNA (Photo credit: Wikipedia)

English: 3d surface model of HindIII dimer com...

English: 3d surface model of HindIII dimer complexed with a DNA fragment from PDB 2E52. Ref.: Watanabe, N., Sato, C., Takasaki, Y., Tanaka, I. Crystal structural analysis of HindIII restriction endonuclease in complex with cognate DNA at 2.0 angstrom resolution to be published (Photo credit: Wikipedia)

English: BglII active site containing residues...

English: BglII active site containing residues that coordinate to a metal ion and water molecules including the nucleophilic water that breaks the scissile phosphodiester bond at the recognition site. (Photo credit: Wikipedia)

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Recurrence Risk for Breast Cancer

Posted in Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Interviews with Scientific Leaders, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , on March 2, 2013| 4 Comments »

Recurrence Risk for Breast Cancer

Reporter: Larry H Bernstein, MD,  FCAP

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Testing recurrence risk for breast cancer
Karen Titus
June 2011 CAP Today

http://www.cap.org/apps/cap.portal_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=cap_today%2F0611%2F0611a_testing_recurrence.html&_state=maximized&_pageLabel=cntvwr

{EXTRACT}

Gene panels for breast cancer recurrence risk have arrived. In fact, they’ve been around since the mid-2000s. And now, like guests at a wedding reception, it’s a matter of figuring out where to seat them.
Like it or not, tests such as Oncotype DX (Genomic Health Inc.), MammaPrint (Agendia), and Mammostrat (Clarient)—to name just a few—are making their presence felt.
Clinicians favor these tests for a simple reason: the results help them decide if patients with breast cancer need chemotherapy. More broadly, the tests reflect a shift in thinking among physicians, one that emphasizes molecular profiling of tumors. They’ve arrived on the scene when physicians are also starting to question the value of lymph node status to help determine treatment.George W. Sledge, MD, finds these changes remarkable. Not all that long ago, he might have pink-slipped a test that would help parse treatment decisions. When the NIH held its consensus development conference on adjuvant therapy with breast cancer in 2000, he recalls, the agreement was, basically, that everyone with a tumor greater than one centimeter ought to be treated with chemotherapy. “There’s no question that resulted in us hugely overtreating patients,” he says. “So I think a test that reduces the quantity of human suffering by half in that group is a useful test,” says Dr. Sledge, professor of medicine and pathology, Indiana University, Indianapolis, and immediate past president of the American Society of Clinical Oncology.
In clinical practice, these tests are functioning like traffic managers. “We now see fewer patients getting chemotherapy who would have gotten it before,” says Thomas Julian, MD, professor of surgery, Drexel University College of Medicine, Philadelphia, and director of breast surgical oncology for the West Penn Allegheny Health Care System, Pittsburgh. “We’re also seeing a few who are getting chemo who might not have gotten it before. So it’s changed in both directions,” says Dr. Julian, who is also senior surgical director for medical affairs for the National Surgical Adjuvant Breast and Bowel Project.
Oncotype DX is a real-time RT-PCR assay measuring RNA expression in 16 cancer-related genes and five reference genes, using paraffin-embedded tissue. Results are given as a recurrence score between zero and 100, which are translated as low risk (a score of 18 or lower), medium risk (19 to 30), or high risk (31 or above). The MammaPrint microarray assay measures expression of 70 genes in fresh tissue; it categorizes patients as either high risk, with a so-called poor signature, or low risk (a so-called good signature) for recurrence. There is no intermediate category. Mammostrat is an immunohistochemistry test measuring five markers: p53, HTF9C, CEACAM5, NDRG1, and SLC7A5. The results are combined into a quantitative risk index: low, moderate, and high. For now, only MammaPrint has FDA clearance.
The test is not useful in patients whose tumors are HER2 positive. The test nearly always will show such patients to be at high risk; moreover, the paradigm for treating such patients is with chemotherapy and trastuzumab (Herceptin). It is used for patients who are lymph node negative, ER positive, and HER2 negative, with “moderate-size tumors—say, tumors that are over a centimeter but less than four or five centimeters. Another consideration is tumor size. The test is most useful for tumors of around five millimeters or greater in size.For patients with very, very small tumors—one, two, three millimeters—there’s no need for the test. Elizabeth Hammond, MD, agrees these tests are useful, although she suspects they may best prove their mettle in second- or third-generation assays. It’s simple biology: phenotypic expression of a genetic alteration of ER or HER2 status is the result of cell-signaling pathway changes. “Looking at multiple expressions of that problem with a gene panel, either by RT-PCR or some other method, will in the long run give us better information.

Comment:  In 1982, labs were running RIA assays for Estrogen Receptor.  It was known for some time that breast cancer is estrogen-dependent.  This was a major discovery by a surgeon at University of Chicago, that led to oophorectomy with resection of the lesion.  The assay was quite elaborate and required a “scatchard plot”.  The assay was no longer used when a good histochemical stain became widely used with a progesterone receptor a few years later.  We went into the 1990’s knowing that if the patient is pre-menopausal, positive ER+/PR+ is likely, and the cancer is aggressive.  If the patient was postmenopausal, the test is more likely ER/PR negative.  This gives us a perspective on how far we have come.

Image representing Genomic Health as depicted ...

Image via CrunchBase

English: Validation chart for Agendia's MammaP...

English: Validation chart for Agendia’s MammaPrint Assay, part of the Symphony Breast Cancer Suite (Photo credit: Wikipedia)

Ovarian and breast cancer patients in a pedigr...

Ovarian and breast cancer patients in a pedigree chart of a family (Photo credit: Wikipedia)

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Attitudes of Patients about Personalized Medicine

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Health Economics and Outcomes Research, Health Law & Patient Safety, International Global Work in Pharmaceutical, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , on March 2, 2013| 4 Comments »

Attitudes of Patients about Personalized Medicine

Curator: Larry H Bernstein, MD, FCAP

Attitudes of Patients With Cancer About Personalized Medicine

SW Gray, K Hicks-Courant, et al.

Personalized Medicine

Personalized Medicine (Photo credit: Wikipedia)

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Understanding the Role of Personalized Medicine

Posted in Health Economics and Outcomes Research, Health Law & Patient Safety, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , on March 2, 2013| 3 Comments »

Understanding the Role of Personalized Medicine

Reporter: Larry H. Bernstein, MD, FCAP

 

Do Patients Understand the Role of Cancer Genetic Testing?

Maurie Markman, MD

Personalized Medicine

Personalized Medicine (Photo credit: Wikipedia)

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Reprogramming Cell Fate

Posted in Biological Networks, Gene Regulation and Evolution, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, International Global Work in Pharmaceutical, Liver & Digestive Diseases Research, Metabolomics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , , , , , on March 2, 2013| 4 Comments »

Reprogramming Cell Fate

 

Reporter: Larry H.Bernstein, MD, FCAP

Kathy Liszewski: reporting Gordon Conference “Reprogramming Cell Fate” meeting
M. Azim Surani, Ph.D., Univ Cambridge
Source unknown: June 21, 2012;32(11)
They report two critical steps both of which are needed for exploring epigenetic reprogramming.  While females have two X chromosomes ,
  • the inactivation of one is necessary for cell differentiation.
  • Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired.

Pluripotent stem cells can generate – any fetal or adult cell type but

    • don’t develop into a complete organism.
Pioneer transcription factors take the lead in – facilitating cellular reprogramming – and responses to environmental cues.
Multicellular organisms consist of
  • functionally distinct cellular types
  • produced by differential activation of gene expression.
They seek out and bind specific regulatory sequences in DNA, even though DNA is coated with and condensed into a thick fiber of chromatin.
The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, endows the competence for gene activity,
  • being among the first transcription factors to
  • engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for
  • induction of the liver program.
    •  nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes.

organ regeneration example from induced plurip...

organ regeneration example from induced pluripotent stem cells(iPS cell) (Photo credit: Wikipedia)

English: Pathway of stem cell differentiation

English: Pathway of stem cell differentiation (Photo credit: Wikipedia)

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Unraveling Retrograde Signaling Pathways

Posted in Biological Networks, Gene Regulation and Evolution, Cell Biology, Signaling & Cell Circuits, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, International Global Work in Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, tagged , , , , , , , , , , , on March 2, 2013| 2 Comments »

Unraveling Retrograde Signaling Pathways

Reporter: Larry H. Bernstein, MD, FCAP

Unraveling Retrograde Signaling

Image Source: Created by Noam Steiner Tomer 8/10/2020

Unraveling retrograde signaling pathways: finding candidate signaling molecules via metabolomics and systems biology driven approaches
C Caldana, AR Fernie, L Willmitzer and D Steinhauser
Front. Plant Sci. 2012; 3:267.                    http://dx.doi.org/10.3389/fpls.2012.00267

http://fpls.com/Unraveling retrograde signaling pathways: finding candidate signaling molecules via
metabolomics and systems biology driven approaches

signals can be generated within organelles, such as chloroplasts and mitochondria,

  • modulating the nuclear gene expression in a process called
    • retrograde signaling.

Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data
of Arabidopsis thaliana, revealed the identification of metabolites which are

  • putatively acting as mediators of nuclear gene expression.

http://fpls.com/unraveling_retrograde_signaling_pathways:_finding_candidate_signaling_molecules_
via_metabolomics_and_systems_biology_driven_approaches

English: Plant Pathology in Arabidopsis thaliana

English: Plant Pathology in Arabidopsis thaliana (Photo credit: Wikipedia)

B0004313 Gene expression in normal and cancer ...

B0004313 Gene expression in normal and cancer cells (Photo credit: wellcome images)

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Modeling Targeted Therapy

Posted in Advanced Drug Manufacturing Technology, Biological Networks, Gene Regulation and Evolution, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, International Global Work in Pharmaceutical, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Industry Competitive Intelligence, Pharmacotherapy and Cell Activity, Statistical Methods for Research Evaluation, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , on March 2, 2013| 3 Comments »

Modeling Targeted Therapy

Reporter: Larry H. Bernstein, MD, FCAP
pharmaceuticalintelligence.com/2013/03/02/modeling-targeted-therapy/

Some Perspectives on Network Modeling in Therapeutic Target Prediction
R Albert, B DasGupta and N Mobasheri
Biomedical Engineering and Computational Biology Insights 2013; 5: 17–24    http://dx.doi.org/BECBI/Albert_DasGupta_ Mobasheri
Key steps of a typical therapeutic target identification problem include synthesizing or inferring the complex network of interactions relevant to the disease, connecting this network to the disease-specific behavior, and predicting which components are key mediators of the behavior
http://www.la-press.com/Some_Perspectives_on_Network_Modeling_in_Therapeutic_Target_Prediction/

Journal of Computational Biology

Journal of Computational Biology (Photo credit: Wikipedia)

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The Heart: Vasculature Protection – A Concept-based Pharmacological Therapy including THYMOSIN

Posted in Atherogenic Processes & Pathology, Bio Instrumentation in Experimental Life Sciences Research, Biomarkers & Medical Diagnostics, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, FDA Regulatory Affairs, Frontiers in Cardiology and Cardiovascular Disorders, HTN, Human Immune System in Health and in Disease, Origins of Cardiovascular Disease, Personalized and Precision Medicine & Genomic Research, Pharmaceutical R&D Investment, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Stem Cells for Regenerative Medicine, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , on February 28, 2013| 14 Comments »

Heart Vasculature – Regeneration and Protection of Coronary Artery Endothelium and Smooth Muscle: A Concept-based Pharmacological Therapy of a Combination Three Drug Regimen including THYMOSIN 

Author & Curator: Aviva Lev-Ari, PhD, RN

 

ABSTRACT

A concept-based original pharmacological therapy was developed for the research results presented in Cell by Wu, Fujiwara, Cibulsky et al. (2006), Moretti, Caron, Nakano, et al. (2006) and for the research results in Nature by Smart, Risebro, Melville, et al. (2007). We propose the following concept-based original pharmacological therapy design for Preoperative and Postoperative management of cardiac injury to heart tissue, smooth muscle, to aorta and coronary artery disease. This is a treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state while restoring its pluripotency.

VIEW VIDEO

What are Induced Pluripotent Stem Cells? (iPS Cells)

 http://www.youtube.com/watch?v=i-QSurQWZo0

Lasker Lecture: Dr. Shinya Yamanaka, 2 of 3:

Induced Pluripotent Stem Cells? (iPS Cells)

http://www.youtube.com/watch?v=DQNoyDwCPzM

Multipotent Embryonic Isl1^+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification

Alessandra A Moretti, Leslie L Caron, Atsushi A Nakano, Jason T JT Lam, Alexandra A Bernshausen,Yinhong Y Chen, Yibing Y Qyang, Lei L Bu, Mika M Sasaki, Silvia S Martin-Puig, Yunfu Y Sun, Sylvia M SM Evans, Karl-Ludwig KL Laugwitz, Kenneth R KR Chien
Cell 127(6):15 (2006), PMID 17123592

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1^+ precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1^+ cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1^+/Nkx2.5^+/flk1^+ defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1^+ cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

http://pubget.com/paper/17123592/Multipotent_Embryonic_Isl1___Progenitor_Cells_Lead_to_Cardiac__Smooth_Muscle__and_Endothelial_Cell_Diversification

 

Thymosin beta 4 (Tβ4)

is a highly conserved, 43-amino acid acidic peptide (pI 4.6) that was first isolated from bovine thymus tissue over 25 years ago. It is present in most tissues and cell lines and is found in high concentrations in blood platelets, neutrophils, macrophages, and other lymphoid tissues. Tβ4 has numerous physiological functions, the most prominent of which being the regulation of actin polymerization in mammalian nucleated cells and with subsequent effects on actin cytoskeletal organization, necessary for cell motility, organogenesis, and other important cellular events.

Recently,

  • Tβ4 was shown to be expressed in the developing heart and found to stimulate migration of cardiomyocytes and endothelial cells, promote survival of cardiomyocytes (Nature, 2004), and most recently
  • to play an essential role in all key stages of cardiac vessel development: vasculogenesis, angiogenesis, and arteriogenesis (Nature 2006).
  • These results suggest that Tβ4 may have significant therapeutic potential in humans to protect myocardium and promote cardiomyocyte survival in the acute stages of ischemic heart disease.

RegeneRx Biopharmaceuticals, Inc. is developing Tβ4 for the treatment of patients with acute myocardial infarction (AMI). Such efforts presented will include the formulation, development, and manufacture of a suitable drug product for use in the clinic, the performance of nonclinical pharmacology and toxicology studies, and the implementation of a phase 1 clinical protocol to assess the safety, tolerability, and the pharmacokinetics of Tβ4 in healthy volunteers.

http://onlinelibrary.wiley.com/doi/10.1196/annals.1415.051/abstract;jsessionid=BB7CC897572B7DDB60370EA64A81FC3F.d01t03?deniedAccessCustomisedMessage=&userIsAuthenticated=false

EXPLORATIONS with THYMOSIN beta4 for INDUCING ADULT EPICARDIAL PROGENETOR MOBILIZATION AND NEOVASCULARIZATION is presented in

Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

http://pharmaceuticalintelligence.com/2012/04/30/93/

EXPLORATIONS with THYMOSIN beta4 for INDUCTION of ARTERIOGENESIS, Prevention and repair of damaged cardiac tissue post MI and other CVD related research projects are presented in

Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel

http://pharmaceuticalintelligence.com/2013/02/27/arteriogenesis-and-cardiac-repair-two-biomaterials-injectable-thymosin-beta4-and-myocardial-matrix-hydrogel/

Recent research results with THYMOSIN beta4 in use for Cardiovascular Disease

appeared in 2010:

Annals of the New York Academy of Sciences, May 2010 Volume 1194 Pages ix–xi, 1–230

http://onlinelibrary.wiley.com/doi/10.1111/nyas.2010.1194.issue-1/issuetoc

appeared in 2012:

  • Thymosins in Health and Disease II: 3rd International Symposium on The Emerging Clinical Applications of Tymosin beta 4 in Cardiovascular Disease

Annals of the New York Academy of Sciences, October 2012 Volume 1270 Pages vii-ix, 1–121.

http://onlinelibrary.wiley.com/doi/10.1111/nyas.2012.1270.issue-1/issuetoc

Allan L. Goldstein, Enrico Garaci, Editors, Thymosins in Cardiovascular Disease, November 2012, Wiley-Blackwell (paperback)

http://www.wiley.com/WileyCDA/WileyTitle/productCd-1573319104.html?cid=RSS_WILEY2_LIFEMED

Selected for this article are the abstracts of the following research projects, all were presented at the 2nd International Symposium, May 2010:

Thymosin β4: structure, function, and biological properties supporting current and future clinical applications

Published studies have described a number of physiological properties and cellular functions of thymosin β4 (Tβ4), the major G-actin-sequestering molecule in mammalian cells. Those activities include the promotion of cell migration, blood vessel formation, cell survival, stem cell differentiation, the modulation of cytokines, chemokines, and specific proteases, the upregulation of matrix molecules and gene expression, and the downregulation of a major nuclear transcription factor. Such properties have provided the scientific rationale for a number of ongoing and planned dermal, corneal, cardiac clinical trials evaluating the tissue protective, regenerative and repair potential of Tβ4, and direction for future clinical applications in the treatment of diseases of the central nervous system, lung inflammatory disease, and sepsis. A special emphasis is placed on the development of Tβ4 in the treatment of patients with ST elevation myocardial infarction in combination with percutaneous coronary intervention, pp.179-189, May 2010.

  

Thymosin β4 and cardiac repair

Hypoxic heart disease is a predominant cause of disability and death worldwide. As adult mammals are incapable of cardiac repair after infarction, the discovery of effective methods to achieve myocardial and vascular regeneration is crucial. Efforts to use stem cells to repopulate damaged tissue are currently limited by technical considerations and restricted cell potential. We discovered that the small, secreted peptide thymosin β4 (Tβ4) could be sufficiently used to inhibit myocardial cell death, stimulate vessel growth, and activate endogenous cardiac progenitors by reminding the adult heart on its embryonic program in vivo. The initiation of epicardial thickening accompanied by increase of myocardial and epicardial progenitors with or without infarction indicate that the reactivation process is independent of injury. Our results demonstrate Tβ4 to be the first known molecule able to initiate simultaneous myocardial and vascular regeneration after systemic administration in vivo. Given our findings, the utility of Tβ4 to heal cardiac injury may hold promise and warrant further investigation, pp. 87-96, May 2010.

 

Thymosin β4 facilitates epicardial neovascularization of the injured adult heart

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans

http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html

After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin β4 (Tβ4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC-derived endothelial-restricted neovascularization constitutes suboptimal “endogenous repair,” following injury, which is significantly augmented by Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage, pp. 97-104, May 2010.

 

Thymosin β4: a key factor for protective effects of eEPCs in acute and chronic ischemia

Acute myocardial infarction is still one of the leading causes of death in the industrial nations. Even after successful revascularization, myocardial ischemia results in a loss of cardiomyocytes and scar formation. Embryonic EPCs (eEPCs), retroinfused into the ischemic region of the pig heart, provided rapid paracrine benefit to acute and chronic ischemia in a PI-3K/Akt-dependent manner. In a model of acute myocardial ischemia, infarct size and loss of regional myocardial function decreased after eEPC application, unless cell pre-treatment with thymosin β4 shRNA was performed. Thymosin ß4 peptide retroinfusion mimicked the eEPC-derived improvement of infarct size and myocardial function. In chronic ischemia (rabbit model), eEPCs retroinfused into the ischemic hindlimb enhanced capillary density, collateral growth, and perfusion. Therapeutic neovascularization was absent when thymosin ß4 shRNA was introduced into eEPCs before application. In conclusion, eEPCs are capable of acute and chronic ischemia protection in a thymosin ß4 dependent manner, pp. 105-111, May 2010.

Clinical Study Data of Thymosin beta 4 Presented

Published on October 3, 2009 at 5:10 AM

REGENERX BIOPHARMACEUTICALS, INC. (NYSE Amex:RGN) today reported on several clinical studies with Thymosin beta 4 (Tβ4) presented the Second International Symposium on Thymosins in Health and Disease, in Catania, Italy. The following are synopses of the presentations:

Myocardial Development of RGN-352 (Injectable Tβ4 Peptide)

David Crockford, RegeneRx’s vice president for clinical and regulatory affairs presented an overview of the biological properties that support Tβ4’s near term and long term clinical applications. Mr. Crockford noted that special emphasis is being placed on the development of RGN-352 for the systemic (injectable) treatment of patients with ST-elevation myocardial infarction (STEMI) in combination with percutaneous coronary intervention, the current standard of care in most western countries for this common type of heart attack. The goal with RGN-352 is to prevent or repair continued damage to cardiac tissue post-heart attack, when such tissue around the damaged site remains at risk.

Dr. Dennis Ruff, vice president and medical director of ICON, and principal investigator, presented the most current results on the Phase I safety study with RGN-352 entitled, “A Randomized, Double-blind, Placebo-controlled, Dose-response Phase I Study of the Safety and Tolerability of the Intravenous Administration of Thymosin Beta 4 and its Pharmacokinetics After Single and Multiple Doses in Healthy Volunteers.” Dr. Ruff discussed key aspects of the study and concluded with, “There were no dose limiting or serious adverse events throughout the dosing period. Synthetic Tβ4 administered intravenously up to 1260 mg, and for up to 14 days, appears to be well tolerated with low incidence of adverse events and no evidence of serious adverse events.”

http://www.news-medical.net/news/20091003/Clinical-study-data-of-Thymosin-beta-4-presented.aspx

RegeneRx Receives Notice of Allowance from Chinese Patent Office for Treatment and Prevention of Heart Disease

RegeneRx Receives Notice of Allowance from Chinese Patent Office for Treatment and Prevention of Heart Disease

February 7, 2013 — Rockville, MD

RegeneRx Biopharmaceuticals, Inc. (OTC Bulletin Board: RGRX) (“the Company” or “RegeneRx”) today announced that it has received a Notice of Allowance of a Chinese patent application for uses of Thymosin beta 4 (TB4) for treating, preventing, inhibiting or reducing heart tissue deterioration, injury or damage in a subject with heart failure disease. Claims also include uses for restoring heart tissue in those subjects. The patent will expire July 26, 2026 http://www.regenerx.com/wt/page/pr_1360265259

Theoretical treatment protocol differential between the Preoperative which may be between 3 to 6 month, and the Postoperative which may prolong to one year.

Proposal for Preoperative Treatment – Three drug combination involves

  • Drug # 1: Thymosin fraction 5 (a sublingual composition)
  • Drug # 2: Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID))
  • Drug # 3: Clevidipine (blood pressure lowering drug, (no effect on heart rate))

 

Proposal for Postoperative Treatment – Three drug combination consists of

  • Drug # 1: Thymosin fraction 5 (a sublingual composition)
  • Drug # 4: ACEI (Captopril (50mg))
  • Drug # 5: Beta Blocker and Diuretic (Metoprolol and hydrochlorothiazide (50 mg/25 mg)) Lopressor HCT

Unprecedented novel paradigm development in the scientific understanding of the origin of

  • (a) myocardial cells
  • (b) smooth muscle cells
  • (c) endothelial cells
  • (d) pace maker cells and
  • (e) heart vasculature: aorta, pulmonary artery and coronary arteries, occurred in 2006.

In a seminal article in Cell, “Developmental Origin of a Bipotential Myocardial and Smooth Muscle Cell Precursor in the Mammalian Heart” Wu, et al., (2006), described their discovery as follows:

“Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood.” To explore the relationship between developmental fate and potential.” They “isolated a cardiac-specific Nkx2.5+ cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5+ cells from in vitro differentiated murine embryonic stem cells and found ~28% of these cells expressed c-kit. These c-kit+ cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell.” They “confirmed these findings by isolating c-kit+Nkx2.5+ cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.”

Another breakthrough article in Cell, “Multipotent Embryonic Isl1+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification” Moretti, et al., (2006) described their discovery as follows:

“Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors.” They “use genetic fate-mapping studies to document that isl1+ precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells”, they “clonally amplified a cellular hierarchy of isl1+ cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1+/Nkx2.5+/flk1+ defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1+ cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.” (Moretti, et al., 2006).

Third scientific breakthrough was reported in Nature on the roles that Thymosin beta4 play in

  • (a) coronary vessel development
  • (b) induction of adult epicardial cell migration
  • (c) cardiomyocyte survival by vascularization which is dependent on Thymosin beta4 and
  • (d) identification of the pro-angiogenic tetrapeptide AcSDKP which is produced by endoproteinase activity of Thymosin beta4 (Smart, et al., 2007).

That new level of understanding has the potential to generate new pharmaco therapies to upregulate biological processes that underlie the function of the various compartments of the cardiovascular system, as new scientific explanations became available in 2006.

We have developed a methodology for discovery of concept-based original pharmacological therapy designs for combination of several drug regimens. We carry out two types of research strategy. Methodology Strategy Type One: we develop an original pharmacological therapy design specialized in addressing medical problems identified in targeted follow up studies on mortality and morbidity of cardiovascular patients. Methodology Strategy Type One is implemented in Lev-Ari & Abourjaily (2006a, 2006b, 2006c). We designed a specialized pharmaco therapy for the research results presented in NEJM, on “Circulating Endothelial Progenitor Cells and Cardiovascular Outcomes” (Werner, Kosiol, Schiegl, et al., 2005a) and the editorial interpretation of these research results by Rosenzweig  (2005). We proposed the following concept-based original pharmacological therapy design for Endogenous Augmentation of circulating Endothelial Progenitor Cells for Reduction of Risk for Macrovascular Cardiac Events.

 

Proposal of Treatment – Three drug combination

  • Inhibition of ET-1, ETA and ETA-ETB (Bosentan)
  • Induction of NO production and stimulation of eNOS (Nebivolol)
  • Stimulation of PPAR-gamma (substitute to Rosiglitazone)

Our Methodology Strategy Type Two involves discovery of concept-based original pharmacological therapy design for combination of several drug regimens for underlying biological processes discovered in the pursuit of basic researchers conducted in wet lab experiments by vascular biologists and molecular cardiologists. Here, we developed a concept-based original pharmacological therapy for the research results presented in Cell by Wu, Fujiwara, Cibulsky et al. (2006), Moretti, Caron, Nakano, et al. (2006) and for the research results in Nature by Smart, Risebro, Melville, et al. (2007). We propose the following concept-based original pharmacological therapy design for Preoperative and Postoperative management of cardiac injury to heart tissue, smooth muscle and to aorta and coronary artery disease. This is a treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state and restoring its pluripotency.

 

Proposal for Preoperative Treatment – Three drug combination

  • Drug # 1:

Thymosin fraction 5 (a sublingual composition)

  • Drug # 2:

Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID))

  • Drug # 3:

Clevidipine (blood pressure lowering drug, no effect on heart rate)

Proposal for Postoperative Treatment – Three drugs combination

  • Drug # 1:

Thymosin fraction 5 (a sublingual composition)

  • Drug # 4:

ACEI (Captopril (50mg))

  • Drug # 5:

HCTBeta Blocker and Diuretic (Metoprolol and hydrochlorothiazide (50 mg/25 mg)) Lopressor

 

Thymosin beta4 Induces Adult Epicardial Progenitor Mobilization and Neovascularization

 

Smart et al. (2007) implicate Thymosine beta4 (Tb4) with the following functions: (a) Tb4 in regulating all three key stages of cardiac vessel development: coronary vasculogenesis, angiogenesis and arteriogenesis – collateral growth; (b) identify the adult epicardium as a potential source of vascular progenitors which, when stimulated by Tb4, migrate and differentiate into smooth muscle and endothelial cells; (c) the ability of Tb4 to promote coronary vascularization both during development and in the adult, enhances cardiomyocyte survival and contributes significantly towards Tb4-induced cardioprotection.

The reaction in the scientific community to these investigative results was most favorable.

“These results are very exciting because most humans suffering from ischemic cardiac events, either acutely or chronically, do not develop the collateral vessel growth necessary to preserve and restore heart tissue. If, in humans, we see the same effects as seen in mice, TB4 would be the first drug to prevent loss of (heart) muscle cells and restore blood flow in this manner and provide a new and much needed treatment modality for these patients,”

commented Deepak Srivastava, M.D., Professor and Director, Gladstone Institute of Cardiovascular Disease, University of California San Francisco, CA. Dr. Srivastava and his colleagues published the first paper on TB4’s effects on myocardial infarction in Nature in November 2004.

http://phx.corporate-ir.net/phoenix.zhtml?c=144396&p=irol-newsArticle&ID=932573&highlight=

VIEW VIDEO

http://www.youtube.com/watch?v=Vjj7LSuSMAo

 

Review of the Chemistry and the Mechanism of action supporting the process by which, N-acetyl-seryl-aspartyl-lysyl- proline (Ac-SDKP) stimulates endothelial cell differentiation from adult epicardium, is presented in

Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

http://pharmaceuticalintelligence.com/2012/04/30/93/

A Concept-based Pharmacologic Therapy of a Combined Three Drug Regimen for Regeneration and Protection of Coronary Artery Endothelium and Smooth Muscle.

This is a treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state and restoring its pluripotency.

 

Preoperative Treatment – Three drugs

  • Drug # 1:
  • Thymosin fraction 5 (a sublingual composition)
  • Drug # 2:
  • Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID)) (25 mg PO bid)
  • Drug # 3:
  • Clevidipine (Blood pressure lowering drug, no effect on heart rate)

 

Postoperative Treatment – Three drugs

  • Drug # 1:
  • Thymosin fraction 5 (a sublingual composition)
  • Drug # 4:
  • ACEI (Captopril (50mg))
  • Drug # 5:
  • Beta Blocker and diuretic (Metoprolol and hydrochlorothiazide (50 mg/25 mg)) Lopressor HCT

Original Drug Therapy Combination Proposed

Drug # 1: Thymosin fraction 5

Drug # 2: Indomethacin

Drug # 3: Clevidipine

Drug # 1:

Sublingual compositions comprising Thymosin fraction 5

United States Patent:  6,733,791

http://www.pharmcast.com/Patents100/Yr2004/May2004/051104/6733791_Sublingual051104.htm

http://www.google.com/patents/US6733791

The compositions comprise a room temperature stable peptide or complex of peptides that may be administered in a dosage of between 0.0001 mg/ml or gm and 600 mg/ml or gm.

Thymosin beta4 is released from human blood platelets and attached by factor XIIIa (transglutaminase) to fibrin and collagen (Huff et al. 2002). They suggest that Thymosin beta4 cross-linking is mediated by factor XIIIa, a transglutaminase that is co-released from stimulated platelets. This provides a mechanism to increase the local concentration of Thymosin beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses (Al-Nedawi, et al., 2004). The beta-Thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G-actin sequestering peptide. There is increasing evidence for extracellular functions of Thymosin beta4. For example, Thymosin beta4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. They show that Thymosin beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, Thymosin beta10 (Huff et al. 2002).

Rationale for selection of Sublingual compositions comprising Thymosin fraction 5

The actin binding motif of Thymosin beta4 is an essential site for its angiogenic activity (Philip, et al. (2003). Thymosin beta4 is presented in Smart, et al. (2007) in the Nature article as a single factor that can potentially couple myocardial and coronary vascular regeneration in failing mouse hearts. They have shown that cells in the heart’s outer layer can migrate deeper into a failing organ to carry out essential repairs. The migration of progenitor cells is controlled by the protein Thymosin beta 4, already known to help reduce muscle cell loss after a heart attack.

http://news.bbc.co.uk/2/hi/health/6143286.stm

The discovery opens up the possibility of using the protein to develop more effective treatments for heart disease. Previously it was thought that cells within the adult heart are in a state of permanent rest and that any progenitor cells that can contribute to heart tissue repair travel into the heart from the bone marrow. See 150 references on that perspective on cEPCs origin and roles, which was the scientific frontier on this topic, prior to the publication of Smart et al., (2007), in Lev-Ari & Abourjaily (2006a, 2006b, 2006c).

However, researchers at University College London have demonstrated that beneficial cells actually reside in the heart itself (Smart et al. (2007). This approach would bypass the risk of immune system rejection, a major problem with the use of stem cell transplants from another source. Allogenic rejection was the main reason for the selection of an endogenous augmentation method for cEPCs using drug therapy by Lev-Ari & Abourjaily  (2006a, 2006b, 2006c). Closer examination revealed that without the Thymosin beta 4 protein, the progenitor cells failed to move deeper into the heart and change the cells needed to build healthy blood vessels and sustain muscle tissue.

http://www.irishhealth.com/clin/cholesterol/newsstory.php?id=10581

Drug # 2:

Indomethacin

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory effects and have been shown to have chemopreventive effects as well. NSAIDs inhibit cyclooxygenase (COX) activity to exert their anti-inflammatory effects, but it is not clear whether their antitumorigenic ability is through COX inhibition. Using subtractive hybridization, Jain et al. (2004) identified a novel member of the transforming growth factor- superfamily that has antitumorigenic activity from Indomethacin-treated HCT-116 human colorectal cancer cells. On further investigation of this library, they now report the identification of a new cDNA corresponding to the Thymosin beta-4 gene. Thymosin beta-4 is a small peptide that is known for its actin-sequestering function, and it is associated with the induction of angiogenesis, accelerated wound healing, and metastatic potential of tumor cells. However, only selective NSAIDs induce Thymosin beta-4 expression in a time- and concentration-dependent manner. For example,

Indomethacin and SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole] induce Thymosin beta-4 expression whereas sulindac sulfide does not.

They show that selective NSAIDs induce actin cytoskeletal reorganization, a precursory step to many dynamic processes regulating growth and motility including tumorigenesis. This is the first report to link Thymosin beta-4 induction with NSAIDs. These data suggest that NSAIDs alter the expression of a diverse number of genes and provide new insights into the chemopreventive and biological activity of these drugs (Jain et al. 2004).

Rationale for Indomethacin selection

 

Inhibitor of prostaglandin synthesis. Inhibits cyclooxygenase (COX) 1 selective.

Suggested dosage: 25 mg PO bid.

Jain et al. (2004) report a link between Thymosin beta-4 induction with NSAIDs. We selected both drugs (drug classes) and anticipate strong synergistic therapeutic effects.

Drug # 3:

Clevidipine

Clevidipine is the first third-generation calcium channel blocker, Dr. Papadakos said. It has what he called an “ultrashort” clinically relevant half-life of about one minute and then is rapidly metabolized. The effect on blood pressure is seen within one to two minutes.

http://www.medpagetoday.com/MeetingCoverage/SCCM/tb/5091

Clevidipine is an investigational agent undergoing late-stage clinical development to evaluate its potential as an innovative, targeted, fast acting intravenous product under investigation for lowering blood pressure before, during and after surgery.

http://www.themedicinescompany.com/products_Clevidipine.shtml

The Medicines Company entered into agreements with AstraZeneca PLC in March of 2002 for the development, licensing and commercialization of Clevidipine. If approved, the product could be an excellent fit with The Medicines Company’s emerging acute cardiovascular care franchise, which is led by Angiomax® (bivalirudin), an anticoagulant approved in the U.S. and other countries for use during coronary angioplasty procedures. If Clevidipine passes further clinical hurdles — phase III trials are under way — the drug may form a useful addition to the medications available to physicians in the perioperative setting

Mechanism of Action

Clevidipine belongs to a well-known class of drugs called dihydropyridine calcium channel antagonists. In vitro studies demonstrated that Clevidipine acts by selectively relaxing the smooth muscle cells that line small arteries, resulting in widening of the artery opening and reducing blood pressure within the artery (Levy, Huraux, Nordlander, 1997, 345-358).

Phase III Clinical Trials

The Medicines Company is currently sponsoring a Phase III clinical program of five studies to evaluate safety and efficacy of Clevidipine:

Early Development

The Medicines Company’s development program for Clevidipine follows upon the data sets generated by AstraZeneca, which completed clinical pharmacology, dose-finding and efficacy studies in almost 300 patients or volunteers. In clinical studies, Clevidipine has shown to provide the desired blood pressure lowering effect without causing an increase in heart rate (Kotrly, et al. 1984). Further studies demonstrate that reductions in blood pressure are dose-dependent, are not associated with an increase in heart rate and cease rapidly after stopping Clevidipine infusions (Ericsson, et al., 2000), (Schwieler, et al., 1999). In clinical studies Clevidipine was rapidly metabolized independent of the liver and the kidneys, allowing rapid clearance of the drug from the bloodstream (Ericsson, et al., 1999a), (Ericsson, et al., 1999b). Therefore, the effects of Clevidipine are short-lived, which translates into a rapid cessation of its effect on reducing blood pressure.

The two efficacy studies are known as ESCAPE-1 and ESCAPE-2. The primary objective of these studies is to determine the efficacy of Clevidipine injection versus placebo in treating pre-operative (ESCAPE-1) and post-operative (ESCAPE-2) high blood pressure. Three safety studies are collectively known as ECLIPSE. The primary objective is to establish the safety of Clevidipine in the treatment of perioperative high blood pressure, as measured by a comparison of the incidences of death, stroke, myocardial infarction and renal dysfunction between the Clevidipine and comparative treatment groups. The comparative treatments are nitroglycerin, sodium nitroprusside and nicardipine.  The ECLIPSE trial randomized 589 patients at 40 centers in the U.S. to get either sodium nitroprusside or Clevidipine. Sodium nitroprusside was administered according to institutional practice; Clevidipine was begun at 2 mg/kg and doubled every 90 seconds until blood pressure was lowered. The primary endpoint was the difference in major clinical events — death, myocardial infarction, stroke, and renal dysfunction 30 days after surgery. The secondary endpoint was blood pressure control during the first 24 hours after surgery.

The study showed no significant differences in the elements of the primary endpoint, except for mortality, Dr. Papadakos said, where 1.7% of Clevidipine patients died, compared with 4.7 of those getting sodium nitroprusside.  The difference was statistically significant at P<0.05, but Dr. Papadakos characterized the improvement as “slight.” On the other hand, the drug did show an important difference in blood pressure control over the first 24 hours, he said:

  • Patients on Clevidipine spent an average of 4.37 minutes per hour outside the desired blood pressure range.
  • Sodium nitroprusside patients spent, on average, 10.5 minutes per hour outside the desired range.
  • The difference was statistically significant at P<0.003.

Dr. Papadakos concluded that Clevidipine is a new drug that is effective, safe, and easy to use. On 2/20/2007, Dr. Deutschman, who moderated the late-breaking session at which Dr. Papadakos spoke, said that a better comparison, would be intravenous nicardipine (Cardene IV), a second-generation calcium channel blocker that is also in wide use and is considered the standard of care. “We don’t know yet if this drug is going to be better than nicardipine,” he said.

http://www.medpagetoday.com/MeetingCoverage/SCCM/tb/5091

Rationale for Clevidipine selection

Clevidipine is an acute care product. Blood pressure management is a major component of care during the 13.4 million inpatient surgeries conducted in the U.S. each year. Blood pressure control, which is managed by an anesthesiologist, is often important in patients with both normal and high blood pressure undergoing surgery or other interventional procedures. Some of these patients require rapid, precise control of blood pressure to avoid compromising key organ function such as the heart, brain and kidney.

CONCLUSION 

This is the first study to design a novel combination drug treatment for Coronary Vasculogenesis, Anti-hypertention (short-acting), Vascular Anti-inflammation (vasculitis), Neovascularization of ischemic tissue and release of adult epicardium from a quiescent state and restoring its pluripotency. This treatment is based on the new three paradigms that were presented in Cell (2006) and Nature (2007). This combination drug therapy of three drugs, one in current use (Indomethacin), and two in clinical trials (Thymosin beta4 & Clevidipine), has not been proposed before. It represents an original concept drug combination design by Lev-Ari & Abourjaily (2007). This combination represents the cutting edge conceptualization of the field of treatment of cardiac injury based on a protein produced in the heart cells, Thymosin beta4, which function as a tissue and artery healer. Its upregulation by drug therapy will revolutionize cardiology and treatment for cardiovascular disease. The combination drug therapy consists of the following drugs:

  • Drug # 1:

Thymosin fraction 5 (a sublingual composition)

  • Drug # 2:

Indomethacin (Nonsteroidal anti-inflammatory drugs (NSAID)) (25 mg PO bid)

  • Drug # 3:

Clevidipine (Blood pressure lowering drug, (no effect on heart rate))

 

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Ericsson, H., Fakt. C., Hoglund. L., et al. (1999a). “Pharmacokinetics and pharmacodynamics of Clevidipine in healthy volunteers after intravenous infusion.” Eur J Clin Pharm., 55 (1), 61-67.

Ericsson, H., Fakt, C., Jolin-Mellgard A., et al. (1999b). “Clinical and pharmacokinetic results with a new ultrashort-acting calcium antagonist, Clevidipine, following gradually increasing intravenous doses to healthy volunteers.” Br J Clin Pharm., 47 (5), 531-538.

Ericsson, H., Bredberg, U., Eriksson, U., et al. (2000). “Pharmacokinetics and arteriovenous differences in Clevidipine concentration following a short and a long-term intravenous infusion in healthy volunteers.” Anesthesiology, 92 (4), 993-1001.

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Heart may be able to repair itself

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http://www.irishhealth.com/clin/cholesterol/newsstory.php?id=10581

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Jain, A.K., Moore, S.M., Yamaguchi, K., Eling, T.E., Baek, S.J. (2004, August). “Selective Nonsteroidal Anti-Inflammatory Drugs Induce Thymosin beta-4 and Alter Actin Cytoskeletal Organization in Human Colorectal Cancer Cells.” Journal of Pharmacology and Experimental Therapeutics, 311 (3) 885-891.

Kotrly, K. J., Ebert, T. J., Vucins, E. et al. (1984). “Baroreceptor reflex control of heart rate during isoflurane anesthesia in humans.” Anesthesiology,  60, 173-179.

Lev-Ari, A. & Abourjaily, P. (2006a) “An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPC) as a Therapeutic Target for Pharmacologic Therapy Design for Cardiovascular Risk Reduction.” Part I: Macrovascular Disease – Therapeutic Potential of cEPCs – Reduction methods for CV risk. Unpublished manuscript.

Lev-Ari, A. & Abourjaily, P. (2006b) “An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPC) as a Therapeutic Target for Pharmacologic Therapy Design for Cardiovascular Risk Reduction.” Part II: Therapeutic Strategy for cEPCs Endogenous Augmentation: A Concept-based Treatment Protocol for a Combined Three Drug Regimen. Unpublished manuscript.

Lev-Ari, A. & Abourjaily, P. (2006c) “An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPC) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction.” Part III: Biomarker for Therapeutic Targets of Cardiovascular Risk Reduction by cEPCs Endogenous Augmentation using New Combination Drug Therapy of Three Drug Classes and Several Drug Indications. A Theoretical Design for Quantification of the Endogenous EPCs Augmentation for Differential Level of CV Risk Reduction and Diagnostic Device Design for Drug Delivery. Unpublished manuscript.

Lev-Ari, A. & Abourjaily, P. (2007). Heart Vasculature – Regeneration and Protection of Coronary Artery Endothelium and Smooth Muscle: A Concept-based Pharmacological Therapy of a Combined Three Drug Regimen. Unpublished manuscript.

Levy, J. H., Huraux, C., Nordlander, M. (1997). “Treatment of perioperative hypertension.” In: Epstein M, Ed. Chapter in Calcium Antagonists in Clinical Medicine. Philadelphiea: Hanely & Belfus, pp. 345-358.

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Protein Discovered That Can Tell Human Heart to Heal Itself

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http://www.cathlabdigest.com/displaynews.cfm?newsid=1122065

Philp D, Huff T, Gho YS, Hannappel E, Kleinman HK. (2003). “The actin binding site on Thymosin beta4 promotes angiogenesis.” FASEB Journal, published on line 9/18/2003.

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Sublingual compositions comprising Thymosin fraction 5 and methods for administration

Retrieved 3/1/2007

http://www.pharmcast.com/Patents100/Yr2004/May2004/051104/6733791_Sublingual051104.htm

 

TMSB4X  Thymosin, beta 4, X-linked

Retrieved on 3/1/2007

http://www.ihop-net.org/UniPub/iHOP/gs/92756.html

Waeckel, L., Jérôme Bignon, J., Jian-Miao Liu, J-M., Markovits, D., Ebrahimian, T.G., Vilar, J., Mees, B., Blanc-Brude, O., Barateau, V., Sophie Le ricousse-Roussanne. S., Duriez, M. Tobelem, G.,  Wdzieczak-Bakala, J., Bernard I Lévy, B.I., Silvestre, J-S. (2006) “Tetrapeptide AcSDKP Induces Postischemic Neovascularization Through Monocyte Chemoattractant Protein-1 Signaling.” Arteriosclerosis, Thrombosis, and Vascular Biology, 26, 773

Wang, D., Oscar A. Carretero, O.A.,Yang, X-Y., Rhaleb, N-E., Liu, Y-H., Liao, T-D., Yang, X-P. (2004). “N-acetyl-seryl-aspartyl-lysyl-proline stimulates angiogenesis in vitro and in vivo.” Am J Physiol Heart Circ Physiol., 287, H2099-H2105.

Werner N, Junk S, Laufs L, Link A, Walenta K, Bohm M, Nickenig G., (2003).  Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res., 93, e17– e24.

Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Böhm M, Nickenig G. (2005a). Circulating Endothelial Progenitor Cells and Cardiovascular Outcomes, NEJM, 353, 999-1007

Werner, N. & Nickenig, G. (2005b). Authors Reply to Correspondence to the Editor on Circulating Endothelial Progenitor Cells. NEJM, 353 (24), 2613-2616

Wu, S.M., Fujiwara, Y., Cibulsky, S.M., Clapham, D.E., Lien, C., Schultheiss, T.M., Orkin, S.H. (2006, December). “Developmental Origin of a Bipotential Myocardial and Smooth Muscle Cell Precursor in the Mammalian Heart.” Cell, 127, 1137-1150.

Other related articles on this Open Access Online Scientific Journal, include the following:

Saha, S. (2012b) Innovations in Bio instrumentation for Measurement of Circulating Progenetor Endothelial Cells in Human Blood.
http://pharmaceuticalintelligence.com/2012/07/08/innovations-in-bio-instrumentation-for-measurement-of-circulating-progenitor-endothelial-cells-in-human-blood/

 

Saha, S. (2012c) Endothelial Differentiation and Morphogenesis of Cardiac Precursor
http://pharmaceuticalintelligence.com/2012/07/17/endothelial-differentiation-and-morphogenesis-of-cardiac-precursors/

Saha, S. (2012e). Human Embryonic-Derived Cardiac Progenitor Cells for Myocardial Repair

http://pharmaceuticalintelligence.com/2012/08/01/human-embryonic-derived-cardiac-progenitor-cells-for-myocardial-repair/

Lev-Ari, A. 12/29/2012. Coronary artery disease in symptomatic patients referred for coronary angiography: Predicted by Serum Protein Profiles

http://pharmaceuticalintelligence.com/2012/12/29/coronary-artery-disease-in-symptomatic-patients-referred-for-coronary-angiography-predicted-by-serum-protein-profiles/

 

Bernstein, HL and Lev-Ari, A. 11/28/2012. Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

http://pharmaceuticalintelligence.com/2012/11/28/special-considerations-in-blood-lipoproteins-viscosity-assessment-and-treatment/

 

Lev-Ari, A. 11/13/2012 Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes

http://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/

 

Lev-Ari, A. 10/19/2012 Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

http://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

 

Lev-Ari, A. 10/4/2012 Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

 

Lev-Ari, A. 10/4/2012 Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

 

Lev-Ari, A. 8/28/2012 Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs

http://pharmaceuticalintelligence.com/2012/08/28/cardiovascular-outcomes-function-of-circulating-endothelial-progenitor-cells-cepcs-exploring-pharmaco-therapy-targeted-at-endogenous-augmentation-of-cepcs/

 

Lev-Ari, A. 8/27/2012 Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

http://pharmaceuticalintelligence.com/2012/08/27/endothelial-dysfunction-diminished-availability-of-cepcs-increasing-cvd-risk-for-macrovascular-disease-therapeutic-potential-of-cepcs/

 

Lev-Ari, A. 8/24/2012 Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

http://pharmaceuticalintelligence.com/2012/08/24/vascular-medicine-and-biology-classification-of-fast-acting-therapy-for-patients-at-high-risk-for-macrovascular-events-macrovascular-disease-therapeutic-potential-of-cepcs/

 

Lev-Ari, A. 7/30/2012 Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-intellectual-property-creation-and-protection-by-pioneer-and-by-biosimilar-manufacturers/

 

Lev-Ari, A. 7/29/2012 Biosimilars: Financials 2012 vs. 2008

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-financials-2012-vs-2008/

 

Lev-Ari, A. 7/29/2012 Biosimilars: CMC Issues and Regulatory Requirements

http://pharmaceuticalintelligence.com/2012/07/29/biosimilars-cmc-issues-and-regulatory-requirements/

 

Lev-Ari, A. 7/19/2012 Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

 

Lev-Ari, A. 4/30/2012 Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

http://pharmaceuticalintelligence.com/2012/04/30/93/

Lev-Ari, A. 5/29/2012 Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes

http://pharmaceuticalintelligence.com/2012/05/29/445/

 

Lev-Ari, A. 7/2/2012 Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

http://pharmaceuticalintelligence.com/2012/07/02/macrovascular-disease-therapeutic-potential-of-cepcs-reduction-methods-for-cv-risk/

 

Lev-Ari, A. 7/9/2012 Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “powerhouse of the cell”

http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

 

Lev-Ari, A. 7/16/2012 Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Proginetor Cells endogenous augmentation

http://pharmaceuticalintelligence.com/2012/07/16/bystolics-generic-nebivolol-positive-effect-on-circulating-endothilial-progrnetor-cells-endogenous-augmentation/

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