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Amy HuangDrugs and Disease – Written Report

A Pharmacogenetic Approach to Reducing the Incidence of Statin-Associated Myopathy

Lipitor (generic: Atorvastatin calcium) is a lipid-lowering drug that is used to treat and prevent

atherosclerosis, hypercholesterolemia, and many other vascular conditions, in conjunction with

diet and exercise. It is a member of a class of drugs called statins, which are 3-hydroxy-3-

methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors that directly inhibit HMG CoA

reductase, an enzyme that catalyzes the formation of melavonate from HMG CoA in the

cholesterol synthesis pathway. The inhibition of this rate-limiting step in the in vivo synthesis of

cholesterol lowers serum cholesterol levels. As one of the best selling drugs in the world, with

more than 100 million prescriptions filled in 2004, Lipitor is well tolerated, has a good safety

profile, and a simple mode of delivery (5).

Despite numerous documentation of efficacy, Lipitor is not without side-effects. Statin use is

associated with an elevated risk of myopathy and rhabdomyolysis, with 1-10% of users

experiencing the muscle weakness and pain of myopathy and 0.1% of users affected by life-

threatening rhabdomyolysis, a condition in which muscle fibers rapidly break down and lead to

kidney failure (8). Though the percentage of patients with statin-associated myopathy may not be

high, the absolute number of patients with myopathic complications is high due to a large

number of statin prescriptions. In addition, many mild cases of myopathy are unreported, leading

to an even greater prevalence. For statin users who exercise, the percentage rises to 25% and

75% in athletes (5). This is a problem, since exercise is an important component of statin

therapy, in addition to diet.

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As Lipitor is well tolerated, effective, and easily administered, there is no need to chemically

alter the drug to increase bioavailability, reduce severe side-effects, or change the mode of

delivery. We therefore propose a pharmacogenetic approach to reducing the incidence of

myopathy by using a blood biomarker of muscle damage, creatine kinase (CK), to establish a

muscle damage/CK level correlation. This would allow us to identify a genetic biomarker for

myopathy through microarray analysis. Knowledge of myopathy risk would help patients

determine if they should commit to Lipitor or switch to an alternative lipid-lowering agent,

saving cost of treatment for side effects and reducing the prevalence of muscle damage.

Pathways in Statin-Induced Myopathy

The mechanisms of statin-induced myopathy are diverse, yet not well understood. One possible

cause of statin-induced myopathy is the reduction of cholesterol content in the plasma membrane

of muscle cells, leading to reduced membrane rigidity and subsequent muscle damage from

mechanical stress. Unfortunately, this does not seem to be a major contributor to myopathy (8).

Another possible mechanism related to the cholesterol synthesis pathway is terpenoid depletion

and apoptotic induction on various types of muscle cells (1). Terpenoids are a diverse class of

lipid compounds that can add hydrophobic prenyl groups to proteins in a process called

prenylation. Terpenoid depletion impairs cell signaling, as critical GTPases like Ras, Rho, and

Rac, require prenylation to function, causing the induction of apoptotic pathway genes such as

atrogin-1, a gene responsible for muscle atrophy in statin-induced myopathy (2).

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Statins may also initiate muscle damage through the generation of reactive oxygen species,

elevated intracellular calcium levels, and mitochondrial dysfunction of muscle fibers (8). The

complexity and multitude of potential pathways to statin-induced myopathy renders research

focus on one pathway or gene for biomarker discovery particularly difficult (see Figure 1). Thus,

using a general enzymatic marker for muscle damage like creatine kinase (CK or CPK) to create

a correlation with muscle weakness and pain associated with statin use, would help us discover a

genetic biomarker for statin-induced myopathy.

Proposal

Creatine kinase is an enzyme normally sequestered in skeletal and heart muscle. When muscle is

damaged or diseased, CK spills into the bloodstream and can be detected through a simple blood

test (4). Elevated CK levels may indicate the presence of various conditions such as myocardial

infarction, muscular dystrophy, and even normal activity such as exercise. In the case of statin-

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associated myopathy, testing for CK levels and correlating them with level of muscle pain or

weakness is the easiest method of determining extent of muscle damage, though it cannot

diagnose myopathy itself. A muscle biopsy will confirm extent of muscle damage

microscopically.

Our wish is to pursue a preventative approach to reducing the incidence of statin-associated

myopathy by developing a diagnostic test to predict myopathic susceptibility in potential users of

Lipitor. Current approaches of patient education are ineffective, as nonadherence to statin

therapy remains high – half of patients prescribed statins continue taking them at 6 months, and

only 30-40% continue to take them at 1 year (3). In addition, merely discontinuing therapy does

not reverse the muscle damage of myopathy. With a preventative approach using the diagnostic

tool, patients can determine if they should proceed with long-term statin treatment or pursue an

alternative. The first step is identifying a reliable biomarker for statin intolerance.

To discover a suitable genetic biomarker, we will implement a control and test group of potential

Lipitor users with atherosclerosis, with the former group receiving a non-statin, lipid-lowering

drug and the latter, varying therapeutic doses of Lipitor. CK levels and self-reported muscle

weakness or pain will be monitored, and myopathy verified by muscle biopsy. A microarray

analysis will then identify potential genes that are highly expressed in myopathic tissue and

genes contributing to myopathic susceptibility.

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A merit of the pharmacogenetic approach is that it leads to a cheap and easy means of informed

decision making. Lipitor, as well as many other drugs, may not be a one-size-fits-all treatment.

This diagnostic test will predict if the patient is able to tolerate statin therapy.

Comparison to Other Drug Proposals

Each groups’ drug proposals fall under one of three categories: 1) To improve drug efficiency 2)

To minimize side-effects 3) To improve mode of delivery. Many of the groups that aim to

increase drug efficiency or minimize side-effects propose to chemically and structurally alter the

drug, while those that aim to improve method of delivery mainly propose to encapsulate the drug

in a polymer system. Our approach is only one of two proposals, the other being that of Gleevec,

that advocates a pharmacogenetic, diagnostic approach.

Gleevec (generic: Imatinib) is a protein-tyrosine kinase inhibitor that is used to treat chronic

myelogenous leukemia (CML) and gastrointestinal stromal tumors (GIST). It functions by

blocking the actions of the BCR-ABL tyrosine kinase responsible for cancer, inhibiting further

proliferation of tumor cells (7). Like Lipitor, Gleevec is well tolerated, effective, and taken

orally.

The Gleevec group uses personalized medicine and pharmacogenetics to combat drug resistance

in Gleevec users. They propose to use Sanger sequencing to subdivide patients into those who

lack natural resistance (NR-) and those who have resistance (NR+). The former group receives

Gleevec, and the latter receives second-generation therapeutics. In addition, they propose to use

deep sequencing to identify patients prone to acquired resistance (AR+); AR+ patients will

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receive personalized dosing of Gleevec. This intricate approach assumes that the biomarkers for

NR and AR, which were mentioned to be mutations in the Gleevec binding regions, are

consistent and accurate – if different laboratories are to test the same patient, the diagnoses

would not differ. The goal of our proposal is to identify biomarkers of similar reliability.

Both proposals for Lipitor and Gleevec focus on diagnostics and personalized medicine. Another

group that proposed the use of personalized medicine, though not diagnostics, is that of Enbrel.

Enbrel (generic: Etanercept) is a tumor necrosis factor (TNF) inhibitor that is used to treat

rheumatoid arthritis and plaque psoriasis. This group proposed to create personalized Enbrel by

obtaining each patient’s TNFR2 sequence, amplifying target exons, and fusing the products to an

Ig1 construct. This patient-specific, recombinant protein will ensure that the drug does not lose

efficacy over time.

Modifying the drug may be feasible in some instances, as with Enbrel, but the Lipitor and

Gleevec groups show that chemical or structural alteration of the drug is unnecessary. If the drug

already has a good safety profile and is effective in treating its target disease, it might also be

advantageous to provide a supplement to reduce common side-effects or to increase drug

efficacy. The Herceptin and Provenge groups both follow this approach – the former group

proposes to supplement Herceptin with NRG1-β, a cardioprotective program, to minimize

cardiotoxicity, and the latter group proposes to supplement Provenge uptake with IL-2, an

immunomodulatory cytokine, to improve the drug efficacy.

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In general, our proposal is comparable to those of the aforementioned drug groups in two main

aspects – 1) decreased reliance on drug modification or 2) pharmacogenetics and personalized

medicine. Though the Gleevec group’s proposal is similar to that of Lipitor’s in that they also

promote a pharmacogenetics and diagnostic approach, we apply a different set of techniques to

accommodate a lack of a known biomarker for statin-induced myopathy. Proxies such as CK

levels and incidence of mutations in the Gleevec-binding domain are used to identify patient

susceptibility to myopathy or Gleevec resistance, respectively. However, we will be using

muscle biopsy and microarray analysis, as opposed to Sanger and deep sequencing, to first

discover a suitable biomarker for statin-induced myopathy. As opposed to the Provenge and

Herceptin proposals, we are not aiming to coadminister supplements to the main therapy to

prevent side-effects during treatment, but to administer a diagnostic test to prevent side-effects

before treatment.

Limitations and Future Approaches

Extensive drug modification is an expensive and lengthy process that involves numerous clinical

trials and a new drug application to the FDA. Alternative approaches must be taken should the

drug be ineffective or have life-threatening side-effects. This would the case with many of the

proposals that involve drug alteration. On the other hand, a diagnostic approach carries a much

less significant risk to the patient. An advantage of using pharmacogenetics to develop a

diagnostic test to determine a patient’s susceptibility to statin-induced myopathy is that the test

itself would cause little to no harm to the patient, while decreasing the prevalence of this

condition if applied correctly. As with the Gleevec approach, the test should be cost-effective

and sensitive.

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Limitations with our proposal are focused on the sensitivity of measuring CK levels as a proxy

for myopathy. Unfortunately, some studies show that a lack of CK elevation does not exclude

lack of muscle injury. Mohaupt et al. conducted a study of 83 patients taking statins, 44 with

statin-associated myopathy, by obtaining muscle biopsies of the patients and performing reverse-

PCR on the tissue to determine extent of muscle injury, while collecting CK measurements.

Many of the patients with evidence of muscle damage had normal CK levels. They concluded a

“lack of a correlation between clinical symptoms and circulating levels of creatine

phosphokinase” and that “damage can occur without increased levels of circulating CPK” (9).

These issues call into the question of how useful are CK measurements as a proxy for muscle

damage in the process of biomarker discovery. However, CK levels can confirm myopathy, but

not rule out lack of muscle damage. An alternative to CK is testing for genetic determinants of

statin intolerance, such as genetic variations in COQ2, a gene that encodes an enzyme in the

biosynthetic pathway for ubiquinone, or coenzyme Q10 (CoQ10) (10). Experiments have

demonstrated that missense mutations in COQ2 increase susceptibility to statin intolerance. A

disadvantage of using COQ2 as a biomarker is that it is useful only for a small subset of patients

who are genetically predisposed to myopathy at this specific allele. Many other genetic

determinants exist, which is why using CK measurements to aid in the discovery of a suitable

biomarker is the best approach, as it allows researchers to find other genetic determinants of

statin-induced myopathy.

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Another difficulty of using pharmacogenetics in our proposal is the multiplicity of metabolic,

pro-inflammatory, and signaling pathways responsible for the muscle damage in statin-

associated myopathy, many of which were previously described. Laaksonen et al. applied a

systems biology approach to identifying major target pathways by performing a bioinformatic

screen of whole genome expression profiling of muscle specimens from high-dose statin

patients, then a lipidomic analysis of plasma samples to identify novel pathways and sensitive

biomarkers for statin-induced myopathy.

A better approach to our proposal is to integrate lipidomic profiling into our microarray analysis

to create a combined bioinformatics and molecular approach. Like the ever-expanding field of

metabolomics, lipidomics allows researchers to study the pathways and metabolites of cellular

lipids, and their interactions with different lipids and proteins. This field is especially useful in

the study of the target diseases of Lipitor, such as stroke, atherosclerosis, and hypertension.

Ekroos et al. states the importance of lipidomics in both personalized medicine and drug

discovery:

“Lipidomics in combination with the appropriate clinical samples and biobank material is used

today to address the many unmet needs of disease diagnostics. In addition to prognostic and

diagnostic value, lipidomics may also find biomarkers that will serve as a read-out of

experimental or approved therapies, whereas the appreciation for the bioactivity of lipids has the

potential for identifying novel drug targets.” (6)

Pharmacogenetics is a useful application in reducing the incidence of statin-associated

myopathy. Though many issues as reliability of CK measurements as a proxy for muscle damage

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and the multiplicity of pathways to myopathy are obstacles to biomarker discovery, a variety of

bioinformatic and molecular approaches can be applied to improve testing outcome.

References

1. Blanco-Colio, LM. et al. (2002). 3-hydroxy-3-methyl-glutaryl coenzyme a reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of bcl-2 expression and rho a prenylation. Atherosclerosis 161, 17-26.

2. Cao, P. et al. (2008). Statin-induced muscle damage and atrogin-1 induction is the result of a geranylgeranylation defect. The FASEB Journal 23, 2844-2854.

3. Chaudhry, HJ and McDermott, B. (2008). Recognizing and improving patient nonadherence to statin therapy. Curr Atheroscler Rep, 10, 19-24.

4. Creatine kinase (ck). (n.d.). Retrieved from http://www.answers.com/topic/creatine-kinase-ck

5. Dirks, A. and Jones, K. (2006). Statin-induced apoptosis and skeletal myopathy. Am J Physiol Cell Physiol 291, 1208-1212.

6. Ekroos, K. (2010). Lipidomics: a tool for studies of atherosclerosis. Curr Atheroscler Rep. 12, 273–281.

7. Imatinib. (2009, February 1). Retrieved from http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH000034

8. Meador, B. and Huey, K. (2010). Statin-associated myopathy and its exacerbation with exercise. Muscle & Nerve 42, 469-479.

9. Mohaupt, M. et al. (2009). Association between statin-associated myopathy and skeletal muscle damage.CMAJ 181, 11-18.

10. Oh, J et al. (2007). Genetic determinants of statin intolerance. Lipids Health Dis. 6, 1-5.