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Posts Tagged ‘Peroxisome proliferator-activated receptor gamma’


Reversal of Cardiac mitochondrial dysfunction

Curator: Larry H Bernstein, MD, FACP

This article is the FOURTH in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

  • Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

  • Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

  • Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

 

Mitochondrial metabolism and cardiac function

There is sufficient evidence to suggest that, even with optimal therapy, there is an

  • attenuation or loss of effectiveness of neurohormonal antagonism as heart failure worsens.

The production of oxygen radicals is increased in the failing heart, whereas

  • normal antioxidant enzyme activities are preserved.

Mitochondrial electron transport is an enzymatic source of oxygen radical generation and

  • can be a therapeutic target against oxidant-induced damage in the failing myocardium.

Therefore, future therapeutic targets

  • must address the cellular and molecular mechanisms that contribute to heart failure.

Furthermore, since  fundamental characteristics of the failing heart are 

  • defective mitochondrial energetics and
  • abnormal substrate metabolism

we might expect that substantial benefit may be derived from the development of therapies aimed at

  • preserving cardiac mitochondrial function and
  • optimizing substrate metabolism.

Nutrition and physiological function

Blockade of electron transport in isolated, perfused guinea pig hearts –
before ischaemia with the reversible complex I inhibitor amobarbital
  • decreased superoxide production and
  • preserved oxidative phosphorylation in cardiac mitochondria,
  • decreased myocardial damage.
But when ascorbic acid was administered orally to chronic heart failure patients, there were improvements
  • in endothelial function but
  • no improvement in skeletal muscle energy metabolism.
Angiotensin I-converting enzyme (ACE) inhibitors with trandolapril treatment  in models of heart failure
  • appear to preserve mitochondrial function
  • improving cardiac energy metabolism and
  • function in rats with chronic heart failure.
Similarly perindopril treatment   – in rat skeletal muscle after myocardial infarction -restored :
  • levels of the mitochondrial biogenesis transcription factors PPARg coactivator-1a and
  • nuclear respiratory factor-2a, and
  • prevented mitochondrial dysfunction
Tissue effects of ACE inhibition, such as
might activate intracellular signalling cascades that
  • stimulate mitochondrial biogenesis and
  • improve energy metabolism.
Clearly, the mechanisms of metabolic regulation by
  • existing cardioprotective agents require further investigation.

Substrate metabolism in the failing heart

Increased sympathetic drive in heart failure patients causes adipose tissue lipolysis, thus
  • elevating plasma FFA concentrations.
Myocardial FFA uptake rates are largely determined by circulating FFA concentrations.
In addition to being a major fuel in heart,
  • fatty acids are ligands for the peroxisome proliferator-activated receptors (PPARs),
    •  members of the nuclear hormone receptor (NHR) family.
One PPAR subtype, PPARa, is highly expressed in heart and skeletal muscle. PPARs regulate gene expression by
binding to response elements in the promoter region of target genes that control fatty acid metabolism, including
It has been known for many years that high plasma FFA concentrations are detrimental to the heart,
  • increasing oxygen consumption for any given workload.
Decreased myocardial oxygen efficiency could result, in part,
  • from the inherent stoichiometric inefficiency of fatty acid oxidation,
  • which accounts for the consumption of 12% more oxygen per ATP synthesized than glucose oxidation.

High levels of plasma FFAs have been associated with increased cardiac UCP3 levels in patients undergoing CABG(Fig) and

  • are believed to activate the uncoupling action of UCP3.

http://htmlimg1.scribdassets.com/8o5pfgywg0lyerj/images/4-244729cb6a.jpg

Fig .  Metabolic modulation of the failing heart can be achieved by inhibiting mitochondrial beta-oxidation with trimetazidine, or
  • free fatty acid (FFA) uptake via the carnitine palmitoyltransferase (CPT) system with perhexiline,
    • giving rise to more oxygen-efficient glucose oxidation.
Alternatively, CPT is inhibited by malonyl-coenzyme A (CoA),
  • synthesized from cytosolic acetyl-CoA by acetyl-CoA  carboxylase.
Pharmacological inhibition, or mutation, of
  • malonyl-CoA decarboxylase, which normally converts malonyl-CoA back to acetyl-CoA,
  • elevates malonyl-CoA levels, inhibiting mitochondrial FFA uptake and thus protects the failing heart.

Nutritional Support for the Mitochondria

Human Studies                                       Animal or In Vitro Studies

Alpha lipoic acid                                                    Resveratrol
Co-Enzyme Q10                                                      EgCG
Acetyl-L-Carnitine                                                Curcumin

Lipoic Acid & Acetyl-L-Carnitine

Alpha lipoic acid is known to be a mitochondrial antioxidant that preserves or improves mitochondrial function.

  •  lipoic acid can prevent arterial calcification, and
  • arterial calcification may be related to mitochondrial dysfunction
  • methods are under study to increase lipoic acid synthase production, the enzyme responsible for making lipoic acid in the body.

Co-Enzyme Q10

It is well known that statin drugs taken for high cholesterol severely reduce CoQ10 levels, and causes other negative cardiovascular side effects.
A  study on CAD patients has shown that over 8 weeks of supplementing with 300mg of CoQ10 reversed

  • mitochondrial dysfunction (as measured by a reduced lactate:pyruvate ratio) and
  • improved endothelial function (as measured by increased flow-mediated dilation)

Other Mitochondrial Antioxidants

Other natural compounds that have been shown to have antioxidant effects in the mitochondria include

  • resveratrol, found in wine and grapes,
  • curcumin from turmeric and
  • EGCG, found abundantly in green tea extract.

But no studies have been conducted for these compounds in CVD.

Metabolic syndrome and serum carotenoids: findings of a cross-sectional study
in Queensland, Australia

Metabolic syndrome and serum carotenoids.

T Coyne, TI Ibiebele, PD Baade, CS McClintock and JE Shaw.
Viertel Center for Research in Cancer Control, Cancer Council Queensland, and School of Public Health,
Queensland University of Technology and University of Queensland, Brisbane, Australia
Several components of the metabolic syndrome are known to be oxidative stress-related conditions
  1. diabetes and
  2. cardiovascular disease,
Carotenoids are compounds derived primarily from plants and several have been shown to be potent antioxidant nutrients.
Both diabetes and cardiovascular disease are known to be oxidative stress-related conditions such that
  • antioxidant nutrients may play a protective role in these conditions.
Several cross–sectional surveys have found lower levels of serum carotenoids among those with impaired glucose metabolism or type 2 diabetes.
Carotenoids are compounds derived primarily from plants, several of which are known to be potent antioxidants.
Epidemiological evidence indicates that some serum carotenoids may play a protective role against the development of chronic diseases such as
  1. atherosclerosis,
  2. stroke,
  3. hypertension,
  4. certain cancers,
  5. inflammatory diseases and
  6. diabetic retinopathy.

The primary carotenoids found in human serum are

  1. α-carotene
  2. β-carotene
  3. β-cryptoxanthin
  4. lutein/zeaxanthin
  5. lycopene.
The aim of this study was to examine the associations between metabolic syndrome status and major serum carotenoids in adult Australians.
Data on the presence of the metabolic syndrome, based on International Diabetes Federation 2005 criteria, were collected from 1523 adults
aged 25 years and over in six randomly selected urban centers in Queensland, Australia, using a cross sectional study design.
The following were determined:
  1. Weight
  2. height
  3. BMI
  4. waist circumference
  5. blood pressure
  6. fasting and 2-34 hour blood glucose
  7. lipids
  8. five serum carotenoids.
Criteria used to assess the number of metabolic syndrome components present in a 171 participant using the
2005 International Diabetes Federation definition are as follows:
Components = 0 -none of the metabolic syndrome components (i.e. abdominal obesity, raised triglyceride,
reduced HDL-cholesterol, raised blood pressure, and impaired fasting plasma glucose) are present;
  1. Components = any 1 one of the five metabolic syndrome components is present ;
  2. Components = 2 – any two of the five components are present;
  3. Components = 3 any three of the components are present;
  4. Components = 4 – any four of the components are present;
  5. Components = 5 = all five metabolic syndrome components are present.
This study investigated the relationships between these five primary serum carotenoids and the metabolic syndrome
in a cross-sectional population-based study in Queensland, Australia.  Distributions of serum carotenoids were skewed
and therefore natural logarithmically transformed to better approximate the normal distribution for regression analyses.
Association between log transformed serum carotenoids as dependent variables and metabolic syndrome status were
assessed using multiple linear regression analysis. Results are reported as back transformed geometric means.
Analysis was performed for each serum carotenoid separately, and the sum of the five carotenoids,
adjusting for the following potential confounders:
  1. age
  2. sex
  3. education
  4. BMI
  5. smoking
  6. alcohol intake
  7. physical activity
  8. vitamin use.
Mean serum alpha-carotene, beta-carotene and the sum of the five carotenoid concentrations were significantly lower (p<0.05)
in persons with the metabolic syndrome (after adjusting for age,sex, education, BMI status, alcohol intake, smoking, physical activity
status and vitamin/mineral use) than persons without the syndrome. Alpha, beta and total carotenoids also decreased significantly
(p<0.05) with increased number of components of the metabolic syndrome, after adjusting for these confounders. These differences
were significant among former smokers and non-smokers, but not in current smokers. Low concentrations of serum
  • alpha-carotene,
  • beta carotene and
  • the sum of five carotenoids
appear to be associated with metabolic syndrome status.
The overall prevalence of the syndrome was 24% and was significantly higher among males than females. As would be expected, significant
differences in prevalence of the syndrome were seen with
  • body mass index
  • waist circumference
  • systolic and diastolic blood pressure
  • blood lipids.
Significant differences were also evident by
  • age group, smoking status, educational status and income.
Income was marginally inversely associated. The prevalence increased with age, and was lower in those with post graduate education.
No significant differences were seen by alcohol intake, physical activity levels,  vitamin usage, or fruit intake. There was actually an
  • inverse relationship between vegetable intake (not fruit) and serum carotenoids.
Those who consumed 4 serves or more of vegetable were less likely to have the metabolic syndrome
  • compared to those who consumed 1 serve or less of vegetables.
The mean concentrations of serum alpha-carotene, beta-carotene and the sum of the five carotenoids were significantly lower for participants
  • with the metabolic syndrome present compared with those without the syndrome, after adjusting for potential confounding variables.
Concentrations of alpha-carotene, beta-carotene and the sum of the five carotenoids decreased significantly as
  • the number of components of the metabolic syndrome increased after adjusting for potential confounding variables.
Similarly there was an inverse association between quartiles of
  • individual and total serum carotenoids and metabolic syndrome status and each of its components.
This study was designed to investigate the association between several serum carotenoids and the metabolic syndrome.
The data from the present population study suggest that several serum carotenoids are inversely related to the metabolic syndrome.
The study showed significantly lower concentrations present among those with the metabolic syndrome of
  1. α-carotene,
  2. β-carotene and
  3. the sum of the five carotenoids
 compared to those without.We also found decreasing concentrations of all the carotenoids tested as

  • the number of the metabolic syndrome components increased.
This was significant for
  1. α-carotene,
  2. β-carotene,
  3. β-cryptoxanthin
  4. total carotenoids.
    (not lycopenes)
These findings are consistent with data reported from the third National Health and Nutrition Examination Survey (NHANES III).
In the NHANES III study, significantly lower concentrations of all the carotenoids, except lycopene, were found among persons
with the metabolic syndrome compared with those without, after adjusting for confounding factors similar to those in our study.

Carnitine: A novel health factor-An overview. 

CD Dayanand, N Krishnamurthy, S Ashakiran, KN Shashidhar
Int J Pharm Biomed Res 2011; 2(2): 79-89.  ISSN No: 0976-0350
Carnitine comprises L-carnitine, acetyl –L-carnitine and Propionyl –L-carnitine. Carnitine is
  • obtained in greater amount from animal dietary sources than from plant sources.
The endogenous synthesis of carnitine takes place in animal tissues like
  • liver
  • kidney
  • brain
using precursor amino acids lysine and methionine by a pathway
  • dependent on iron, vitamin C, niacin, pyridoxine .
This is the basis of vegans generally depending on carnitine in larger proportion
  • through in vivo synthesis than omnivorous subjects.
The concentration of tri-methyl lysine residues and the tissue specificity of  butyro-betaine dehydrogenase
  • plays a significant role in regulating the carnitine biosynthesis.
Carnitine transport from the site of synthesis to target tissue occurs via blood.
The measurement of plasma carnitine concentration represents –
  • the balance between the rate of synthesis and rate of excretion
    • through specific transporter proteins.
The cellular functional role of carnitine depends on the uptake into cells through
  1. carnitine transport proteins and
  2. transport into mitochondrial matrix.
The function of carnitine is to traverse Long-chain Fatty Acids across inner mitochondrial membrane
  • for β-oxidation, thereby, generating ATP.
Carnitine deficiency results in muscle disorders.  The deficiency states are primary and secondar.
The primary is of systemic or myopathic, characterized by a defect of high affinity organic cation transporter protein (CTP)
  • present on the plasma membrane of liver and kidney and
  • also due to dysfunction of carnitine reabsorbtion through
    • similar transport proteins in renal tubules.
Secondary carnitine deficiency is associated with
  1. mitochondrial disorders and also
  2. defective β-oxidation such as CPT-II and acyl CoA dehydrogenase.
In recent times, carnitine has been extensively studied in various research activities to explore the therapeutic benefit.
Thus, carnitine justifies as a novel health factor.

Propionyl-L-carnitine Corrects Metabolic and Cardiovascular Alterations in
Diet-Induced Obese Mice and Improves Liver Respiratory Chain Activity

C Mingorance,  L Duluc, M Chalopin, G Simard, et al.
PLC improved the insulin-resistant state and reversed the increased total cholesterol
but not the increase in free fatty acid, triglyceride and HDL/LDL ratio induced by high-fat diet.
Vehicle-HF exhibited a reduced

  • cardiac output/body weight ratio,
  • endothelial dysfunction and
  • tissue decrease of NO production,

all of them being improved by PLC treatment.
The decrease of hepatic mitochondrial activity by high-fat diet was reversed by PLC.

Oral administration of PLC improves the insulin-resistant state developed by obese animals and
decreases the cardiovascular risk associated with the metabolically impaired mitochondrial function.

Omega-3 Fatty Acid and cardioprotection

The Benefits of Flaxseed    

By Elaine Magee, MPH, RD    WebMD Expert Column
Some call it one of the most powerful plant foods on the planet. There’s some evidence it may help reduce your risk of

  • heart disease, cancer, stroke, and diabetes.

That’s quite a tall order for a tiny seed that’s been around for centuries.

Flaxseed was cultivated in Babylon as early as 3000 BC. In the 8th century, King Charlemagne believed so strongly in the
health benefits of flaxseed that he passed laws requiring his subjects to consume it. Now, thirteen centuries later, some
experts say we have preliminary research to back up what Charlemagne suspected.

http://img.webmd.com/dtmcms/live/webmd/consumer_assets/site_images/article_
thumbnails/features/benefits_of_flaxseed_features/375x321_benefits_of_flaxseed_features.jpg

Not only has consumer demand for flaxseed grown, agricultural use has also increased.
Flaxseed is what’s used to feed all those chickens that are laying eggs with higher levels of omega-3 fatty acids.
Although flaxseed contains all sorts of healthy components, it owes its primary healthy reputation to three of them:

  1. Omega-3 essential fatty acids, have been shown to have heart-healthy effects.  1.8 grams of plant omega-3s/tablespoon ground.
  2. Lignans, which have both plant estrogen and antioxidant qualities.  75 to 800 times more lignans than other plant foods.
  3. Fiber. Flaxseed contains both the soluble and insoluble types.

Omega-3 Polyunsaturated Fatty Acids and Cardiovascular Diseases

CJ Lavie, RV Milani, MR Mehra, and HO Ventura.
J. Am. Coll. Cardiol. 2009;54;585-594.   http://dx.doi.org/10.1016/j.jacc.2009.02.084
Fish oil is obtained in the human diet by eating oily fish, such as
  • herring, mackerel, salmon, albacore tuna, and sardines, or
  • by consuming fish oil supplements or cod liver oil.
Fish do not naturally produce these oils, but obtain them through the ocean food chain from the marine microorganisms
  • that are the original source of the omega-3 polyunsaturated fatty acids (ω-3 PUFA) found in fish oils.
Numerous prospective and retrospective trials from many countries, including the U.S., have shown that moderate
  • fish oil consumption decreases the risk of major cardiovascular (CV) events, such as
  1. myocardial infarction (MI),
  2. sudden cardiac death (SCD),
  3. coronary heart disease (CHD),
  4. atrial fibrillation (AF), and most recently,
  5. death in patients with heart failure (HF).
Most of the evidence for benefits of the ω-3 PUFA has been obtained for
  • eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the long-chain fatty acids in this family.
There is support for a benefit from alpha-linolenic acid (ALA),
  • the plant-based precursor of EPA.
The American Heart Association (AHA) has currently endorsed the use of ω-3 PUFA in patients with documented CHD

  • at a dose of approximately 1 g/day of combined DHA and EPA, either in the form of fatty fish or fish oil supplements
The health benefits of these long chain fatty acids are numerous and remain an active area of research.
Omega-3 polyunsaturated fatty acid (ω-3 PUFA) therapy continues to show great promise in primary and,
  • particularly in secondary prevention of cardiovascular (CV) diseases.
This portion of discussion summarizes the current scientific data on the effects of the long chain ω-3 PUFA
  • in the primary and secondary prevention of various CV disorders.
The most compelling evidence for CV benefits of ω-3 PUFA comes from 4 controlled trials
  • of nearly 40,000 participants randomized to receive eicosapentaenoic acid (EPA)
  • with or without docosahexaenoic acid (DHA) in studies of patients
    • in primary prevention,
    • after myocardial infarction, and
    • with heart failure (HF).
The evidence from retrospective epidemiologic studies and from large randomized controlled trials
show the benefits of ω-3 PUFA, specifically EPA and DHA, in primary and secondary CV prevention
and provide insight into potential mechanisms of these observed benefits.

Background Epidemiologic Evidence

During the past 3 decades, numerous epidemiologic and observational studies have been published on the CV benefits of ω-3 PUFA.
As early as 1944, Sinclair described the rarity of CHD in Greenland Eskimos, who consumed a diet high in whale, seal, and fish.
More than 30 years ago, Bang and Dyberg reported that despite a diet low in fruit, vegetables, and complex carbohydrates but
high in saturated fat and cholesterol, serum cholesterol and triglycerides were lower in Greenland Inuit than in age-matched residents
of Denmark, and the risk of MI was markedly lower in the Greenland population compared with the Danes. These initial observations raised
speculation on the potential benefits of ω-3 PUFA (particularly EPA and DHA) as the protective “Eskimo factor”.
Potential EPA and DHA Effects   
  1. Antiarrhythmic effects
  2. Improvements in autonomic function
  3. Decreased platelet aggregation
  4. Vasodilation
  5. Decreased blood pressure
  6. Anti-inflammatory effects
  7. Improvements in endothelial function
  8. Plaque stabilization
  9. Reduced atherosclerosis
  10. Reduced free fatty acids and triglycerides
  11. Up-regulated adiponectin synthesis
  12. Reduced collagen deposition
The target EPA + DHA consumption should be at least 500 mg/day for individuals without underlying overt CV disease
  • and at least 800 to 1,000 mg/day for individuals with known coronary heart disease and HF.
Further studies are needed to determine optimal dosing and the relative ratio of DHA and EPA ω-3 PUFA that
  • provides maximal cardioprotection in those at risk of CV disease
  • as well in the treatment of atherosclerotic, arrhythmic, and primary myocardial disorders.
Lavie et al.  Omega-3 PUFA and CV Diseases  J Am Coll Cardiol 2009; 54(7): 585–94

Assessing Appropriateness of Lipid Management Among Patients With Diabetes Mellitus

Moving From Target to Treatment.   AJ Beard, TP Hofer, JR Downs, et al. and Diabetes Clinical Action Measures Workgroup
Performance measures that emphasize only a treat-to-target approach may motivate ove-rtreatment with high-dose statins,
  • potentially leading to adverse events and unnecessary costs.
We developed a clinical action performance measure for lipid management in patients with diabetes mellitus that is designed
  • to encourage appropriate treatment with moderate-dose statins while minimizing over-treatment.
We examined data from July 2010 to June 2011 for 964 818 active Veterans Affairs primary care patients ≥18 years of age with diabetes mellitus.
We defined 3 conditions as successfully meeting the clinical action measure for patients 50 to 75 years old:
  1.  having a low-density lipoprotein (LDL) <100 mg/dL,
  2. taking a moderate-dose statin regardless of LDL level or measurement, or
  3. receiving appropriate clinical action (starting, switching, or intensifying statin therapy) if LDL is ≥100 mg/dL.
We examined possible over-treatment for patients ≥18 years of age by examining the proportion of patients
  • without ischemic heart disease who were on a high-dose statin.
We then examined variability in measure attainment across 881 facilities using 2-level hierarchical multivariable logistic models.
Of 668 209 patients with diabetes mellitus who were 50 to 75 years of age, 84.6% passed the clinical action measure:
  1. 67.2% with LDL <100 mg/dL,
  2. 13.0% with LDL ≥100 mg/dL and either on a moderate-dose statin (7.5%) or with appropriate clinical action (5.5%), and
  3. 4.4% with no index LDL on at least a moderate-dose statin. Of the entire cohort ≥18 years of age, 13.7% were potentially over-treated.
Use of a performance measure that credits appropriate clinical action indicates that almost 85% of diabetic veterans 50 to 75 years of age
  • are receiving appropriate dyslipidemia management.

Exercise training and mitochondria in heart failure

The beneficial effects of exercise in the rehabilitation of patients with heart failure are well established,
with improvements observed in
  • exercise capacity,
  • quality of life,
  • hospitalization rates and
  • morbidity/mortality.
There is no evidence of training-induced
improvements in cardiac energy metabolism or
  • mitochondrial function, and
  • no modification of myocardial oxidative capacity,
  • oxidative enzymes, or
  • energy transfer enzymes
in exercising rats with experimental heart failure, but there is  evidence of
There are also improvements in
  • skeletal muscle oxidative capacity with
  • increased mitochondrial density
following endurance training in heart failure patients associated with alleviation of symptoms such as
  • exercise intolerance and
  • chronic fatigue.
The mechanism underlying improvements in mitochondrial function may perhaps be a result of
  • more effective peripheral oxygen delivery following training,
  • alleviating tissue hypoxia and oxidative stress.

Treating Type 2 diabetes, and lowering cardiovascular disease risk

Treating Diabetes and Obesity with an FGF21-Mimetic Antibody
Activating the βKlotho/FGFR1c Receptor Complex

IN Foltz, S Hu, C King, Xinle Wu, et al.  Amgen and Texas A&M HSC, Houston, TX.
Sci Transl Med  Nov 2012; 4(162), p. 162ra153
http://dx.doi.org/10.1126/scitranslmed.3004690

Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on

  1. lipid
  2. body weight
  3. glucose metabolism

A monoclonal antibody, mimAb1, binds to βKlotho with high affinity and specifically

  • activates signaling from the βKlotho/FGFR1c (FGF receptor 1c) receptor complex.

Injection of mimAb1 into obese cynomolgus monkeys led to FGF21-like metabolic effects:

  1. decreases in body weight,
  2. plasma insulin,
  3. triglycerides, and
  4. glucose during tolerance testing.

Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements

  • in glucose metabolism and body weight.

mimAb1 depends on βKlotho to activate FGFR1c, but

  • it is not expected to induce side effects caused by activating FGFR1c alone.

The results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated

  • the essential role of FGFR1c in FGF21 function and
  • suggest fat as a critical target tissue for the cytokine and antibody.

This antibody activates FGF21-like signaling through cell surface receptors, and  provided

  • preclinical validation for an innovative therapeutic approach to diabetes and obesity.

Influencing Factors on Cardiac Structure and Function Beyond Glycemic Control
in Patients With Type 2 Diabetes Mellitus (T2DM)

R Ichikawa, M Daimon, T Miyazaki, T Kawata, et al.     Cardiovasc Diabetol. 2013;12(38)

We studied 148 asymptomatic patients with T2DM without overt heart disease.
Early (E) and late (A) diastolic mitral flow velocity and early diastolic mitral annular velocity (e’)

  • were measured for assessing left ventricular (LV) diastolic function.

In addition

  • insulin resistance,
  • non-esterified fatty acid,
  • high-sensitive CRP,
  • estimated glomerular filtration rate,
  • waist/hip ratio,
  • abdominal visceral adipose tissue (VAT),
  • subcutaneous adipose tissue (SAT)

In T2DM (compared to controls),

  • E/A and e’ were significantly lower, and
  • E/e’, left atrial volume and LV mass were significantly greater

VAT  and age were independent determinants of

  • left atrial volume (β =0.203, p=0.011),
  • E/A (β =−0.208, p=0.002), e’ (β =−0.354, p<0.001) and
  • E/e’ (β=0.220, p=0.003).

Independent determinants of LV mass were

  • systolic blood pressure,
  • waist-hip ratio (β=0.173, p=0.024)
  • VAT/SAT ratio (β=0.162, p=0.049)

Excessive visceral fat accompanied by adipocyte dysfunction may play a greater role than

  • glycemic control in the development of diastolic dysfunction and LV hypertrophy in T2DM

Inhibition of oxidative stress and mtDNA damage

Novel pharmacological agents are needed that

  • optimize substrate metabolism and
  • maintain mitochondrial integrity,
  • improve oxidative capacity in heart and skeletal muscle, and
  • alleviate many of the clinical symptoms associated with heart failure.

The evidence for the attenuation or loss of effectiveness of neurohormonal antagonism as heart failure worsens

  • indicates future therapeutic targets must address the cellular and molecular mechanisms that contribute to heart failure.

Pharmacological Targets of oxidative stress and mitochondrial damage

Defective mitochondrial energetics and abnormal substrate metabolism are fundamental characteristics of CHF.

A significant benefit may be derived from developing therapies aimed at

  • preserving cardiac mitochondrial function and
  • optimizing substrate metabolism.
Oxidative stress is enhanced in myocardial remodelling and failure. The increased production of oxygen radicals in the failing heart
  • with preserved antioxidant enzyme activities suggests
  • mitochondrial electron transport as a source of oxygen radical generation
  • can be a therapeutic target against oxidant-induced damage in the failing myocardium.
Chronic increases in oxygen radical production in the mitochondria
  • leads to mitochondrial DNA (mtDNA) damage,
  • functional decline,
  • further oxygen radical generation, and
  • cellular injury.
MtDNA defects may thus play an important role in the
  • development and progression of myocardial remodelling and failure.
Reactive oxygen species induce
  1. myocyte hypertrophy,
  2. apoptosis, and
  3. interstitial fibrosis
  4. by activating matrix metallo-proteinases,
  5. promoting the development and
  6. progression of maladaptive myocardial remodelling and failure.
Oxidative stress has direct effects on cellular structure and function and
  • may activate integral signalling molecules in myocardial remodelling and failure (Figure).
ROS result in a phenotype characterized by
  • hypertrophy and apoptosis in isolated cardiac myocytes.
Therefore, oxidative stress and mtDNA damage are good therapeutic targets.
Overexpression of the genes for
  • peroxiredoxin-3 (Prx-3), a mitochondrial antioxidant, or
  • mitochondrial transcription factor A (TFAM),
    • could ameliorate the decline in mtDNA copy number in failing hearts.
Consistent with alterations in mtDNA, the
  • decrease in mitochondrial function was prevented,
  • proving that the activation of Prx-3 or TFAM gene expression
  • could ameliorate the pathophysiological processes seen
  1. in mitochondrial dysfunction and
  2. myocardial remodelling.
Inhibition of oxidative stress and mtDNA damage
  • could be novel and effective treatment strategies for heart failure.
Proposed mechanisms through which overexpression of the
  • mitochondrial transcription factor A (TFAM) gene prevents
  • mitochondrial DNA (mtDNA) damage,
  • oxidative stress, and
  • myocardial remodelling and failure.
In wild-type mice, mitochondrial transcription factor A
  • directly interacts with mitochondrial DNA to form nucleoids.
Stress such as ischaemia causes mitochondrial DNA damage, which
  1. increases the production of reactive oxygen species (ROS)
  2. leading to a catastrophic cycle of mitochondrial electron transport impairment,
  3. further reactive oxygen species generation, and mitochondrial dysfunction.
TFAM overexpression may protect mitochondrial DNA from damage by
  1. directly binding and stabilizing mitochondrial DNA and
  2. increasing the steady-state levels of mitochondrial DNA
ameliorating mitochondrial dysfunction and thus the development and progression of heart failure.

Conclusion

Heart failure is a multifactorial syndrome that is characterized by
  • abnormal energetics and substrate metabolism in heart and skeletal muscle.
Although existing therapies have been beneficial, there is a clear need for new approaches to treatment.
Pharmacological targeting of the cellular stresses underlying mitochondrial dysfunction, such as
  • elevated fatty acid levels,
  • tissue hypoxia and oxidative stress and
  • metabolic modulation of heart and skeletal muscle mitochondria,
    • appears to offer a promising therapeutic strategy for tackling heart failure.
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Delarue J, Magnan C. Free fatty acids and insulin resistance. Curr Opin ClinNutr Metab Care 2007; 10:142
Lee L, Campbell R, Scheuermann-Freestone M, et al. Metabolic modulation with perhexiline in chronic heart failure: a randomized, controlled trialof short-term use of a novel treatment. Circulation 2005; 112:3280
Tsutsui H, Kinugawa S, Matsushima S. Mitochondrial oxidative stress and dysfunction in myocardial remodelling. Cardiovasc Res. 2009;81(3):449-56. http://dxdoi.org/10.1093/cvr/cvn280.
C Maack, M Böhm. Targeting Mitochondrial Oxidative Stress in Heart Failure. J Am Coll Cardiol. 2011;58(1):83-86. http://dx.doi.org/10.1016/j.jacc.2011.01.032

 References

Mitochondrial dynamics and cardiovascular diseases    Ritu Saxena
https://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress   larryhbern
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation   larryhbern
http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Ca2+ signaling: transcriptional control     larryhbern
http://pharmaceuticalintelligence.com/2013/03/06/ca2-signaling-transcriptional-control/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified  Aviva Lev-Ari
http://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function    larryhbern
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis  larryhbern
http://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar
http://pharmaceuticalintelligence.com/2012/11/09/low-bioavailability-of-nitric-oxide-due-to-misbalance-in-cell-free-hemoglobin-in-sickle-cell-disease-a-computational-model/

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure    larryhbern
http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Reversal of Cardiac mitochondrial dysfunction, Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

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

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation, Aviva Lev-Ari, PhD, RN 10/4/2012
https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

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, Aviva Lev-Ari, PhD, RN 10/4/2012
https://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/

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013, L H Bernstein, MD, FACP and Aviva Lev-Ari,PhD, RN  3/7/2013
https://pharmaceuticalintelligence.com/2013/03/07/genomics-genetics-of-cardiovascular-disease-diagnoses-a-literature-survey-of-ahas-circulation-cardiovascular-genetics-32010-32013/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production, Aviva Lev-Ari, PhD, RN 7/19/2012 https://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/

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis. Aviva Lev-Ari, PhD, RN 10/30/2012
https://pharmaceuticalintelligence.com/2012/10/30/cardiovascular-risk-inflammatory-marker-risk-assessment-for-coronary-heart-disease-and-ischemic-stroke-atherosclerosis/

Cholesteryl Ester Transfer Protein (CETP) Inhibitor: Potential of Anacetrapib to treat Atherosclerosis and CAD.     Aviva Lev-Ari, PhD, RN 4/7/2013
https://pharmaceuticalintelligence.com/2013/04/07/cholesteryl-ester-transfer-protein-cetp-inhibitor-potential-of-anacetrapib-to-treat-atherosclerosis-and-cad/

Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN  4/4/2013  https://pharmaceuticalintelligence.com/2013/04/04/hypertriglyceridemia-concurrent-hyperlipidemia-vertical-density-gradient-ultracentrifugation-a-better-test-to-prevent-undertreatment-of-high-risk-cardiac-patients/

Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore.
Aviva Lev-Ari, PhD, RN 4/3/2013
https://pharmaceuticalintelligence.com/2013/04/03/fight-against-atherosclerotic-cardiovascular-disease-a-biologics-not-a-small-molecule-recombinant-human-lecithin-cholesterol-acyltransferase-rhlcat-attracted-astrazeneca-to-acquire-alphacore/

High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk.   Aviva Lev-Ari, PhD, RN 3/31/2013
https://pharmaceuticalintelligence.com/2013/03/31/high-density-lipoprotein-hdl-an-independent-predictor-of-endothelial-function-artherosclerosis-a-modulator-an-agonist-a-biomarker-for-cardiovascular-risk/

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

Sulfur-Deficiciency and Hyperhomocysteinemia, L H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-and-hyperhomocusteinemia/

Structure of the human mitochondrial genome.

Structure of the human mitochondrial genome. (Photo credit: Wikipedia)

English: Treatment Guidelines for Chronic Hear...

English: Treatment Guidelines for Chronic Heart Failure (Photo credit: Wikipedia)

English: Oxidative stress process Italiano: Pr...

English: Oxidative stress process Italiano: Processo dello stress ossidativo (Photo credit: Wikipedia)

Diagram taken from the paper "Dissection ...

Diagram taken from the paper “Dissection of mitochondrial superhaplogroup H using coding region SNPs” (Photo credit: Asparagirl)

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Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation 

Author and Curator of an Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

A Three Component Method for Endogenous Augmentation of cEPCs

Macrovascular Disease: The  Therapeutic Potential of cEPCs

Observations on Intellectual Property Development For an Unrecognized Future Fast Acting Therapy for Patients at High Risk for Macrovascular events

ElectEagle represents a discovery of a novel “multimarker biomarker” for cardiovascular disease that innovates on four counts.

First, it proposes new therapeutic indications for acceptable drugs.

Second, it defines a specific combination of therapeutic agents, thus, it put forth a proprietary drug combination.

Third, it targets receptor systems that have not been addressed in the context of cEPCs augmentation methods. Chiefly, modulation of the following three-targeted receptor systems: (a) inhibition of ET-1, ETA and ETA-ETB receptors by antagonists (b) induction of eNOS, by agonists and NO stimulation and (c) upregulation of PPAReceptor-gamma by agonists (TZD). While (b) and (c) are implicated as having favorable effects of cEPCs count, each exerting its effect by a different pathway, it is suggested in this project that (a) might be identify to be the more powerful of the three markers. Our method, ElectEagle is the FIRST to postulate the following: (1) time concentration dependence on eNOS reuptake (2) dose concentration dependence on NO production (3) time and dose concentration dependence for ET-1, ETA and ETA-ETB inhibition, and (4) dose concentration dependence on PPAReceptor-gamma. Points First, Second and Third are covered in Part II where a special focus is placed on ET-1, ETA and ETA-ETB receptors.

Fourth, ElectEagle proposes a platform with triple modes of delivery and use of the test, as described in Part III. The triple modes are as follows: (A) an automated platform from a centralized lab with integration to Lab’s information management system. (B) a point-of-care testing device with appropriate display of test results (small benchtop analyzers in PCP office). (C) a device used for home monitoring of analytes (the hand-held device facilitates rapid read of scores and their translation to drug concentration of each of the three therapeutic agents, with computation of the three drug concentrations done by the device. Thus, it offers quicker optimization of treatment.  ElectEagle is the FIRST to propose a CVD patient kit, hand-held device, which calculates on demand an adjustable therapeutic regimen as a function of cEPCs count biomarker. In this regard, a similarity to the pump, in management of blood sugar in DM patients, exists. Since there is a high co-morbidity between DM and CVD, our methods, ElectEagle may eventually become a targeted therapy for the DM Type 2 population.

Postulates of Multiple Indications for the Method Presented: Positioning of a Therapeutic Concept for Endogenous Augmentation of cEPCs

Potential Therapeutic Indications for ElectEagle

ElectEagle can become the drug therapy of choice for the following indications:

  •       CAD patients
  •       Endothelial Dysfunction in DM patients with or without Erectile   Dysfunction
  •       Atherosclerosis patients: Arteries and or veins
  •       pre-stenting treatment phase
  •       post-stenting treatment phase
  •       if stent is a Bare Metal stent (BMS)
  •       if stent is Drug Eluting stent (DES)
  •       if stent is EPC antibody coated (the ElectEagle method increase cEPCs generation in vitro) so availability of cEPCs is increased
  •       post CABG patients (the ElectEagle enhances healing by endogenous augmentation of cEPCs)
  •       target sub segments of CVD patients on blood thinner drugs (the ElectEagle does not require treatment with antiplatelet agents, it is suitable for all patients on Coumadin. This population have a counter indication for antiplatelet agents which is a follow up treatment after stent implantation for 30 days, with stent-eluting long term regimen of antiplatelet agents, 6 months and in some cases indefinitely (Tung, 2006).
  •       ElectEagle is based on systemic therapeutics (versus the localized stent solution requiring multiple and even overlapping stents)
  •       ElectEagle will be having potential in two contexts

1.  Coronary disease

2.  Periphery vascular disease

Comparative analysis of endogenous and exogenous cEPCs augmentation methods:

A. endogenous augmentation method properties:

  •    temporal – while drug therapy in use – drug action is interruptible
  •    time concentration on eNOS reuptake
  •    dose concentration on NO production
  •    time and dose concentration manner for ETB inhibition
  •    dose concentration on PPAR-gamma

B.  cell-based and other exogenous methods

  • permanent colonization till apoptosis if no repeated attempts of re-transfer, re-implantation as the protocol usually has several stages

ElectEagle will be resulting in potential delay of stenting implantation. Patients that are target for stenting may benefit form ElectEagle that will facilitate and accelerate healing after the stent is in place. EPC antibody coated stents will work if and only if the patient has more that just low cEPCs, most patient undergoing stenting tend to have low level of cEPC. The ElectEagle method can be coupled with that type of new stents, called Genous, now in clinical trials (HEALING II, III). These stents enhance the body ability in mobilization of cEPCs, only. However, if the initial population of cEPCs is low, an endogenous fast acting cell augmentation method is needed for pretreatment before the PCI procedure with Genous is scheduled.

Mechanism of action (MOA) for ElectEagle‘s component 1

Inhibition of ET-1, ETA and ETA-ETB

Source for vasodilators substances in the endothelium are PGI2 and NO. A potent vasoconstrictor peptide is the endothelin family, first isolated in the aortic endothelial cells.

Endothelins: Biosynthesis, Structure & Clearance

Three isoforms of endothelin (ET) have been identified. ET-1, ET-2 and ET-3. Each isoform is the product of a different gene and is synthesized as a prepro form that is processed to a propeptide and then to the mature peptide. Endothelin-converting enzyme (ECE) converts a prepro into a mature peptide. Each ET is a 21-amino-acid peptide containing two disulfide bridges. ETs are widely distributed in the body. ET-1 is the predominant ET secreted by the vascular endothelium. It is also produced by neurons and astrocytes in CNS and in endometrial, renal mesangial, sertoli, breast epithelial and other cells. ETs are present in the blood in low concentrations, they act locally in a paracrine or autocrine fashion rather than as circulating hormones.

Expression of ET-1 gene is increased by Growth Factors and cytokines, transforming factor-beta (TGF-beta) and interleukin 1 (IL-1), vasoactive substances including angiotensin II and vasopressing and mechanical stress. Expression is inhibited by NO, prostacyclin and ANP (source for vasodilators substances in the endothelium are PGI2 and NO.) Clearance of ETs from the circulation is rapid and involves enzymatic degradation by NEP 24.11 and clearance by the ETB receptor.

Endothelins: Action

ET exerts many actions on the body. In particular dose-dependent vasoconstriction in most vascular beds. Intravenous administration of ET-1 causes a rapid decease in BP followed by a prolonged increase. The depressor response results PGI2 and NO release from the vascular endothelium. The pressor response is due to direct constriction of vascular smooth muscle. ETs exert direct positive inotropic and chronotropic actions on the heart and are potent coronary vasoconstrictors. ETs actions on other organ is described in (Reid, 2004). ETs interact with several endocrine systems, increase secretion of renin, aldosterone, vasopressin and Atrial natriuretic peptide (ANP.) Action exerted on CNS and PNS, GI system, liver, GU, reproductive system, eye, skeletal and skin. ET-1 is a potent mitogen for vascular smooth muscle cells, cardiac myocytes and glomerular mesangial cells.

ET receptors are present in many tissues and organs, blood vessel wall, cardiac muscle, CNS, lung, kidney, adrenal, spleen, and GI. The signal transduction mechanism triggered by binding of ET-1 to its receptors, ETA & ETB includes effects of stimulation of phospholipase C, formation of inositol triphosphate and release of calcium from the ER which results in vasoconstriction. Stimulation of PGI2 and NO synthesis result in decreased intracellular calcium concentration and vasodilation.

Two receptor subtypes, ETA & ETB have been cloned and sequenced. ETA receptors have a high affinity for ET-1 and a low affinity for ET-3 and are located on smooth muscle cells, where they mediate vasoconstriction. ETB receptors have an equal affinity for ET-1 and ET-3 and are located on vascular ECs, where they mediate release of PGI2 and NO. Both receptor types belong to the G protein-coupled seven-transmembrane domain family of receptors.

Inhibitors of Endothelin Synthesis & Action

ETs can be blocked with receptor antagonists and with drugs that block the Endothelin-converting enzyme (ECE), Endothelin-converting enzyme inhibitors (ECEI). Two receptor subtypes, ETA & ETB can be blocked selectively, or both can be blocked with nonselective ETA – ETB antagonists. Bosentan is a nonselective antagonist, available both intravenously and orally. It blocks the initial transient depressor (ETB ) and the prolonged pressor (ETA) responses to intravenous ET. Oral ET antagonists are available for research purposes. The formation of Endothelin-converting enzyme (ECE) can be blocked with Phosphoramidon. The therapeutic potential of ECEI is similar to that of the ET receptor antagonist, Bosentan, an active competitive inhibitor of ET [it has teratogenic and hepatotexic effects].

Physiologic & Pathologic Roles of Endothelin Antagonists

Systemic administration of ET receptor antagonists or ECEI causes vasodilation and decreases arterial pressure in human and in experimental animals. Intra-arterial administration of the drugs also causes slow-onset forearm vasodilation in humans. This is an evidence that the endothelin system participates in the regulation of vascular tone, even under resting conditions (Reid, 2004).

There is evidence that ETs participate in CVD, including hypertension, cardiac hypertrophy, CHF, atherosclerosis, CAD, MI. ETs have been implicated in pulmonary diseases, PA HTN, asthma, renal diseases. Increased ET levels was found in the blood, increased expression of ET mRNA in endothelial or vascular smooth muscle cells and the responses to administration of ET antagonists. ET antagonists have potential for treatment of these diseases. In clinical trials, Bosentanand other nonselective antagonists as well as ETA selective antagonists produce beneficial effects on hemodynamics and symptoms of CHF, PA HTN and essential HTN (Sütsch et al., 1998), (Haynes, 1996), (Lahav et al., 1999). Currently, it is approved for use in pulmonary hypertension (Benowitz, 2004).

ElectEagle Project Drug combination Therapy has selected Bosentan or other nonselective ET antagonists as well as ETA selective antagonists to enhance the effects an eNOS agonist and a PPAR-gamma agonist will have on CVD patient’s propensity to achieve beneficial effects for endogenous augmentation of cEPCs. The impact the ETs have on the body is of a very wide range and of a most important from a physiological point of view, respectively, we did not leave Big ET-1 out of the therapeutic treatment design.

Proposed integration plan for ElectEagle’s Version I with CVD patients current medication regimen for selective medical diagnoses

Blood Pressure Medicine:

Beta blockers, Verapamil (Calan), Reserpine (Hydropes), Clonidine (Catapres), Methyldopa (Aldomet)

Diuretics:

Thiazides, Spironolactone (Aldactone), Hydralazine

Antidepressants:

Prozac, Lithium, MOA’s, Tricyclics

Stomach Medicine:

Tagamet and Zantac, plus other compounds containing Cimetidine and Ranitidine or associated compounds in Anticholesterol Drugs

Antipsychotics:

Chlorpromazine (Thorazine), Pimozide (Orap), Thiothixine (Navane), Thiordazine (Mellaril), Sulpiride, Haloperidol (haldol), Fluphenazine (Modecate, Prolixin)

Heart Medicine:

Clofibrate (Atromid), Gemfibrozil, Diagoxin

Hormones:

Estrogen, Progesterone, Proscar, Casodex, Eulexin, Corticosteroids Gonadotropin releasing antagonists: Zoladex and Lupron

Cytotoxic agents:

Cyclophosphamide, Methotrexate, Roferon Non-steroidal anti-inflammatories

Others

Alprazolam, Amoxapine, Chlordiazepoxide, Sertraline, Paroxetine, Clomipramine, Fluvoxamine, Fluoxetine, Imipramine, Doxepine, Desipramine, Clorprothixine, Bethanidine, Naproxen, Nortriptyline, Thioridazine, Tranylcypromine, Venlafaxine, Citalopram.

INTERACTIONS for Nebivolol

Calcium Antagonists:

Caution should be exercised when administering beta-blockers with calcium antagonists of the verapamil or diltiazem type because of their negative effect on contractility and atrio-ventricular conduction. Exaggeration of these effects can occur particularly in patients with impaired ventricular function and/or SA or AV conduction abnormalities. Neither medicine should therefore be administered intravenously within 48 hours of discontinuing the other.

Anti-arrhythmics:

Caution should be exercised when administering beta-blockers with Class I anti-arrhythmic drugs and amiodarone as their effect on atrial conduction time and their negative inotropic effect may be potentiated. Such interactions can have life threatening consequences.

Clonidine:

Beta-blockers increase the risk of rebound hypertension after sudden withdrawal of chronic clonidine treatment.

Digitalis:

Digitalis glycosides associated with beta-blockers may increase atrio-ventricular conduction times. Nebivolol does not influence the kinetics of digoxin & clinical trials have not shown any evidence of an interaction.

Special note: Digitalisation of patients receiving long term beta-blocker therapy may be necessary if congestive cardiac failure is likely to develop. The combination can be considered despite the potentiation of the negative chronotropic effect of the two medicines. Careful control of dosages and of individual patient’s response (notably pulse rate) is essential in this situation.

Insulin & Oral Antidiabetic drugs:

Glucose levels are unaffected, however symptoms of hypoglycemia may be masked.

Anaesthetics:

Concomitant use of beta-blockers & anaesthetics e.g. ether, cyclopropane & trichloroethylene may attenuate reflex tachycardia & increase the risk of hypotension

Testing ElectEagle’s a-priori postulates presented in Part I

a-priori postulates presented in Part I for Component 1:ET-1, ETA and ETA-ETB inhibition

  • time and dose concentration dependence for ETA and ETA-ETB inhibition

 In the literature we found evidence for dose concentration dependence manner (Reid, 2004).

 

ETA and ETA-ETB inhibitor time concentration dependence manner dose concentration dependencemanner time and dose dose  
Bosentan   (Reid, 2004)   62.5, 125 mg tablets

a-priori postulates presented in Part I for Component 2: NO, eNOS induction and stimulation

  • time concentration dependence on eNOS reuptake
  • dose concentration dependence on NO production

In the literature we found evidence for dose concentration dependence manner

Ach, Histamine, Genistein, ACEI, Fenofibrates, NEBIVOLOL, Calcium channel blocker, Enzyme S-nitrosylation

In the literature we found evidence for time concentration dependence manner:

Ach, BRL37344, a 3-adrenoceptor agonist

In the literature we found evidence for time and dose concentration dependence manner:

Histamine

NO, eNOS AgonistsStimulate phosphorylation of eNOS at serine 1177, 1179, 116 Conversion of L-arginine toL-citrulline time concentration dependence manner dose concentration dependencemanner time and dose dose (nmol·mg

of protein-1)

Grovers et al., (2002)

A23187       (5µM)
Acetylcholine Xu et al., (2002) Sanchez et al., (2006)   (1µM)
5-Hydroxytryptamine       (1µM)
VEGF (       (20ng/ml)
Bradykinin       (1µM)
Histamine   McDuffie et al., (1999) McDuffie et al., (2000) (10µM)
genistein   Liu et al., (2004)   (1µM)
ACEI   Skidgel et al., (2006)    
Fenofibrates   Asai et al., (2006)    
BRL37344, a 3-adrenoceptor agonist Pott et al., (2005)      
NEBIVOLOLß1-selective adrenergic receptor antagonist with nitric oxide (NO)–mediation for vasodilation

 

  Ritter et al., (2006)    
Calcium channel blocker   Church and Fulton, (2006),    
Enzyme S-nitrosylation   Erwin et al., (2006)    

 

a-priori postulates presented in Part I for Component 3: PPAR-gamma

  • dose concentration dependence on PPAReceptor-gamma – confirmed by a study for Rosiglitazone and a study for Ciglitazone
PPAReceptor-gamma agonists time concentration dependence manner dose concentration dependencemanner time and dose dose  
Rosiglitazone   Polikandriotis et al., (2005)   maximum recommended daily dose of 8 mg to 2,000 mg.
Ciglitazone Polikandriotis et al., (2005)    


Development of an Experimental Treatment Protocol for

ElectEagle Version I

Therapeutic Strategy for cEPCs Endogenous Augmentation for measuring the number of circulating Endothelial Progenitor Cells (cEPCs) before and after a newly design treatment with Pharmacological agents

Component 1: Inhibition of ET-1, ETA and ETA-ETB

Bosentan (Tracleer) Oral: 62.5, 125 mg tablets

 

Component 2: Induction of NO production and stimulation of eNOS

Nebivolol – ß1-selective adrenergic receptor antagonist with nitric oxide (NO)– mediation for vasodilation

A single daily dose of 5 mg was appropriate, with no evident advantage at 10 mg (Van Nueten et al.,1997)

Component 3: Treatment Regime with PPAR-gamma agonists (TZD)

A Substitute for Rosiglitazone, 2-8 mg once daily

The combination drug therapy for endogenous augmentation of cEPCs in CVD patients for achievement of reduction in risk for macrovascular events is recommended to be applied for Clinical Trial Phase One in the following regimen:

Use the following combination of drugs for the following Stages

Bosentan (Tracleer), Oral: 62.5 mg tablets

Nebivolol, Oral: 5mg once daily

A substitute for Rosiglitazone, 8 mg once daily

 

Stage 1: ET-1 Antagonist Effect on eEPC

1.0 Measurement of the Baseline of number of cEPC

1.1 Administer ET-1 antagonist for 10 days

1.2 Measurement of number of cEPC after 10 days of treatment with ET-1 antagonist

Stage 2: Nitric Oxide Effect on cEPC

2.0 Measurement of number of cEPC obtained in 1.2

2.1 Administer Nitric Oxide Agonist for 10 days

2.2 Measurement of number of cEPC after 10 days of

treatment with Nitric Oxide Agonist

Stage 3: Comparison of ET-1 and NO Effects on cEPC Proliferation

3.0 Comparison of number of cEPC in 1.2 to 2.2

¨     IF number of cEPC in 1.2 > number of cEPC in 2.2

-> continue 1.1 only

[ET-1 antagonist more effective for proliferation of cEPC than NO Agonist]

3.1.1      Measurement of number of cEPC every 10 days

¨     IF number of cEPC in 1.2 < number of cEPC in 2.2

-> continue 2.1 only

[ET-1 antagonist less effective for proliferation of cEPC than NO Agonist]

3.2.1      Measurement of number of cEPC every 10 days

¨     IF number of cEPC in 1.2 = number of cEPC in 2.2

-> continue 1.1 AND 2.1

[ET-1 antagonist equal NO Agonist in effectiveness for proliferation of cEPC]

-> Administer a Combination therapy of ET-1 antagonist and NO Agonist for 10 days

3.3.1      Measurement of number of cEPC every 10 days

Stage 4: ET-1 and/or NO Effect on Cardiovascular (CV) Events

q      After 12 months Comparison of CV events in patient population in

Stage 3.1, 3.2, 3.3

  • Cardiovascular events in patients in 3.1
  • Cardiovascular events in patients in 3.2
  • Cardiovascular events in patients in 3.3

Conclusions

  •       Most favorable and unexpected to us was finding in the literature new indications for TDZs as stimulators of eNOS, in addition to the new indication for atherosclerosis besides the classic indication in pharmacology books, being in the reduction of insulin resistance. Reassuring our selection of a substitute for Rosiglitazone.
  •       Most favorable and unexpected to us was finding in the literature new indications for beta blockers as NO stimulant, nebivolol, a case in point, thus, fulfilling two indications in one drug along the direction of the study to identify eNOS agonists.
  •       The following combination of drugs was selected for ElectEagle Version I

Bosentan (Tracleer), Oral: 62.5 mg tablets

Nebivolol, Oral: 5mg once daily

A Substitute for Rosiglitazone, 8 mg once daily

  •       We confirmed time and dose concentrations postulating apriori in most cases. Additional literature searches will benefit the project for the three drugs selected
  •       We have identified Inhibition of ET-1, ETA and ETA-ETB as one of the agent in the drug combination. The entire literature on cEPCs does not implicate Endothelin with impact on eEPCs while it is known that mechanical stress increase its secretion, this type of stress is implicated with hypertension. To leave out ET-1 from the cEPCs function in CVD risk equates to leaving out Thrombin from the coagulation cascade. ElectEagle Version I corrects that ommission. 

REFERENCES

Benowitz, NL., (2004). Antihypertensive Agents. Chapter 11 in Katzung, BG., Basic & Clinical Pharmacology. McGraw-Hill, 9th Edition, pp. 160-183.

Haynes WG, Ferro CJ, O’Kane KP, Somerville D, Lomax CC, Webb DJ, (1996). Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans. Circulation, 15;93(10):1860-70. 

N S Kirkby, P W F Hadoke, A J Bagnall, and D J Webb (2008)

The endothelin system as a therapeutic target in cardiovascular disease: great expectations or bleak house? Br J Pharmacol. 2008 March; 153(6): 1105–1119.

Ohkita Mamoru, Masashi Tawa, Kento Kitada and Yasuo Matsumura (2012). Pathophysiological Roles of Endothelin Receptors in Cardiovascular Diseases,  J Pharmacol Sci 119, 302 – 313 (2012)

Reid, Ian A., (2004). Vasoactive Peptides. Chapter 17 in Katzung, BG., Basic & Clinical Pharmacology. McGraw-Hill, 9th Edition, pp. 281 – 297, in particular, Endothelins, pp. 290-293.

  For a comprehensive Bibliography on the Three Therapeutic Componenets and the pathophysiology of Cardiovascular Disease, follow this link:

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

https://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/

 Other aspects of Nitric Oxide involvement in biological systems in humans are covered in the following posts on this site:

Nitric Oxide in bone metabolism July 16, 2012

Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/16/nitric-oxide-in-bone-metabolism/?goback=%2Egde_4346921_member_134751669

 

Nitric Oxide production in Systemic sclerosis July 25, 2012

Curator: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/07/25/nitric-oxide-production-in-systemic-sclerosis/?goback=%2Egde_4346921_member_138370383

 

Nitric Oxide Signalling Pathways August 22, 2012 by

Curator/ Author: Aviral Vatsa, PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/22/nitric-oxide-signalling-pathways/?goback=%2Egde_4346921_member_151245569

 

Nitric Oxide: a short historic perspective August 5, 2012

Author/Curator: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/05/nitric-oxide-a-short-historic-perspective-7/

 

Nitric Oxide: Chemistry and function August 10, 2012

Curator/Author: Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/08/10/nitric-oxide-chemistry-and-function/?goback=%2Egde_4346921_member_145137865

 

Nitric Oxide and Platelet Aggregation August 16, 2012 by

Author: Dr. Venkat S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/?goback=%2Egde_4346921_member_147475405

 

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure August 20, 2012

Author: Larry Bernstein, MD

https://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Nitric Oxide: The Nobel Prize in Physiology or Medicine 1998 Robert F. Furchgott, Louis J. Ignarro, Ferid Murad August 16, 2012

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/16/nitric-oxide-the-nobel-prize-in-physiology-or-medicine-1998-robert-f-furchgott-louis-j-ignarro-ferid-murad/

 

Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents August 13, 2012

Author: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

 

Nano-particles as Synthetic Platelets to Stop Internal Bleeding Resulting from Trauma

August 22, 2012

Reported by: Dr. V. S. Karra, Ph.D.

https://pharmaceuticalintelligence.com/2012/08/22/nano-particles-as-synthetic-platelets-to-stop-internal-bleeding-resulting-from-trauma/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production July 19, 2012

Curator and Research Study Originator: Aviva Lev-Ari, PhD, RN

https://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/

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

July 2, 2012

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

Curator: Aviva Lev-Ari, PhD, RN

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

 

Bone remodelling in a nutshell June 22, 2012

Author: Aviral Vatsa, Ph.D., MBBS

https://pharmaceuticalintelligence.com/2012/06/22/bone-remodelling-in-a-nutshell/

Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part, September  

AuthorL Aviral Vatsa, PhD, September 23, 2012

https://pharmaceuticalintelligence.com/2012/09/23/targeted-delivery-of-therapeutics-to-bone-and-connective-tissues-current-status-and-challenges-part-i/

Calcium dependent NOS induction by sex hormones: Estrogen

Curator: S. Saha, PhD, October 3, 2012

https://pharmaceuticalintelligence.com/2012/10/03/calcium-dependent-nos-induction-by-sex-hormones/

 

Nitric Oxide and Platelet Aggregation,

Author V. Karra, PhD, August 16, 2012

https://pharmaceuticalintelligence.com/2012/08/16/no-and-platelet-aggregation/

Bystolic’s generic Nebivolol – positive effect on circulating Endothelial Progenitor Cells endogenous augmentation

Curator: Aviva Lev-Ari, PhD, July 16, 2012

https://pharmaceuticalintelligence.com/?s=Nebivolol

 

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation

Author: Aviva Lev-Ari, PhD, 10/4/2012

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

 

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

Curator: Aviva Lev-Ari, 10/4/2012.

https://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/

 

Nitric Oxide Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, 10/7/2012.

https://pharmaceuticalintelligence.com/2012/10/07/no-nutritional-remedies-for-hypertension-and-atherosclerosis-its-12-am-do-you-know-where-your-electrons-are/

Drug Information

Component 1: Inhibition of ET-1, ETA and ETA-ETB

Bosentan (Tracleer)

BACKGROUND: Although local inhibition of the generation or actions of endothelin-1 has been shown to cause forearm vasodilatation, the systemic effects of endothelin receptor blockade in healthy humans are unknown. We therefore investigated the cardiovascular effects of a potent peptide endothelin ETA/B receptor antagonist, TAK-044, in healthy men. METHODS AND RESULTS: Two randomized, placebo-controlled, crossover studies were performed. In nine subjects, TAK-044 (10 to 1000 mg IV over a 15-minute period) caused sustained dose-dependent peripheral vasodilatation and hypotension. Four hours after infusion of the highest dose (1000 mg), there were decreases in mean arterial pressure of 18 mm Hg and total peripheral resistance of 665 AU and increases in heart rate of 8 bpm and cardiac index of 0.9 L x min(-1) x m(-2) compared with placebo. TAK-044 caused a rapid, dose-dependent increase in plasma immunoreactive endothelin (from 3.3 to 35.7 pg/mL within 30 minutes after 1000 mg). In a second study in eight subjects, intravenous administration of TAK-044 at doses of 30, 250, and 750 mg also caused peripheral vasodilatation, and all three doses abolished local forearm vasoconstriction to brachial artery infusion of endothelin-1. Brachial artery infusion of TAK-044 caused local forearm vasodilation. CONCLUSIONS: The endothelin ETA/B receptor antagonist TAK-044 decreases peripheral vascular resistance and, to a lesser extent, blood pressure; increases circulating endothelin concentrations; and blocks forearm vasoconstriction to exogenous endothelin-1. These results suggest that endogenous generation of endothelin-1 plays a fundamental physiological role in maintenance of peripheral vascular tone and blood pressure. The vasodilator properties of endothelin receptor antagonists may prove valuable therapeutically (Haynes et al., 1996).

http://www.tracleer-pph.com/

http://www.medicinenet.com/script/main/art.asp?articlekey=44221&pf=3&page=1

GENERIC NAME: BOSENTAN – ORAL (boh-SEN-tan)

BRAND NAME(S): Tracleer

WARNING: This medication may cause serious liver problems. Your doctor should monitor your liver function closely to decrease your risk of liver-related side effects. Tell your doctor immediately if you notice any of these symptoms of liver problems: nausea, vomiting, stomach pain, unusual tiredness, and yellowing eyes or skin. These effects, if they occur, may go away over time (are reversible). This medication must not be used during pregnancy because it can cause fetal harm (e.g., birth defects). See the pregnancy warning information below (in Precautions section).

USES: Bosentan is used to treat a condition of high blood pressure in the lungs (pulmonary arterial hypertension). It works by causing the blood vessels (arteries) in the lungs to relax and expand, thus decreasing the pressure.

HOW TO USE: Before using, review the bosentan Medication Guide for information on the safe use of this medicine. Take this medication by mouth usually twice daily in the morning and evening with or without food; or as directed by your doctor. The dosage is based on your medical condition and response to therapy. Your doctor may recommend to gradually increase your dose over time so your body may better adjust to the effects of this drug. Do not stop taking this medication without consulting your doctor. Some conditions may become worse when the drug is abruptly stopped. Your dose may need to be gradually decreased.

SIDE EFFECTS: Headache, nose/throat irritation, itching, flushing, or stomach upset may occur. If any of these effects persist or worsen, notify your doctor or pharmacist promptly. Tell your doctor immediately if any of these unlikely but serious side effects occur: irregular heartbeat, unusual tiredness and weakness, swelling of the feet or ankles, trouble breathing, dizziness or lightheadedness. If you notice any of the following very serious side effects of liver problems, stop taking bosentan and consult your doctor immediately: vomiting, stomach pain, yellowing eyes or skin. A serious allergic reaction to this drug is unlikely, but seek immediate medical attention if it occurs. Symptoms of a serious allergic reaction include: rash, itching, swelling, dizziness, severe trouble breathing. If you notice other effects not listed above, contact your doctor or pharmacist.

PRECAUTIONS: Tell your doctor your medical history, especially of: liver problems, blood disorders (e.g., anemia), any allergies. Caution is advised when using this drug in the elderly because they may be more sensitive to the effects of the drug. This medication must not be used during pregnancy because it may cause fetal harm. If you are pregnant or think you may be pregnant, do not take this medication and consult your doctor immediately. It is recommended that you use two reliable forms of birth control while taking this medicine. It is also recommended to have a pregnancy test done before treatment and every month during treatment with this drug. It is not known whether this drug passes into breast milk. Because of the potential risk to the infant, breast-feeding while using this drug is not recommended.

DRUG INTERACTIONS: This drug is not recommended for use with: cyclosporine, glyburide. Ask your doctor or pharmacist for more details. Tell your doctor of all prescription and nonprescription medication you may use, especially: azole antifungals (e.g., itraconazole, ketoconazole), statins for high cholesterol (e.g., lovastatin, simvastatin), HIV protease inhibitors (e.g., indinavir, ritonavir), tacrolimus. This medication may decrease the effectiveness of combination-type birth control pills. This can result in pregnancy. You may need to use an additional form of reliable birth control while using this medication. Consult your doctor or pharmacist for details. Do not start or stop any medicine without doctor or pharmacist approval.

OVERDOSE: If overdose is suspected, contact your local poison control center or emergency room immediately. US residents can call the US national poison hotline at 1-800-222-1222. Canadian residents should call their local poison control center directly.

NOTES: Do not share this medication with others. Laboratory and/or medical tests (e.g., liver function tests- LFT’s, blood tests) will be performed to monitor your progress and for side effects.

MISSED DOSE: If you miss a dose, use it as soon as you remember. If it is near the time of the next dose, skip the missed dose and resume your usual dosing schedule. Do not double the dose to catch up.

STORAGE: Store at room temperature between 68 and 77 degrees F (20 and 25 degrees C) away from light and moisture. Brief storage between 59 and 86 degrees F (15 and 30 degrees C) is permitted.

MEDICAL ALERT: Your condition can cause complications in a medical emergency. For enrollment information call MedicAlert at 1-800-854-1166 (USA), or 1-800-668-1507

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