Caco-2 cells of lower passage numbers (*22) have been shown to have a shorter doubling time than those of higher passage number (*72) purchase periactin 4 mg line, resulting in an increased number of cells per monolayer and thus an increased amount of membrane protein order periactin overnight. However quality periactin 4mg, several reports have stated that Caco-2 cells at higher passage numbers (>90) contain significantly more P-gp than those at lower passage numbers cheap periactin 4mg overnight delivery. P-gp expression in the Caco-2 cells has been shown to be stable, and this allows relatively accurate comparison of data from various monolayers as long as they represent a relatively narrow range of passage numbers. Expression of specific proteins can be induced in Caco-2 cells using simple culturing techniques. Overexpression of P-gp can also be achieved in the Caco-2 cell line by culturing with vinblastine, verapamil, and celiprolol (358,359). No morphological differences were noticed for vinblastine cultured cells with respect to appearance, formation of tight monolayers, and the corresponding transepithelial resistance (359). Both have been used to follow the passive diffusion of compounds across monolayers. The model’s considerable advantages have led to it being increas- ingly used as the model of choice to screen for P-gp efflux liability. These cultured cells have been shown to retain many morphological and biochemical properties of their in vivo counterparts, including distinguishable luminal and abluminal membrane domains that are functionally and biochemically distinct (371–381). The comparable leakiness of the system can also make it difficult to quantify differences in transport that may be mediated by transporter activity. Several examples have demonstrated the usefulness of this system to study polarized efflux via P-gp. For example, the influence of P-gp expressed in brain capillary endothelial cells on the transport of cyclosporin A (388,389), vincris- tine (381), protease inhibitors (amprenavir, saquinavir, and indinavir) (245,390), rhodamine 123 (211,383), opioid peptides (211,391,392), and the b-blocking agent bunitrolol (393) have all been determined using this system. Experimental Methods Used with Tissue Culture Transport Models to Study P-gp Efflux The use of appropriate experimental design can provide definitive evidence that P-gp-mediated efflux is altering the transport of a compound and can provide further mechanistic information regarding the transport of a compound. Recently it has been appreciated that P-gp efflux can be a potential source for drug interactions and in vitro experimentation can be very helpful to understand potential liability. The techniques described in this section can be used with any tissue culture transport model. Transport across cell monolayers can be easily determined using a bicameral system, such as the 1 Transwell system, in which the compartments are separated by the polarized cell monolayer (attached to a porous filter support). One of the most significant advantages of this experimental system is that the appearance rather than the disappearance of the compound can be easily quantified to yield a permeability value. Comparison of the per- meability values provides a true measure of how P-gp affects the transport of the substrate across polarized epithelium and correctly identifies if the transport is subject to P-gp-mediated efflux activity (vs. This experimental format allows an assessment of how significantly P-gp efflux attenuates or enhances absorptive versus secretory transport, respec- tively (394). Another well-established metric used to identify P-gp substrates is the efflux ratio, in which secretory permeability is compared with absorptive permeability. An efflux ratio greater than one can imply apically directed transport polarity, suggesting that the compound is a substrate for efflux transport (395). It is important to note that apically directed transport as determined by efflux ratio does not provide unambiguous evidence that P-gp is responsible for the efflux of the compound (transporters other than or in combination with P-gp may be respon- sible for transport polarity). Although the efflux ratio can, under the proper experimental construct, be useful to identify The Role of P-Glycoprotein in Drug Disposition 397 P-gp substrates, it does not quantify the functional activity of P-gp and furthermore cannot be used to understand how P-gp affects absorptive or secretory transport (206,394,396). For example, although digoxin and rhodamine 123 have similar efflux ratios, P-gp affects these compounds in much different ways; P-gp efflux affects digoxin absorption, but not rhodamine 123 absorption, and affects rhod- amine 123 secretion greatly but digoxin secretion modestly (396). There is one major caveat of using the tissue culture transport experiment to study P-gp efflux that cannot be overlooked—P-gp efflux is not directly determined in this experiment. Rather, the effects of P-gp-mediated efflux activity and changes to this activity are inferred from the resulting overall transport data. Particularly with regards to substrate identification, there is the potential for false negatives. For a compound to be affected by P-gp-mediated efflux, it must reach P-gp’s binding site that is within the cell. Compounds with poor membrane (transcellular) per- meability are not likely to be identified as substrates (395,397). Conversely, compounds with very high passive membrane permeability can saturate P-gp efflux at low micromolar concentrations and are often not identified as substrates (206,395,397). The tissue culture transport study is a powerful tool, but the reasons listed above make it an absolute necessity to incorporate proper controls while performing and making conclusions from these studies. Increasingly, efforts are being made to quantify the inhibitory potency of new molecular entities against P-gp-mediated efflux using interaction studies performed in vitro. In particular, several efforts have specifically focused on determination of inhibitory potency against P-gp efflux of digoxin, a substrate with a narrow therapeutic window with kinetics known to be determined in part by P-gp (398,399). The transport study using a probe substrate such as digoxin, verapamil, or taxol can be con- ducted in the presence of a test compound over a series of concentrations to determine the inhibitory potency of the test compound (199,201,359,398,399). A comparison of this inhibitory potency to expected systemic concentrations can provide some insight into potential interactions that may be seen following coadministration of the compounds of interest. These compounds can be used in any competition assay in which the test compound is added with these dyes. Any reduction in the dye efflux would be indicative of the inhibitory properties of the test compounds toward P-gp. The amount of P-gp inhibition can be directly correlated with the amount of intracellular fluorescence. It is important to note that P-gp inhibition by a compound for the efflux of any of these ligands does not directly correlate with the ability of P-gp to efflux the compound of interest (177). Such is the case with paclitaxel, which is con- sidered to be an excellent P-gp substrate but a poor inhibitor as determined by the dye-efflux method. The converse is seen with progesterone, which is a good inhibitor of P-gp-mediated efflux and yet is a poor substrate. Furthermore, the false negatives due to poor permeability noted for transport assays can also produce false negatives in these interaction assays. Membrane vesicles are typically formed from intact cells and require some skill for their preparation. Given this relative limitation, the use of membrane vesicles as a rapid screen for P-gp efflux activity has not been extensive and has proven a better tool for studying the microscopic aspects of P-gp-mediated efflux. Additionally, these vesicles have been used to study microscopic aspects of P-gp-mediated efflux, such as the relationship of P-gp function to the membrane fluidity (137). In the intestinal segment study, the intestine is removed and either mounted in a diffusion apparatus (Ussing chamber) or everted to make an everted sac (234,414–416). The transport characteristics of verapamil were determined for each region of the intestine as well as the colon with this model system. The duodenum and jejunum showed the most P-gp activity followed by lower activity in the colon and, surprisingly, none in the ileum (416). Polarized transport of quinidine due to P-gp efflux was demonstrated by using intestinal segments mounted in Ussing chambers (414). Further studies using everted sacs showed that P-gp inhibition by quinidine caused an altered drug absorption of digoxin and explained the interaction seen with coadministration of these agents (234). Metabolism and P-gp-mediated efflux of the macrolide antibiotic tacrolimus were studied in perfusion studies and in everted sacs (415). It was shown that inhibiting P-gp with miconazole (a P-gp inhibitor) greatly increased the amount of tacrolimus in the tissue (415). The results of these experiments provided evidence that P-gp is active in limiting tissue exposure to drugs and also that the intestinal metabolism of certain compounds can be significant. P-gp, expressed in these in vitro systems, is thought to function normally (analogous to function seen in in vivo systems) even though the former lacks glycosylation at N-terminal. Despite the normal functional activity of P-gp, researchers found it difficult to use P-gp expressed in E. To solve this problem, Beja and Bibi developed a method to express P-gp in ‘‘leaky’’ E. The results of these assays may be significantly different than those obtained in studies performed with 400 Troutman et al. A membrane product pre- pared from baculovirus infected insect cells containing this activity is now commercially available from Gentest Corp. By determination of inorganic phosphate liberated in the reaction containing a P-gp preparation and a test compound, in the presence and absence of vanadate, one can determine if the test compound is a substrate/inhibitor of P-gp (123,422). In Situ and In Vivo Models Whereas in vitro models are the tool of choice to identify P-gp substrates and to specifically study molecular aspects of P-gp-mediated efflux activity, extrapolation of these data to predict relevance in vivo can sometimes be difficult. Indeed, P-gp-mediated efflux activity is often one of a multitude of parameters that ultimately combine to confer substrate disposition; these exact relationships between key parameters are complex and remain to be resolved.

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By the time she had it filled cheap periactin express, the next day buy periactin 4mg with visa, her lesion had stopped enlarging cheap periactin 4 mg overnight delivery, and she could reduce her supplements buy periactin 4mg mastercard. Her ratio of segmental to lymphocyte white blood cells was low, evidence for a chronic viral condition. She stopped using tooth- paste (strontium), salt, deodorant, detergents (aluminum). She got the metal out of her mouth and eliminated her radon problem by opening crawl space vents. Fatigue Fatigue, whether minor or extreme, is always associated with blood sugar disturbances. We have three organs that do most of the sugar regu- lating: our adrenals, the liver, and the islets in the pancreas. In severe fatigue, that keeps you partly bedridden, all three organs are heavily parasitized. Killing the viruses is not as important as killing the larger parasites and getting your organs functioning for you again. The adrenals (the outer layer called the cortex) help to regulate the blood sugar in a complex way. The heart of sugar regulation is in your pancreas in the tiny islands of cells that secrete insulin, called the islets of Langer- hans. There is wood alcohol in store-bought drinking water, fruit juice, powders meant to be stirred into bev- erages, even if they are health food varieties. The only beverage you can safely buy (not safe unless you sterilize it, though) at a grocery store is milk. Your first step toward curing your fatigue syndrome is to kill the pancreatic fluke and all other living invaders of the pancreas, liver, adrenals and thyroid. Your energy can bounce back in a few weeks by attending your liver, adrenals and pancreas. Take these supplements for three weeks, then cut the dose in half, and take on alternate days only, as a hedge against possible pollution in these. Although your energy may be normal in three weeks, you are at higher risk for fatigue than the average person. Reinfection with anything will put the new parasites right back where the old ones were. Other bacteria, solvents and toxins will head for the pancreas, liver and adrenals again because these are weakened organs. It could take two years to build your health to its previous level, but is well worth it to have youth, initiative, and a beautiful appearance again. Going back to school is a good use of your time when your initiative has returned but your physical strength is still not up to housework or a job. When your energy comes back to you, it is tempting to overwork: to clean the whole house or to get into some gardening. Our test showed her body was full of bismuth (fragrance) and silver (tooth fillings) especially in the ovaries. She cleansed her kidneys and killed parasites but could not make up her mind to do the expensive dental work. Her skin, kidneys, breasts, brain, ovaries and pancreas were all loaded with mercury, platinum and other metals. Before the moving date arrived she had cleansed kidneys, killed parasites and done dental work and was feeling noticeably better. She immediately was very fatigued again and worried that the move had been in vain. This time she had a liver full of Salmonella and a return of phosphate crystals in her kidneys. But it was easy to clear up and it was a very useful lesson to her to avoid unsterilized dairy products. Her tissues were full of arsenic from pesticide; her urinalysis showed kidney crystals and her eosinophil count was high 5. She had sheep liver flukes and stages in her pancreas due to a buildup of wood alcohol there. In four months after killing parasites and doing a kidney cleanse she was much improved. She had Ascaris and pancreatic flukes in her pancreas and reacted to sugar in her diet quite strongly, so avoided it. In 6 weeks she had done everything except the mercury removal and was feeling much better. She cleaned her home and cleansed kidneys, killed parasites, and did two liver cleanses. Meanwhile, though, her infertility problem got solved (she got pregnant) and this encouraged her to continue the battle against fatigue after the baby was born. Hector Garcia, age 14, was getting gamma globulin injections every three weeks for his chronic fatigue syndrome. He had pancreatic flukes in his pancreas, sheep and human liver flukes in his liver and intestinal fluke in his intestine. He had a buildup of benzene, propanol, and carbon tetrachloride as well as aflatoxin from his granola breakfasts. He killed parasites with a frequency generator and went off the solvent polluted items in the propyl alcohol and ben- zene lists. Dana Levi, age 16, had chronic fatigue syndrome and dizziness; he was not in school. He had pancreatic fluke in his pancreas, sheep, human and intestinal flukes in his liver! As soon as the para- sites were killed (with a frequency generator) and he changed a lot of his products, he felt better but soon lost his improvement. At the next visit, our tests showed a buildup of vanadium (from burning candles in his bedroom). But getting a taste of normal energy gave him the determination to get himself well! His lungs and trachea had accumulated seven heavy metals: va- nadium, palladium, cerium, barium, tin, europium, beryllium. The gas leak was fixed (vanadium), the garage was sealed off from the house to eliminate barium and beryllium but the other toxic elements came from his dental retainer. As soon as his retainer came out, and they stopped using flea powder on their dog, his energy became normal and sinuses cleared up. Evelina Rojas, age 12, was having extreme fatigue with mood problems and sudden fevers. She killed Ascaris and sheep liver flukes with the parasite program but promptly got them back due to a benzene buildup I believe due to using products containing an herbal oil. Her high levels of Streptococcus pneumoniae (cause of fevers), Staphylococcus aureus and Nocardia could not be eliminated until her three baby teeth (with root canals) were pulled. She was toxic with arsenic, a substance that replaces en- ergy with nervous excitement and exhaustion. She also had a backlog of antimony (using baby oil), aluminum, rhenium (hair spray), benzalkonium (toothpaste) and radon. In four months, she had the arsenic and three other toxins eliminated and already had more energy. He had the mirac- idia of the intestinal fluke, sheep liver fluke, and pancreatic fluke in his thyroid! He had been drinking a great deal of regular tea, which let oxalate crystals deposit in his kidney and slow down the excretion of toxins. The parasites were killed with a frequency generator, he changed his diet to get rid of solvents. Change all detergents (for dishes, laundry, and body use) to borax and/or washing soda. Whether you have cysts or not, it is always a good idea to use borax and washing soda instead. If you test positive for it, stop all commercial soap and detergent for all possible uses. The fungus is hosted by another parasite but finds your skin quite satisfactory for a home, at least while your skin immunity is low. It may be low from wearing metal jewelry, having metal tooth fillings, aluminum (from lotions and soaps), cobalt (from shaving supplies), and zirconium (from deodorant.

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Other respiratory depressant drugs such as narcotics effective periactin 4 mg, opiates purchase cheap periactin line, and analgesics can also have additive effects (77) periactin 4 mg otc. In one study cheap periactin 4 mg otc, it was found that fen- tanyl and alfentanil pretreatment have also reduced the dose of thiopental required for anesthesia induction (78). Also, the simultaneous administration of morphine and phenothiazines can result in significant hypotension (15). Alprazolam mediated analgesic effects, most probably via a µ opiate mechanism of action (79). For instance, sertaline increases the plasma methadone concentration significantly in depressed patients on methadone (80). Several lines of evidence suggest that benzodiazepines and methadone may have synergistic interactions and that opiate sedation or respiratory depression could be increased. In a study, meperidine and chlorpromazine com- pared to meperidine and placebo resulted in significantly increased lethargy and hypo- tension (81). Opiate abusers use amphetamines to in- crease the effects obtained from poor quality heroin (82). Braida and coworkers demonstrated that cannabinoids produce reward in conditioned place preference tests and interconnection of opioid and cannabinoid systems (83). Muscle Relaxants Patients recovering from relaxant anaesthesia are especially vulnerable to the respiratory depressant effects of morphine. Respiratory acidosis, secondary to acute hypercapnia, can result in reactivation of the long-acting relaxant on the completion of anesthesia, resulting in further depression of respiration. The combination of muscle relaxant and morphine could result in a rapidly progressing respiratory crisis (15). Adrenergic Drugs Agmatin (an endogenous polyamine metabolite formed by the carboxylation of L-arginine) potentiates antinociception of morphine via an alpha2 adrenergic receptor- mediated mechanism. This combination may be an effective therapeutic strategy for the medical treatment of pain (92). Yohimbine (an alpha2 antagonist) tends to limit opiate antinociception and the additive potential of µ and delta opioid agonists (93). Clonidine (4 and 10 µg/kg) in cats had a differential degree of inhibition in the order of analgesia, much greater than hypotension, greater than bradycardia. Fur- thermore, pain suppression of clonidine appeared to be independent of vasodepression and cardio inhibition (94). When administered with pentazocine, clonidine caused a statistically signif- icant increase in pentazocine analgesia (95). Clonidine induced dose- and time-depen- dent suprasensitivity to norepinephrine, similar to that produced by morphine. Thus, clonidine and morphine possess comparable properties on the antagonism of chronic morphine tolerance; and this maybe the therapeutic basis for clonidine’s clinical appli- cation in the treatment of opiate addicts (96). Heroin and Alcohol There have been numerous reports of the enhancement of acute toxicity and fatal outcome of overdose of heroin by ethanol. There is evidence that systemic disease may be more preva- lent in users at greatest risk of overdose. It is suggested that pulmonary and hepatic dys- function resulting from such disease may increase susceptibility to both fatal and nonfatal overdose (97). In one study, at all ranges of free-morphine concentrations, there was a greater percentage of heroin deaths when ethanol was present (98). Toxicological evi- dence of infrequent heroin use was more common in decedents with blood ethanol con- centration greater than 1 µg/mL than in those with lower concentrations (99). Opioids and Opiates 141 Table 4 Drug Interactions of Some Opioids Object Precipitant Drug(s) Drug(s) Interaction Ref. Genotoxic Damage and Immunosuppression Opiate addicts have higher chromosome damage and sister chromatid exchange frequencies. These interactions of opiates with T lym- phocytes may regulate metabolism and could thereby be responsible for the sensitivity of cells from opiate addicts to both genotoxic damage and immunological effects (100). Before attributing death due to narcotism purely on the basis of circumstantial evidence, it is essential to exclude other natural or unnatural causes of death such as 142 Moallem, Balali-Mood, and Balali-Mood spontaneous intracranial hemorrhage, occult subdural hemorrhage, or evidence of non- narcotic drugs. Postmortem Appearances The appearances could be divided into external and internal: 1. The postmortem lividity is intense, almost black, and is better seen in a fair-skinned body. There is froth at the mouth and nose, but neither so fine nor as copious as in drowning. Internal: the stomach may show the presence of small, soft, brownish lumps of opium and the smell of drug may be perceived. The internal organs, especially the trachea, bronchi, lungs, and brain, exhibit a marked degree of venous congestion. In addition, the trachea and bronchi are covered with froth and the lungs are edematous. Associated with edema of the lungs, the intense lividity of the face almost approaching to blackness should make one suspicious of opium poisoning as the cause of death. At autopsy of an individual who has died of an overdose of heroin, the lungs are heavy and show congestion, though the classic pulmonary edema mentioned in some of the other textbooks is not always present. Microscopic examination of the lungs commonly reveals foreign-body granulomas with talc crystals and cotton fibers. Sam- ples of the venous blood, urine, stomach and contents, liver, and in some circum- stances, additional samples such as bile, cerebrospinal fluid and vitreous humor, kidney, and brain, may be taken. When the drug has been injected, an ellipse of skin around the injection mark extending down through the subcutaneous tissue to the muscle should be excised, along with control area of skin from another noninjected site (102). Toxicological Analyses Various analytical methods for the estimation of morphine and its derivatives have been reported. The most reliable methods are gas chromatography–mass spec- trometry and radioimmunoassay. Blood and urine as well as the other samples such as gastric contents and the organ tissue extracts may be analyzed. In order to identify a certain opiate or opioid, a highly specific method should be used to determine the parent drug as well as the metabolites. For instance, if both morphine and monoace- tylmorphine are detected in the blood, then, the individual took heroin. Plasma concentrations of some opiates such as methadone correlated well with the intake doses. Plasma methadone concentration appears to increase by 263 ng/mL for every mg of methadone consumed per kilogram of body weight (103). Interpretation of the Results Interpretation of the results of toxicological analyses is very important in both clinical and forensic toxicology. History of drug use and abuse, overdose, and clinical and postmortem findings should be considered for the evaluation and interpretation of the results. Opioids and Opiates 143 As with all deaths from toxic substances, the interpretation of analytical results may present considerable difficulties. There might be a long delay between the intake of a drug and death, during which time the blood, urine, and even tissue levels may decline, or even disappear. Many drugs break down rapidly in the body and their metab- olites may be the only recognizable products of their administration. In some cases, data on lethal blood levels may be imperfectly known and great variations in personal susceptibility may make the range of concentrations found in a series of deaths so wide as to be rather unhelpful. If a person dies rapidly after the first episode of taking a normal dose of a drug, because of some ill-understood personal idiosyncrasy, the quantitative analysis may not assist. Where habituation and tolerance has developed, drug users may have concentra- tions in their body fluids and tissues far higher than lethal levels published for non- dependence. In general, the great usefulness of toxicological analysis is both qualitative and quantitative. The qualitative tests will show what drugs have been taken in the recent past; the length of time that drugs or their metabolites persist in different fluids and tissues varies widely. The quantitative analysis can be useful, especially when the results reveal high levels—into the toxic or lethal ranges. These ranges are usually obtained anecdotally from surveys of large number of deaths but, as stated, can differ in terms of minimum and maximum values from different laboratories. Interaction of other drugs and alco- hol or both, delayed death, and abnormal sensitivity are other problems that should be considered.

Other than equipment requirements buy periactin 4 mg with mastercard, the caffeine breath test is 13 simple to perform and for [ C]-(N-3-methyl) caffeine buy periactin 4 mg on-line, a commercial kit is available for this purpose discount periactin 4mg with visa. Typically buy generic periactin 4mg, exhaled breath is collected at several intervals up to one to eight hours following an oral dose of labeled caffeine. The caffeine breath test appears to be reproducible, although extensive testing of this characteristic has not been reported. Early studies demonstrated the feasibility of this approach and its potential application to evaluating hepatic function (65,66). No extensive validation was attempted, so it is difficult to determine how well this test reflects the enzyme’s intrinsic clearance, rather than perhaps some other determinant, such as liver blood flow. However, the situation appears to be moot since phenacetin is no longer an approved drug worldwide because of its renal side effects following chronic dosing; accordingly, further studies of this approach are unlikely. These metabolites account for about 20%, 40%, and 15%, respec- tively, of the urinary recovery of caffeine-derived products. In addition, approxi- mately 10% of 17X is excreted unchanged and another 20% is hydroxylated to form 1,7-dimethylurate (17U). Theobromine (37X) is in part excreted unchanged (10%), and about 20% is metabolized to 3-methylurate (37U) and approximately 50% to 7-methylxanthine (7X). About 10 to 15% of theophylline (13X) is excreted into urine, with about 50% of this primary metabolite being metabolized to 1,3-demethylurate (13U) and some 23% to 1U. Finally, a small amount of caffeine is excreted unchanged in urine, and some additional minor metabolites are formed (45,51). Thus, the metabolism of caffeine results in a complex urinary recovery profile involving multiple primary and secondary metabolites as well as unchanged drug. A major difficulty in the application of these phenotypic trait measures is that they are essentially all empirical, and until recently their limitations were not understood or, more importantly, appreciated. A rigorous sensitivity analysis based on a phar- macokinetic model of caffeine’s metabolism and urinary excretion profile identi- fied a number of confounding variables that contributed to this situation (51). Experimental investigations have subsequently confirmed these theoretical findings. For example, significant correlations were obtained between Ratio 4 and caffeine’s oral clearance (r ¼ 0. As a result, con- clusions drawn from the interpretation of such flawed data may be inaccurate. Moreover, numerous studies based on the determination of caffeine’s plasma clearance in large numbers of subjects have not provided any evidence of discrete subgroups with either low or high values within a log- normal distribution. Modeling analysis also indicates the likelihood that the polymodal distribution could be an artifact (51); this is supported by the observations that despite the fact that the frequency distributions of Ratio 4 and caffeine clearance were unimodal, the distribution for Ratio 2 in the same subjects was bimodal (70). The metabolism of theophylline (1,3-dimethylxanthine) is similar to that of caffeine but less complex (vide supra). However, potential analytical sen- sitivity problems and, more importantly, safety considerations do not suggest that theophylline has any advantage over caffeine for this purpose (86). The gold standard approach depends on determination of the drug’s oral clearance following a single phenotyping dose under dietary caffeine-free conditions. Alternatively, a caffeine breath test can similarly provide such within-subject information. Activity is localized mainly in the liver; however, extrahepatic distribution is also present, especially in the nasal epi- thelium and lung. The 7-hydroxylation of coumarin (1,2-benzopyrone) is a major urinary metabolic pathway that accounts for about 60% of an orally administered dose (102). The phenotypic trait measure is simply the percentage of a 5-mg dose of coumarin excreted in urine as 7-hydroxycoumarin over the zero- to two-hours period following oral administration in the fasted state (102). Because the 7-hydroxy metabolite is excreted mainly as a conjugate, urine is pretreated with b-glucuronidase prior to analysis, and a methodology based on chromatographic separation would appear to be preferable to one using solvent extraction (103). Application of this phenotyping procedure to various population groups has shown that the trait measure exhibits considerable interindividual variability, and it is unimodally distributed in a normal fashion (102–104). Accordingly, it would be expected that in the general population all three phenotypes (extensive, intermediate, and poor) would be present. First, is the fact that the trait value is entirely empirical and has been validated and characterized to only a very limited extent. As expected, severe but not mild liver disease reduces the urinary recovery of 7-hydroxycoumarin, but, not unexpect- edly, renal dysfunction has also been found to affect the trait value (109). This is because of the extreme analytical difficulties associated with measuring plasma coumarin levels because of its relatively high volatility, and this problem is further compounded by the low dose used for phenotyping (5 mg). Coumarin is also excreted in the urine as a result of dietary and environmental exposure through fragrances and other sources. Such daily exposure may be as high as 25 mg (110), which probably accounts for the finding that in certain subjects the urinary molar recovery of 7-hydroxycoumarin exceeds the molar dose of cou- marin administered to determine the trait value (103,110). An additional con- sideration, especially in North America, is the absence of an available approved formulation containing coumarin, which was removed from the market 45 years ago because of its hepatotoxicity and carcinogenic properties in animals (111). More recently, limited use of coumarin in certain types of cancer has been investigated (112), but the strength of the available tablet is 100 mg, i. The frequency distribution of this trait measured in 103 subjects identified two individuals with values markedly greater than the remainder of the population. Moreover, within the major subgroup, there was evidence of overlapping bimodality. The major human urinary metabolites of nicotine are cotinine, nicotine N -0 oxide, and trans-3 -hydroxycotinine0 (113). One reported approach is based on the 30-minute intravenous infusion of a 50:50 mixture (2 mgbase/kg)of3,3 -dideuterium-labeled0 0 nicotine and 2,4,5,6-tetradeutero cotinine followed by serial blood sampling over the following 96 hours and a 0- to 8-hour urine collection (116,117). Using gas chromatography–mass spectrometric–based assays, the levels of nicotine and cotinine derived from each stable-labeled form are measured. Appropriate pharmacokinetic anal- ysis then allows estimation of nicotine’s formation clearance to cotinine and also the latter’s clearance. To date, this methodology has been applied primarily to investigating nicotine’s metabolism within the context of cigarette smoking and addiction (116,117). Despite the need for stable-labeled drugs and the associated sophisticated instrumentation for their measurement, such an approach would provide a gold-standard against which alternative trait measures such as the coumarin index or others could be evaluated and validated. Possibly, a simpler, single-point plasma- or urine-based measure could be developed using nicotine/cotinine. Accordingly, these two isoforms have received the most attention with regard to the development and application of in vivo probes. Such drugs for which the isoform catalyzes the formation of a principal metabolite include phenytoin, tolbutamide, fluoxetine, losartan, S-warfarin, torsemide, valproic acid, and many nonsteroidal anti-inflammatory agents (diclofenac, ibuprofen, naproxen, piroxicam, suprofen, and tenoxicam). Furthermore, this difference has also been noted to be present in patients receiving warfarin therapy, where a gene-dose effect leads to reduced clearance of the anticoagulant’s S-enantiomer (120–122). However, for safety and analytical reasons, it is unlikely that the anticoagulant could be used as an in vivo probe in healthy subjects. Tolbutamide The metabolism of tolbutamide (l-butyl-3-p-tolysulfonylurea) in humans in- volves a single pathway, with the initial and rate-limiting step being tolyl methyl-hydroxylation to form hydroxytolbutamide, which is further oxidized to carboxy-tolbutamide by alcohol and aldehyde dehydrogenases. Since the drug’s half-life ranges between 4 and 12 hours, this approach requires not only multiple blood samples but collection over a considerable time period (24–36 hr). However, such use is not without problems, in par- ticular, the safety issue associated with the hypoglycemic response produced by tolbutamide administration. However, in fasted individuals, blood glucose levels may be sig- nificantly reduced by tolbutamide and require reversal using glucose supple- mentation (130); use of a lower dose (250 mg) may obviate this problem. Unfortunately, validation of neither of these putative trait measures using diclofenac has been reported. Such preliminary information will obviously require appropriate substantiation before the descri- bed trait measures will be widely accepted. Although, its substrate specificity was originally thought to be limited to related anticonvulsant agents, the in vivo metabolism of an increasing number of structurally unrelated drugs appear to be mediated by this isoform. These include R-mephobarbital (4 -hydroxylation)0 (135), hexobarbital (3 -hydroxylation) (136),proguanil (ring cyclization)(137),omeprazole, and related0 proton pump inhibitors (5 -methylhydroxylation)0 (138–140), diazepam (N-demethylation) (141), certain tricyclic antidepressants (N-demethylation) (142–144), carisoprodil (N-dealkylation) (145), citalopram (N-demethylation) (146), moclobemide (C-hydroxylation) (147), propranolol (side-chain oxidation) (148), and nelfinavir (methylhydroxylation) (149). A similar low prevalence rate is also present in Africans and African Americans (161–163). By contrast, a much higher frequency (13–23%) is found in indigenous populations living in Southeast Asia, such as Chinese, Japanese, and Koreans (138,164–168). These factors have led to the development of in vivo probes to classify individuals according to phenotype. Subsequently, two alternative phenotyping procedures were developed that have been widely used throughout the world by numerous investigators.

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