Testing Pharmacogenetic Hypotheses

No hard and fast rules can be quoted to guide the investigator who wishes to explore human drug responsiveness for altered pharmacogenetic mechanisms. There are, however, some general principles that are widely applicable as E. Bright Wilson explains in his book An Introduction to Scientific Research (1952). First, an experiment is almost inevitably based on a main hypothesis, and one or more auxiliary hypotheses, that the experimenter attempts to disprove; the novice investigator may, in fact, proceed with only a vague notion of the hypothesis

Table 3.3 Pharmacologic Mechanisms Responsible for Human Pharmacogenetic Traits

Pharmacologic mechanism Drug or other substance

Comment

Altered pharmacokinetic mechanisms Drug entry

Increased first pass Hydralazine, isoniazid metabolism

Drug inactivation Increased

Increased

Decreased

Decreased

Decreased

Decreased

Decreased

Drug conversion Increased

Increased

Isoniazid

Many drugs

Nicotine

Many drugs

6-Mercaptopurine, 6-thioguanine, 8-azathioprine Methotrexate

5-Fluorouracil

Isoniazid

Benzidine and other carcinogenic arylamines

Rapid acetylator phenotype predisposes to drug failure

Rapid acetylator phenotype predisposes to drug failure CYP2D6 ultra rapid metabolizer phenotype predisposes to drug failure CYP2A6 poor metabolizer phenotype protects against cigarette smoking CYP2C19 and CYP2D6 poor metabolizer phenotype predisposes to drug toxicity Thiopurine methyltransferase deficient variants predispose to neutropenia Amplification of dihydrofolate gene protects against methotrexate therapy Pyrimidine dehydrogenase deficiency predisposes to drug toxicity

Slow acetylator phenotype predisposes to isoniazid hepatitis Slow acetylator phenotype predisposes to bladder urinary

Increased Decreased

Drug interaction Decreased enzyme activity

Drug targeted enzyme Increased abundance

Cooked food mutagens (heterocyclic amines) Acetophenetidin

Drugs that compete for the same drug-metabolizing enzyme

Many drugs

Increased abundance Succinylcholine

Polymorphic acetylator variants may predispose to colorectal cancer Decreased CYP catalyzed O-dealkylation predisposes to methemoglobinemia

CYP2D6, CYP2C19, CYP2C9 poor metabolizer variants and other polymorphic drug metabolizing enzyme targets predispose to interactions

CYP2D6 and CYP2C19 ultra rapid metabolizer phenotype predispose to drug failure Cynthiana serum cholinesterase variant predisposes to drug failure

(continued )

cancer

Pharmacologic mechanism Drug or other substance

Comment

Increased abundance Aldosterone

Decreased activity Succinylcholine

Decreased activity Many oxidant drugs

Decreased activity

Decreased activity

Decreased activity

Decreased activity Decreased activity Decreased activity

Decreased affinity

Decreased affinity

Enzyme (CYP2C9) inhibition

Enzyme (CYP2D6) inhibition

Many drugs

Smoking

Parathion

Decreased activity Fructose

Epoxides of benzopyrene, aflatoxin, and styrene

Trimethylamine

Licorice

(glycyrrhentic acid)

Succinylcholine Vitamin B6 Phenytoin/isoniazid

Quinidine/propafenone

Enzyme (CYP2C19) R and S enantiomers substrate competition

Ectopic overexpression of chimeric aldosterone synthase variant causes early-onset, dexamethasone-remediable hypertension Silent serum cholinesterases predispose to prolonged apnea G6PD deficiency predisposes to spontaneous and drug-induced hemolysis CYP2C19, CYP2C9 and CYP2D6 poor metabolizer phenotypes predispose to drug toxicity a1-Antitrypsin null variants predispose smokers (and non-smokers to a lesser degree) to premature emphysema Low-activity paraoxonase variant predisposes to parathion toxicity Low-activity aldolase B variant predisposes to fructose intolerance Glutathione-S-transferase null mutants predispose to cancer of various tissues Fish malodor syndrome due to an FMO3 variant predisposes to odor of rotting fish Licorice ingestion pseudoaldosterone causes state with hypertension attributed to b-hydroxylase deficiency or a glycrrhentic acid-induced inhibition Atypical serum cholinesterase predisposes to prolonged apnea Aminolevulinate synthase variant predisposes to anemia Slow acetylator phenotype predisposes to phenytoin toxicity from phenytoin/ isoniazid interaction Quinidine inhibition of CYP2D6 EM variants produces PM phenocopies that may predispose to toxicity or therapeutic ineffectiveness of many CYP2D6 drug substrates CYP2C19 PM variants

Pharmacologic mechanism Drug or other substance

Comment

Altered pharmacodynamic mechanisms

Drug entry

Decreased uptake

Increased excretion

Vitamin B12 Alkylating agents

Drug-targeted receptor

Decreased abundance Insulin

Decreased abundance Vasopressin

Decreased abundance Glucocorticoids, estrogen

Increased function Cyproterone,

Decreased function

Decreased function

Decreased function

Decreased down-regulation

Decreased affinity

Decreased affinity Decreased affinity hydroxyflutamide, nilutamide All-irans-retinoic acid

Decreased function Halothane/succinylcholine

Decreased function Not applicable

Endotoxin

(lipopolysaccharide, LPS)

Quinidine, psychotropics, antihistamines

Albuterol

Altered function Spironolactone

Dicumarol / warfarin Insulin

Glucocorticoids

B12 transporter variant predisposes to B12 deficiency Multidrug resistance transporter predisposes to therapeutic ineffectiveness

Insulin-resistant diabetes mellitus Vasopressin-resistant diabetes insipidus Resistance to steroid hormones Androgen receptor variant causes a paradoxical response to prostate cancer antiandrogens Chimeric retinoic acid receptor blocks terminal differentiation of myeloid cells to cause retinoic acid resistance Ryanodine-receptor variant of skeletal muscle predisposes to malignant hyperthermia CCR5 coreceptor A32 variant protects against, and slows progression, of HIV-1 infection TLR4 variant predisposes to endotoxin-mediated disorders such as asthma and septic shock Ion channel variants predispose to spontaneous and drug-induced ventricular arrhythmias and sudden death Nocturnal asthmatics with the Gly variant of the b2-adrenergic receptor (Arg16Gly) may be more resistant to albuterol treatment than those with the Arg isoform Mineralocorticoid receptor variant predisposes to early-onset hypertension; progesterone and clinically used antagonists lacking 21-hydroxy groups such as spironolactone become agonists VKORCI variants predisposes to anticoagulant resistance Insulin-resistant diabetes mellitus Glucocorticoid receptor variant predisposes to glucocorticoid resistance being tested, but skilled investigators know that it is advantageous to have thought about it explicitly. Auxiliary hypotheses are set forth as reasonable alternatives in case the main hypothesis proves to be untrue. Second, investigators should keep in mind that for every discovery made with innovative approaches, many important advances are made simply by recognizing that an established method or familiar technique devised for one application can be employed for another. And third, in some cases, it is possible to design a single experiment that decides the fate of a given hypothesis.

An authentic example of pharmacogenetic variation may provide a more ready understanding of important principles. Consider an episode that took place in the 1970s at St. Mary's Medical School in London during a small trial with debri-soquine, a new therapeutic agent for treating high blood pressure. A number of healthy human volunteers were each given a small dose of the drug, and afterward one of the volunteers suddenly collapsed because of a drastic fall in his blood pressure. None of the other volunteers experienced anything out of the ordinary. After several days in the hospital the unusually sensitive man had fully recovered, but the reaction in a healthy person was so startling that more tests were run to find the reason for it. The hyperresponsive individual was found to process the drug in a way that turned a normal dose into a massive overdose because he did not make the normal enzyme required to hydroxylate the drug and eliminate it from his system. Further tests led to the discovery of two medical students at St. Mary's who also were unable to hydroxylate debrisoquine. The occurrence of such an unusual response in three individuals among so few persons tested raised the possibility that there could be many others who would respond similarly; indeed, larger studies revealed at least one person in ten in the British population at large possessed the same trait. Later, family studies demonstrated that persons who are sensitive to debrisoquine carry a double dose of the defective gene responsible for the trait. This dramatic discovery of the CYP2D6 polymorphism is described by Moyra Bremner in the Sunday Times (London), October 2, 1983.

Presumably, hyperresponsive individuals had developed an elevated concentration of the debrisoquine in plasma (i.e., at receptors) despite receiving only a therapeutic dose of the drug, but had reacted as though they had received an overdose. The investigators reasoned that a defect in the pharmacokinetics (absorption, distribution, or elimination) of debrisoquine would account for the excessive response; also, it was reasonable to propose auxiliary hypotheses that a defect in the receptor for debrisoquine, or a defect in the regulation (signal transduction) of the receptor, could be responsible if the main hypothesis proved untrue. Being experienced investigators, they knew that safe, noninvasive methods for characterizing the pharmacokinetics of debrisoquine in plasma and urine were available that could be used to obtain the information they sought and that values for most of the pharmacokinetic parameters that were necessary for their analysis could be determined by simple arithmetic or computer techniques.

The investigators had previously shown that debrisoquine was mainly converted to a single, biologically inactive metabolite, and they set out to disprove the main hypothesis by examining the urinary metabolite pattern. They found that debrisoquine hyperresponders excreted a much greater quantity of unmetabolized (active) debrisoquine as well as a much smaller quantity of the inactive metabolite than normal responders. From this observation, they could conclude that their main hypothesis was substantially correct, namely that one of the phar-macokinetic mechanisms for disposing of debrisoquine was defective in the hy-perresponder. The next step, to get a more fundamental explanation of this trait, was carried out by other investigators more than 10 years later when techniques for identifying, cloning, and sequencing genes were available.13 The defect was demonstrated to be a variant form of the enzyme that failed to convert debriso-quine to the main biologically inactive product (see CYP2D6, also known as the debrisoquine/sparteine oxidation polymorphism) confirming the main hypothesis.{

Suppose, on the other hand, that a normal pattern of urinary metabolites for debrisoquine had been found in the hypersensitive responder instead of the abnormal pattern. This finding would strongly suggest that a pharmacokinetic defect was unlikely, and that the main hypothesis proposed was false. The alternative possibility speaks to a functional change in the receptor that might be responsible, as proposed in the auxiliary hypothesis. How might the investigators test for such a change in the debrisoquine receptor? Two possibilities might be proposed: there might be an altered receptor with an increased affinity for debrisoquine, or an overabundance of the unaltered debrisoquine receptor. To test the validity of these hypotheses, investigations would assume a direction similar to that taken to investigate the mechanism of warfarin resistance described in connection with graded dose-response relationships. If the affinity of the receptor was increased, the concentration of debrisoquine needed to attain a given response would be decreased; if a more complete graded dose-response curve were obtained, theoretically the curve would be shifted to the left as shown in Figure 3.1B (curve 2A). In that case, an ordinary dose would produce a hyperresponse. If the abundance of unaltered receptors was increased, a therapeutic dose would result in a hy-perresponse, as shown in Figure 3.1A (curve 1B). Determination of graded dose-response curves for abnormal and normal responders to debrisoquine would thus help the investigators decide whether a change in the abundance or in the affinity of the debrisoquine receptor had occurred.

If an increased affinity or an overabundance of debrisoquine receptors were found in abnormal responders compared to normal responders, additional studies would be undertaken to obtain a more detailed view of the receptor. They would probably include the isolation, cloning, sequencing, and expression of the genes

{Many years may elapse between the observation of a phenomenon and its explanation. Mendel singled out the seven contrasting characters for his study of inheritance in pea plants—one of them being seed shape, either round (the dominant form) or wrinkled. Plant scientists in England showed with recombinant DNA techniques that the wrinkling of peas is due to a deficiency of starch branching enzyme I, which results in a high sucrose content and, consequently, a high osmotic pressure. This leads to the accumulation of water, but when the seed dries out, the skin collapses into a mass of wrinkles. Peas homozygous for the defective "wrinkled" gene completely lack the starch branching enzyme, whereas the normal enzyme is active in peas with the "round" gene.14

for the receptor from both responder phenotypes. The goals of these studies would include a search for structural and regulatory differences of receptor synthesis, stability, and targeting (to ascertain the mechanism for overabundance), and a search for overexpression of the receptor gene and for structural change in the receptor molecules (to determine the mechanism for increased affinity). Quite possibly, the abundance of receptors, or the receptor affinity, for debrisoquine for hyperresponsive subjects might not be found to differ from those of normal responders. Although these findings would disprove the auxiliary hypothesis, they would not preclude the possibility of other, associated alterations that could overamplify the signal from debrisoquine and generate intracellular changes resulting in the dramatic fall in blood pressure in abnormal responders. These include (1) an altered signal transduction system such that the coupling between the drug-receptor complex and stimulation of an effector protein (e.g., adenylyl cyclase) is enhanced; (2) an altered effector protein that overamplifies the signal generated by the coupling (e.g., overactive adenylyl cyclase or underactive phosphodiesterase); and (3) an altered protein kinase or substrate for the kinase that mediates the drug effects, and so on.

The precise nature of the receptor-related defect responsible for the hyperre-sponse to therapeutic concentrations of debrisoquine could then be sought by correlating the molecular characteristics with the functional properties of the expressed receptors within each phenotype, and then by comparing the receptors, or the signal transduction systems, for normal responders and the abnormal responders.

Diabetes 2

Diabetes 2

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