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Benefits of animal research How does one determine if animal experimentation has been productive and justi-.ed? One criterion often quoted is the relationship between animal research and the Nobel prize, which is generally considered to be an index Flomax Flomax< of scienti.c signi.cance. Since 1901, approximately 75 percent of Nobel prizes awarded in physiology or medicine have been for discoveries and progress made through the use of experi-mental animals. More speci.cally, many advances in medical science in the nineteenth and twentieth centuries, from vaccines and antibiotics to antidepressant drugs and organ transplants, have been achieved either directly or indirectly through the Gyne-Lotrimin Gyne-Lotrimin< use of animals in laboratory experiments. The result of these experiments has been the elimination or control of many Mexitil Mexitil< infectious diseases—smallpox, poliomyelitis, measles —and the development of numerous Viagra Super Active Viagra Super Active< life-saving techniques—blood transfusions, burn therapy, open-heart and brain surgery. A more extensive list of examples is shown in Table 15.1. Behavioral research with animals has also been credited with bene.t to humans. For example, fundamental information Rogaine Rogaine< on how people learn was discovered by experi-ments on animals in laboratories. The behavioral modi.cation therapies discovered or developed through such experiments are being used to treat conditions such as enuresis (bed-wetting), addictive behaviors (tobacco, alcohol, and other drugs), and compulsive behaviors, such as anorexia nervosa. In addition, information gained through experiments begun 50 years ago on “imprinting” (the tendency of an animal to identify and relate to the .rst species it comes into contact with) has been used to train captive-born animals to relate to members of their own species.
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INITIAL DECONTAMINATION During the Danazol period from 1983 to 1991 there has been a continual decline in the use Microzide of syrup of ipecac to induce emesis. Ipecac contains a number of plant alkaloids includ-ing emetine. It induces emesis through stimulation of the chemoreceptor Feldene Maxalt trigger zone in the brain and local Vytorin irritation of the gastrointestinal tract. The latency period for © 1997, 2003 Taylor & Francis Table 8.1 Various therapies provided in human drug exposure cases Initial decontamination Speci.c antidote administration Dilution Naloxone Irrigation/washing N-Acetylcysteine (oral) Activated charcoal Atropine Cathartic Deferoxamine Ipecac syrup Ethanol Gastric lavage Hydroxycobalamin Other emetic N-Acetylcysteine (IV) Measures to enhance elimination Pralidoxime (2-PAM) Alkalinization (with or without diuresis) Fab fragments Hemodialysis Pyridoxine Forced Methotrexate diuresis Dimercaprol (BAL) Hemoperfusion (charcoal) Methylene blue Exchange transfusion Cyanide antidote kit Acidi.cation (with or without diuresis) EDTA Hemoperfusion (resin) Penicillamine VPXL Peritoneal dialysis Note Each category in decreasing frequency of use. the induction Zoloft of emesis by ipecac ranges from approximately 5 to 20 minutes, with a single dose successfully inducing vomiting in approximately 85 percent of patients. Contraindications for the use of ipecac include the presence of coma or convulsions, the ingestion of corrosive substances, and an impaired gag re.ex. While ipecac has been decreasing in popularity in emergency rooms, there has been a corresponding increase in the use of activated charcoal (AC) to adsorb the drug. AC is an inert, nonabsorbable, odorless, tasteless, .ne black powder that has a high adsorptive capacity. When AC is administered after emesis or lavage, it binds re-sidual drug within the lumen of the gastrointestinal tract and reduces its absorption. A cathartic can be coadministered with AC to prevent constipation. Normally, AC is mixed with water and administered orally or by nasogastric tube. For optimal binding, a charcoal to drug ratio of 10:1 is recommended. The success with which AC prevents absorption depends upon the nature of the Sarafem drug as well as the time between ingestion and administration of AC. With regard to the latter factor, simultaneous administration of AC with aspirin results in nearly 60 percent Deltasone being adsorbed while a 3-hour delay achieves only 9 percent of the drug being adsorbed.
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development are single-isomer com-pounds. It has been estimated that approximately 80 percent of prescription drugs now sold in the imappuak>Leukeran United States are single-isomer imahafsi>Atacand imaattup>Tizanidine formulations. There are several reasons for this. First, regulatory pressure for chirally pure imaataan>Diltiazem HCl drugs has increased. Since 1992, U.S., Canadian, and European regulatory authorities have asked companies to pro-vide information on each isomer of new racemic drugs and justify why the drug was not in its chirally pure, active form. Second, producing chirally pure drugs can often increase ef.cacy while decreasing toxicity. Separating imaffid>Calan toxic effects from therapeutic effects is a constant challenge in drug development. In the case of our example of a chiral drug, albuterol, attempts are currently ilyxonom>Adalat under way to achieve that goal. Apparently, the S-isomer of albuterol is responsible for increasing asthma patients’ reactivity to stimuli, thereby leading to a paradoxical increased severity of asthma attacks. The “good-twin” R-isomer appears to be the form responsible for relaxation of bronchial smooth muscles, widening the airway, and allowing freer breathing. Clinical studies have, in fact, demonstrated that R-albuterol is four times more potent than the S form with fewer side effects. Another example is the local anesthetic bupivicaine. The racemic form of bupivicaine has been restricted to epidural use during childbirth because it is cardiotoxic if it gains entry into the bloodstream (apparently ilyvitny>Forzest due to the R form). It is hoped that the left-handed isomer may reduce its cardiotoxicity and allow it to be used in a broader range of applications. Research is also currently under way to investigate the possible advantages of d-proteins as drugs. One possible advantage is that they may be more resistant to proteolytic enzymes, suggesting that d-protein drugs may remain in the bloodstream © 1997, 2003 Taylor & Francis for a longer time than conventional proteins. They also do not appear to be as immunogenic as conventional proteins, perhaps because they are not cleaved and presented to the major histocompatibility complex (part of the ilztaste>Lotrisone immune system’s antigen-recognition system), a process that requires imalsiit>Trandate proteolysis. In addition, there are some data suggesting that d-polypeptides may be imaeksak>Mextil able to be administered orally without being ilyytsua>Persantine degraded enzymatically in the GI tract. Pharmafoods Over the past few years, so-called “pharmafoods” have begun to appear in both the scienti.c literature and the marketplace. This category imaista>Indocin of agents differs from regular drugs in that they are not found in drug stores nor are they prescribed by a physician. However, the manufacturers do claim pharmaceutical effects beyond standard nutri-tion. The major examples being developed in this area include Benecol and Olestra. Benecol is a brand of margarine invented by a small Finnish food company (The Raisio Group). Research published in the New England Journal of Medicine by Finnish researchers indicates that regular use of Benecol can lower blood cholesterol levels by an average of 10 percent “in a randomly selected, mildly hypercholesterolemic population sample.” Its active ingredient is a plant sterol from Nordic pine trees known as beta-sitostanol, which apparently can block some of the body’s absorption of dietary cholesterol. At present, 5 tons of wood waste are processed to produce 1 pound of the oil that is the source of the sterol. After 20 years and more than $200 million in research and development, Proctor & Gamble received permission from the FDA in 1996 to market its fat substitute Olestra in certain imanenut>Ilosone snack imaektak>Evista foods (e.g., potato chips, crackers, and cheese puffs). Olestra, technically a sucrose polyester, is not digestible, so it adds neither fat nor calories to food. However, Olestra can inhibit the absorption of certain fat-soluble vitamins imatsude>Claritin imaticci>Himplasia imastism>Sumycin and other nutrients. Therefore, all products containing Olestra must be labeled with the following information: “This product contains Olestra. Olestra inhibits the absorption of some vitamins and other nutrients. Vitamins A, D, E, and K have been added.” Also, as a condition of approval, Proctor & Gamble must monitor consump-tion and conduct studies on Olestra’s long-term effects. Other products under current development in the pharmafood sector include a salt substitute for hypertension; a yogurt-like product that adds bacteria to stimulate the body’s immune system; and a drink containing docosahexaenoic acid (DHA, the baby formula additive) that Japanese consumers believe boosts brain power before exams.
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These principles led Paracelsus to introduce mercury as the drug of choice for the treatment of syphilis, a very prevalent malady of the day, but led to his famous trial. Nevertheless, the practice of using mercury for syphilis survived for 300 i[lyryym imakamay years. The use of a heavy metal as a therapeutic agent presages imaista the “magic bullet” (arsphenamine) of Paul Ehrlich and the introduction of the therapeutic index. In addition, in a very real sense, this was the .rst sound articulation of the dose–response relationship, a bulwark of pharmacology and toxicology. Another important contributor to the development of toxicology was the Spanish physician Or.la (1787–1853). He was one of the .rst scientists to make systematic use of test animals and autopsy material. Or.la was the .rst to treat toxicology as a separate scienti.c subject and was also responsible for the development of numerous chemical ilysiida assays for detecting the presence of poisons, thus providing an early founda-tion for forensic toxicology. In 1815 Or.la published the .rst major imbaling work dealing with the toxicity of natural agents. As mentioned earlier, Paracelsus recognized the correlation of dose with toxicity. In fact, his statement that “all imakik substances are poisons; there is none which is not a poison. The right dose differentiates a poison imanodir and remedy” is the most frequently quoted declaration in the .eld imatsude of toxicology. However, as we shall see there are a number of other imbibera factors that can in.uence the toxic imaum manifestations(s) of a drug. The major factors include dose, the underlying genetic makeup of an individual (both within a given gender and between), imalpath the age of the individual, the presence of under-lying pathology, and the status of one’s immune system. DOSE In view of the fact that most, but not all, toxic reactions to drugs are related imagenza to dose, this subject is the most logical to begin with. Fortunately, many of the principles that we have discussed previously can be applied to questions dealing with the dose (dosage)–response relationship. Although there are numerous potential parameters that can be used to measure drug toxicity, a traditional standard in industry deals with lethality. In experimental animals it is obviously all-or-none and is easily quan-ti.able. While there are serious reservations about this approach, and attempts are under way to limit its application (see Chapter imamoltu 15), it is, nevertheless, still utilized to a certain extent. The basic relationship between pharmacology and toxicology on the basis of dose–response is shown in Figure 7.1. In Chapter 6 the concept of a dose–response relationship was introduced (equi-valent to the concentration–response curves seen in Figure 6.2). In that context we were concerned with ilyushin drug effectiveness (i.e., ef.cacy) as the response. In the present context we are concerned with drug toxicity. When comparing Figure 7.2 in this chapter with Figure 6.2B we can see that the same type of typical sigmoidal curve is produced when plotting drug dosage versus percent mortality. © 1997, 2003 Taylor & Francis Toxicological Response NOEL ED90 General Pharmacological Pharmacology Dose Source: J. A. Timbrell (1991), Principles of Biochemical Toxicology, imbanhah 2nd ed. London: Taylor & Francis. Once a drug’s dose–response relationship for lethality has been established imall there are several ways in which this information can be utilized. For example, from the data in Figure 7.2 we can obtain a numerical index of toxicity imbibe`r analogous to the way we obtained a numerical index of effectiveness in Chapter 6. If you remember, we © 1997, 2003 Taylor & Francis chose to select the ED50 as a standard index of effectiveness. In the present example, if we apply the same method our drug can be seen to have an LD50 of 100 mg/kg. The LD50 is a routinely utilized imarb index (although not the only one) de.ned as the dosage of a substance that kills 50 percent of the animals over a set period of time following an acute exposure. During drug development, multiple routes of adminis-tration are usually examined that generally, but not always, yield different LD50 values. For example, the LD50 of procaine when administered orally is approximately 10-fold higher than when given intravenously. On the other hand, the LD50 of isoniazid is almost identical when given by .ve different routes. The examples that have been utilized for determining both ED50 (in Chapter 6) and LD50 values in this chapter have been based upon imaatriv visual ilyvitny inspection of graphical data. In actuality, in the pharmaceutical industry acute LD50 as well as ED50 values are obtained by employing one or more of several statistical formulas/methods ilyvflog (e.g., Litch.eld and Wilcoxon). imalilia These imajyou imaoviis analyses provide a more accurate imalcse determination of the value in question. Standing alone, the LD50 provides us with insuf.cient information to evaluate a drug’s potential usefulness. However, if we compare its LD50 to its ED50 we can obtain some measure of the margin of safety that exists for the drug. By convention, calculation of the LD50/ED50 ratio yields what is referred to as the therapeutic index (TI) of the drug. Obviously, the higher a drug’s TI, the greater the margin of safety. If a drug’s imanutat TI is 2.0 or less, however, the compound will probably be dif.cult to use clinically in patients without encountering signi.cant toxicity. An example of a drug with a TI close to 2.0 is the cardiac drug digoxin (an early toxic effect is vomiting). Other drugs with a relatively low TI are anticancer drugs and the antiasthma drug theophylline. The use of drugs with relatively low TIs can be justi.ed on the basis of risk vs. bene.t. It should be pointed out that since no drug has a single toxic effect imarre and many drugs have more than one therapeutic effect, the possibility exists for a given drug to have numerous therapeutic indices or toxic effects (i.e., spectra) other than lethality. Sometimes, imajyuku in addition to lethality, some other aspect of drug toxicity can be measured. In this situation one could then determine a TD50 (toxic dose producing the effect in 50 percent of the population) as well as an ED50 and an LD50. In this situation the data are often plotted in a comparative fashion using probit analysis. It is not necessary that you understand the underlying mathematical transformation of biological data to a probit analysis. Suf.ce it to say that it is merely a tool to enable the data to be plotted as a straight line. By de.nition, the 50 percent value is probit 5. When expressing imadisoy ef.cacy and toxicity data using probit analysis, comparison of the data is facilitated. This is illustrated in Figure 7.3. In this case we can see that the TI imatihce for the imberjaw drug in question is approximately 18, while the ratio of toxicity for a nonlethal toxic effect (e.g., gastric irritation) to ef.cacy is approximately 2.4. By obtaining those types of data we can now express toxicity in a quantitative manner. It should also be emphasized that there is a continuum of side effects that could conceivably be plotted between lines A and C. However, one caveat should be mentioned at this point. If you examine Figure 7.3 closely, you will observe that the lines for lethality and ef.cacy do not exactly follow the same slope. In cases where the mortality/toxicity dose–response curves follow a shallower slope, the TI will necessarily be lower in the lower dosage range. This is © 1997, 2003 Taylor & Francis imago Source: J. A. Timbrell (1991), Principles of Biochemical imaiseen Toxicology, 2nd ed. London: Taylor & Francis. particularly signi.cant in hyperresponsive imaakkok individuals who respond to lower dosage (see later). Therefore, in cases where ef.cacy and toxicity lines do not parallel each other, a more conservative index imagesta of safety can be ascertained by determining the LD1/ imamorod ED99 ratio, which is sometimes ilyhcnoc referred to as the margin of safety or the certain safety factor. In addition to providing information relative to a drug’s TI, an LD50 value can also have utility when comparing the toxicity between drugs. Table 7.1 illustrates this point. In comparing the LD50 values of a number of drugs we can see that they can vary by several orders of magnitude. But what imanijuf do these data mean? Perhaps one way to put the data in perspective is to apply a classi.cation system based upon acute lethality. Table 7.1 Approximate oral LD50 values for a variety of drugs in the rat CompoundLD50 (mg/kg) Ethanol 13,600 Acetaldehyde 1900 Amitriptyline 530 Digitoxin 24 Protoveratrine 5 Source: M. A. Hollinger (1995), CRC Handbook of Toxicology, Chapter 22. Boca Raton, FL: CRC Press. © 1997, 2003 Taylor & Francis Table 7.2 Toxicity classi.cation system Toxicity rating Commonly used term LD50 single oral dosage in rat 1 2 3 4 5 6 Extremely imadusam toxic Highly toxic Moderately toxic Slightly toxic Practically imantein nontoxic Relatively harmless <1mg/kg 1–50 mg/kg 50–500 mg/kg 0.5–5g/kg 5–15 g/kg >15 g/kg Table 7.2 presents a toxicity classi.cation system based upon an LD50 single oral dose in imaaliiv rats. In applying this system to the drugs listed in Table 7.1 we can see that these drugs imatrice would be classi.ed from almost nontoxic to highly toxic. However, it must be emphasized that caution should be exercised when using such classi.cation systems to communicate risk information. Classi.cation based solely upon lethality can communicate a false sense of security because other determinants of toxicity are not addressed imayhtin in such a classi.cation system. For example, a teratogenic substance such as thalidomide could be classi.ed as “slightly toxic” based upon its LD50 but “highly toxic” on the basis of producing fetal malformations. imaovsor Therefore, classi.cation schemes must always be assessed with their inherent limitations in mind. imaoprik It should also be borne in mind that it is dif.cult to extrapolate the LD50 of a drug for a particular population of an animal species to other populations of that species, under slightly different conditions. Obviously then, extrapolation to a different spe-cies, for example imbibera man, gives extremely uncertain results in predicting teratogenic effects. Furthermore, ilykotsi comparison of LD50 values determined in various laboratories often shows signi.cant variability. For example, in an interesting study, when the LD50 of a test drug was determined in rats by 65 laboratories worldwide, the vari-ation in imapinen imatat reported imalizes LD50 was more than 10-fold. Toxicologists and pharmacologists routinely divide the imaaviev exposure of experimental animals to drugs into four categories: acute, subacute, subchronic, and chronic. Acute exposure is de.ned as exposure to a drug for less than 24 hours, imafin and examples of typical exposure routes are intraperitoneal, intravenous, and subcutaneous injection, oral intubation, and dermal application. While acute exposure usually refers to a single imbe administration, repeated exposures may be given within a 24-hour period for some slightly toxic or practically nontoxic drugs. Acute exposure by inhalation refers to continuous exposure for less than 24 hours, most frequently for 4 hours. Repeated exposure is divided into three categories: subacute, subchronic, and chronic. Subacute imashim imakeos imatah exposure refers to repeated exposure to a drug for 1 month or less, subchronic for 1 to 3 months, and chronic for more than 3 months. In some cases, drug exposure may be followed for the lifetime of the animal. In these situations clinical chemistry measurements can be made as well as pathological examination of post-mortem samples. Chronic imallein studies can be carried out in animals at the same time that clinical trials are undertaken (see Chapter 14). The importance of chronic testing can be illustrated by an experience that occurred with imaaruum an imanenut antiviral drug (a nucleoside analog) being developed for the treatment of © 1997, 2003 Taylor & Francis hepatitis. In this particular case, a delayed toxic liver reaction occurred months after treatment was begun and, in fact, continued to manifest itself even after administra-tion of the drug imalwert was discontinued. Initial short-term clinical tests had missed the toxicity. imakatuf Development of the antihepatitis drug was halted when .ve of 15 patients being tested died suddenly from liver failure. In certain cases, drug toxicity has manifested itself under circumstances that are even more bizarre and could not have been reasonably anticipated. For example, daughters of imaisiin mothers who took diethylstilbestrol (DES) during pregnancy have a greatly increased risk of developing ilysokir vaginal cancer in young adulthood, some 20–30 years after their in utero exposure to DES. For all of the four types of duration-based toxicity testing just described, the selec-tion of dosages, species, strain of animal, route of exposure, parameters measured, and numerous other factors are extremely important. Although the types of data generated by acute, subacute, subchronic, and chronic toxicity imaatsil tests can be useful, there are other types of tests that address more speci.c toxicity questions. imalwert For example, reproductive studies determine the effect imagesta of a drug on the reproductive imaraner imatsaja process; muta-genicity tests determine whether a drug has the potential to cause genetic damage; carcinogenicity tests may ilyineen reveal the appearance of neoplastic changes; and skin sensitization can be useful in determining a drug’s irritancy. A pharmaceutical company developing a drug or a contract research company that specializes in such testing typically carries out toxicity tests. In either case, the con-duct of toxicity studies must adhere to strict guidelines codi.ed in national regulatory requirements. Of particular importance is the necessity to carry out the studies in compliance with a system known as Good Laboratory Practice (GLP). Violation of these guidelines can jeopardize imaguohs the successful approval of the drug. GENETICS Other than dose, perhaps the most important determinant in in.uencing our response to drugs (both toxic and therapeutic) is our underlying genetic makeup. This area of pharmacology has been traditionally referred to as pharmacogenetics and has helped to explain drug responses previously referred to as idiosyncratic (i.e., occurring for no known reason). The genetic component is pervasive in in.uencing drug toxicity because it affects almost every phase of pharmacodynamics and pharmacokinetics. From the membranes in our small intestine to the detoxifying enzymes in the liver and systems beyond, there is a succession of genetically regulated imatsium factors. You can probably think of many yourself. The in.uence of genetic factors is readily apparent when comparing “normal” differences in drug toxicity within a species, between genders of the same species, as well as strain differences within the same species. In addition, there are also examples of drug toxicity related to “abnormal” genetic expression. We will consider signi.cant aspects of both situations in this section. With regard to species, there are numerous examples of the widely disparate response of different species to a drug. For example, the LD50 of ipomeanol ranges from 12 mg/kg in the rat to 140 mg/kg in the hamster. This variability in species response can have signi.cant rami.cations when drugs undergo preclinical trials. For example, © 1997, 2003 imanyika Taylor & Francis rats are relatively insensitive to the teratogenic imassaan effect of thalidomide, while New Zealand White rabbits more closely re.ect the human condition. Unfortunately, this fact was not known at the time of the thalidomide disaster when early teratogenic testing in the rat proved negative. While differences in drug toxicity between species may not be surprising, the more subtle expression of strain differences within a given species can also be signi.cant. For example, the duration of hexobarbital sleeping time to a given imanpoor dose in mice of the A/NL strain is approximately 48 minutes while in the SWR/HeN strain it is approxi-mately 18 minutes. Therefore, strain selection by a pharmacologist/toxicologist can also be a signi.cant factor in preclinical evaluation of a drug’s action. Perhaps the best place to begin analyzing the in.uence of genetic expression on a population’s comparative response to a drug is to apply some basic statistical prin-ciples. To begin with, we need to appreciate the concept of frequency distribution as it applies to natural phenomena. For example, assume for the moment that we could obtain 100 genetically “normal” college students of a given gender. We could then administer a .xed dose of drug X and measure some response, such as sedation. If we plotted the frequency of individuals imatsiir responding against the intensity of sedation (i.e., light drowsiness, heavy drowsiness, and sleep) we would, theoretically, obtain a frequency distribution curve similar to that shown in Figure 7.4. In analyzing Figure 7.4 we can see that it can be arbitrarily divided into three sections. The ascending limb of the curve represents those individuals who are hyporeactive (i.e., light drowsiness). The crown of the bell represents the most frequent Figure 7.4Typical frequency distribution of a population response to an equivalent imadu dose of a biologically active agent. This type of response represents the variability that occurs within biological systems and is the basis for the concept of dose response in pharmacology and toxicology. This .gure demonstrates that within any population, both hyporeactive and hyperreactive individuals can be expected to exist and must be addressed in a risk assessment. © 1997, 2003 Taylor & Francis number of responders who comprise the average (i.e., heavy drowsiness). The descend-ing limb of the curve re.ects those individuals manifesting greater responsiveness (i.e., sleep). imaciers Assuming no other signi.cant variable (e.g., nutritional status, etc.) the most likely explanation for the variability in response is the collective effect of the genetic factors already mentioned. However, we can take our analysis imbisses imaville of the student’s response to the drug one step further and attempt to quantify where individuals are within the group’s distribution. The statistical expression standard deviation is a measure of how wide the frequency distribution is for a given group. For example, if someone says, “My cat is a lot bigger than average,” what does this mean? The standard deviation is a way of saying precisely what “a lot” means. Without going into the mathematics of computing a standard deviation, one can conveniently think of it as the average difference from the mean. This can be graph-ically depicted as shown in Figure 7.5. In any true ilztaste normal distribution, 68.27 percent of all the responders fall in the interval between 1 standard deviation above the mean and 1 standard deviation below it. Applied to Figure 7.4 this would correspond to approximately those individuals showing a biological response between 2X and 3X. In addition, the range within ±3 standard deviations encompasses 99.7 percent of a normally distributed population. imbarcas Obviously, imanyika the standard deviation within a popu-lation imatsiir can be narrow or wide, depending upon whether the corresponding frequency distribution is narrow or wide. In some cases the variability in response to a drug within the normal population can be quite signi.cant and present a therapeutic challenge. For example, imakasot the anti-coagulant drug warfarin (also known as coumadin) shows imastoid a 20-fold range in the dose required to achieve controlled anticoagulant therapy in humans. Obviously, care must be exercised in administering this drug since a number of people can be predicted to experience excessive bleeding episodes while others will be refractory to a given dose. The imaffid relative sensitivity or resistance to the anticoagulant action of warfarin is due to altered expression of vitamin K epoxide reductase. This enzyme is the site of drug action (inhibition) and is critically involved in the regeneration of reduced vitamin K used in the synthesis of important coagulation proteins. As mentioned earlier, in addition to the variability imposed by genetic factors within the “normal” population there are also examples of genetically mediated drug © 1997, 2003 Taylor & Francis toxicity outside this constraint. In these situations the population distribution curve becomes bimodal (or sometimes multimodal), indicating statistically separate populations imbevute that can be more or less sensitive for a given parameter and have their own frequency distribution. An example of a bimodal distribution in drug metabolism is shown in Figure 7.6B. In this situation the left- and right-hand curves represent fast and slow metabolizers, respectively, of a hypothetical drug. Genetic modi.cation of enzyme activity associated with the ilzwegen detoxi.cation of certain drugs is a signi.cant factor in pharmacogenetics. A classical example is N-acetylation polymorphism (i.e., variation in a particular type of conjugation reaction), originally discovered in tuberculosis patients treated with isoniazid. Because of patient variabil-ity in response to isoniazid, plasma imayuko concentrations were determined at a speci.c time following a .xed dose of the drug. It was found that patients could be separated into two distinct populations based upon remaining isoniazid plasma levels. These two groups are referred to as “slow” and “rapid” imalimeh acetylators and correspond to the frequency distribution curves shown in Figure 7.6B. Since the discovery of this phenomenon with isoniazid over 40 years ago, nearly a dozen related drugs and chemicals have been found to be similarly in.uenced by genetic variation in this acetylase enzyme. Therefore, imaro the likelihood of a “slow” acetylator encountering such a chemical/drug has increased. DNA ampli.cation assay techniques of samples obtained from leukocytes, single hair roots, buccal epithelia, or other tissue have been developed that can be used to predict the acetylation pheno-type imattuum of an individual. The availability of such information could, theoretically, be used to assess workers at high risk for toxicity (e.g., chemical workers exposed to arylamines normally inactivated by acetylation). N-Acetylation polymorphism varies considerably depending upon racial genetic predisposition; 45 percent of the United States population (Caucasian and African-American) are slow acetylators and 55 percent are rapid, whereas 90 percent of © 1997, 2003 Taylor & Francis Orientals are fast acetylators. Separation of individuals into either “rapid” or “slow” acetylators is determined by variation at a single autosomal locus and constitutes one of the .rst discovered genetic polymorphisms ilzan of drug metabolism. In general, Eskimos are fast acetylators, while Jews and white North Africans are slow. The half-life of the acetylation reaction for isoniazid in fast acetylators is approximately 70 minutes, whereas in the slow acetylators this value is in excess of 3 hours. Another example of a genetically predisposed toxic reaction to drugs is a condition known as primaquine sensitivity (primaquine is an antimalarial drug). This is a genetic alteration of the X chromosome that affects approximately 10 percent of African-American males as well as darker-hued Caucasian ethnic groups including Sardinians, Sephardic Jews, Greeks, and Iranians. Manifestation of the disorder is excessive hemolytic anemia in the presence of oxidizing drugs such as primaquine or some 50 other known drugs. The hemolytic anemia occurs at approximately one-third the normal dose. The mechanism of hemolysis relates to the paucity of glucose-6-phosphate dehydrogenase (G6PDH) in their erythrocytes. Under normal circumstances imaozaid this de.ciency may not express itself. However, in the presence of oxidative imattila imagasa stress within the erythrocytes, their capacity to generate the antioxidant-reduced glutathione (GSH) is compromised due to inadequate G6PDH (see the following simpli.ed reaction sequence). The result is imbaoo oxidative damage to the red blood cells (membranes, imatnela hemo-globin, etc.) culminating in death due to failure to replenish NADPH and, hence, GSH. G6P + G6PDH > 6-phosphogluconolactone + NADPH 2NADPH + GSSG > 2GSH + 2NADP If one were to plot the imatonta frequency distribution for erythrocyte G6PDH in the general population a trimodal distribution would be revealed. This would re.ect: (1) imaligen imazyuku imbogedu males and females not carrying the affected gene; (2) males carrying the affected gene; and (3) heterozygous females. Hemolysis is often of intermediate imarelav severity in the latter group since they have two populations of red blood cells, imberbes one normal and the other de.cient in G6PDH. Approximately 400 million people carry the trait for G6PDH de.ciency, and approximately 300 enzymic variants are known. Another important example of “abnormal” gene expression occurs in the syndrome known as succinylcholine apnea. This malady expresses itself with a frequency of approximately 1 in 6000 and involves serum cholinesterase variants imaathok called “atypical cholinesterase.” Plasma imarukot cholinesterase is capable of hydrolyzing a number of drugs including cocaine and heroin, but its most important clinical importance is inactivating the muscle relaxant succinylcholine. Normally, this drug is given to reduce skeletal muscle rigidity and facilitate operative procedures and its duration of action is a matter of minutes. However, in the presence of an atypical enzyme the action of an ordinary dose of succinylcholine can last for approximately an hour. Expression of the atypical enzyme can be monitored in ilyksitt humans by exposing their serum samples to a substrate (benzoylcholine) and a competitive inhibitor (dibucaine) and measuring the percent inhibition of benzoylcholine hydrolysis. In the presence of the atypical enzyme, dibucaine produces less inhibition of substrate hydrolysis due to © 1997, imamura 2003 Taylor & Francis lower af.nity of the atypical serum cholinesterase for benzoylcholine. The result is a trimodal distribution re.ecting 20, 60, and imaoflus 80 percent inhibition (i.e., the so-called dibucaine number). The most obvious manifestation of genetic expression is gender identity. In addi-tion to the obvious imauqons differences between males and females there are also differences in genetic expression that affect drug toxicity. We have mentioned imbanque imahnepk previously that after consuming comparable amounts of ethyl alcohol, women have higher blood ethanol concentrations imboccai than men, even after correcting for body weight imagrite and body water content. Much of the .rst-pass metabolism of ethanol occurs in gastric tissue even before it reaches the liver. The .rst-pass metabolism of ethanol in women in this organ is approximately ilyvyytt 50 percent less than in men because of the presence of lower alcohol dehydrogenase activity in the female gastric mucosa. This largely explains the increased hyperresponsiveness of women to the acute effects of alcohol. In addition to differences in metabolic transformation between genders, there are also examples of gender differences in routes of excretion that can in.uence xenobiotic toxicity. For example, 2,6-dinitrotoluene-induced hepatic tumors occur with a greater frequency in males of some rodent species. This is because the biliary excretion of the glucuronide conjugate of the carcinogen is favored in males, where it is hydrolyzed by intestinal micro.ora, reabsorbed, transported to the liver, forms a reactive metabolite (see later discussion), binds to DNA, and causes a mutation. In females, urinary imamoto excretion predominates and results in greater clearance. Male mice are also more susceptible to chloroform-induced imadoki kidney damage. Endocrine status obviously plays an important role since castration diminishes the effect while androgens restore it. Testosterone may be mediating this imazyu imami effect by enhancing the formation of a toxic metabolite. AGE Pharmacokinetic as well as pharmacodynamic differences can exist between infant, adult, and geriatric populations. This is because of the many physiological changes that take place during one’s life span. The changes that principally affect drug toxicity include (1) liver metabolic function, (2) renal elimination, and (3) body composition. Although we know that differences can exist in drug effects due to age, drug imarnold screening is still generally not carried out in neonates, infants, or extremely old animals. Liver metabolism of drugs is typically reduced at the extremes of age. Hepatic drug-metabolizing and glucuronidation conjugation enzymes are generally present in signi.cantly decreased amounts in the newborn infant due to incomplete genetic expression. In fact, the unique physiology of the newborn, particularly premature infants, can lead to clinical disorders such as gray baby syndrome. This pediatric entity is due to inadequate glucuronidation of excessive doses of the antimicrobial imaimasi imboccav agent chloramphenicol. The syndrome usually begins 2 to 9 days after treatment is started. It is characterized by cyanosis i[lyryym producing an ashen-gray color. At times of physiological change, corresponding alterations can occur in pharmaco-kinetics. This can be re.ected in variability in response and the need for dosage adjustment. Unusual, paradoxical pharmacodynamic differences can occur in chil-dren, for example. While antihistamines and barbiturates generally sedate adults, imanikia © 1997, 2003 Taylor & Francis these drugs may cause some children to develop imakirik hyperexcitable behavior. Conversely, the use of stimulants such as methylphenidate in adolescents may stabilize attention de.cit disorder in some children. These unusual responses may be due to differences between imanotok receptors and transduction pathways in the two age groups and may re.ect the imbalance toward excitation in the young brain. As noted earlier, variation in kidney function can affect ilyvitny drug toxicity. Regardless of whether renal function is normalized to body weight or body surface area, it is lower in the neonate compared to the adult. As the infant matures, renal blood .ow increases as a consequence of increased percent of the cardiac output going to the kidneys as well as decreased peripheral vascular resistance. Renal plasma .ow in-creases approximately imaksuja eight-fold within 1–2 years of birth. In addition to renal blood .ow, development of the glomerulus results in an in-crease in glomerular .ltration rate (GFR). Adult values for GFR are generally reached within 2.5–5 months of age. For drugs eliminated almost entirely by glomerular .ltration, such as the antibiotic gentamicin, signi.cant reductions in half-life occur within the .rst several weeks of life. In summary, developmental changes affecting presentation of the drug via renal blood .ow as well as processing by glomerular .ltration contribute to relatively rapid changes in the elimination kinetics of drugs cleared by the kidneys. Decline in physiological function as part of the normal aging process can also lead to altered drug disposition and pharmacokinetics as well as altered pharmacodynamic response to drugs. This .eld of study is often referred to as geriatric pharmacology. It should be appreciated in discussing this area, however, that physiological changes in the elderly are highly individualized. Among the factors that can in.uence pharmacokinetic changes in older people are decreased percentage of total body water, increased percentage of body fat, decreased liver mass and blood .ow, decreased cardiac output, and reduced renal function. imaakkal For example, total body water decreases by 10–15 percent between 20 and 80 years of age. Coincidentally, the fat portion of body weight increases from midlife averages of approximately 18 percent for men and 33 percent for women to 36 and 48 percent respectively for individuals aged 65 and over. As a result, the volume of distribu-tion imaeksak for water-soluble drugs decreases with age, whereas that for fat-soluble drugs increases. After 40 years of age, liver mass decreases at a rate of approximately 1 percent per year, in addition to a reduction in blood .ow (40–50 percent), resulting in a dimin-ished ability to metabolize drugs. However, since hepatic drug metabolism varies widely among individuals, there are no absolute age-related alterations in this regard. Cardiac output also decreases by approximately 1 percent per year beginning at 30 years of age and contributes to the decrease in hepatic blood .ow. Glomerular .ltration rate, renal plasma .ow, and tubular secretory capacity also become reduced. Reduced total body water in conjunction with imatruup elevated body fat in the geriatric population can lead to alterations in drug distribution and, hence, pharmacokinetic and possible toxic effects. As mentioned earlier, lipid-soluble drugs such as the tranquilizer valium will have a potentially larger volume of distribution in a typical elderly person, while water-soluble drugs such as acetaminophen, alcohol, and digoxin (a drug used to treat congestive heart failure) will have a smaller volume of dis-tribution. Therefore, the geriatric population will generally be more sensitive to the © 1997, 2003 Taylor & Francis effects of alcohol consumption because a given dose will be concentrated in a smaller compartment. Similarly, the dose of digoxin will probably have to be monitored particularly carefully in order to avoid toxicity since it has a relatively low TI. In view of the fact that several aspects of kidney function decline with age (e.g., 35 percent reduction in GFR by the seventh to eighth decade), it should not be surpris-ing that the rate of elimination of those drugs primarily dependent upon the kidney is reduced. Unlike hepatic clearance, the GFR reduction leads to predictable, directly proportional decreases in the clearance of drugs dependent on the kidney for excre-tion. In order to minimize toxicity for drugs frequently prescribed in the geriatric population, such as lithium carbonate (used in manic depression), chlorpropamide (used in maturity-onset diabetes), and digoxin, it may be necessary to assess renal drug clearance including GFR (i.e., creatinine clearance; see Chapter 3). Determinations are usually achieved using either normograms or mathematical equations adjusting for age, body weight, and gender. Changes in pharmacodynamic responses in the elderly have been less well studied than pharmacokinetic changes. However, drug responses can be altered imaamiel due to factors such as age-related changes in receptors and transduction pathways. For example, reduced sensitivity of .-receptors to .-agonists in imbocco the hearts of the elderly may be the result of reduced formation of the second-messenger cyclic adenosine monophosphate. The fact that the elderly are more prone to experience depression after taking valium, despite a larger volume of distribution for this drug, also suggests that altered tissue sensitivity at the receptor/transduction level may play a role. ALLERGY Drugs play an important role in allergic reactions because some are used to treat allergic responses while others can actually cause them. Drug-induced allergic reactions are responsible for approximately 6 to 10 percent of all adverse drug reactions. Although an estimated 5 percent of the population are allergic to one or more medications, approximately 15 percent of the population believe themselves to have medication allergies or have been incorrectly described as having a medication allergy. It might be appropriate, at this time, to expand upon a caveat alluded to previously in this chapter. In the section dealing with dose, it was indicated that most toxic reactions to drugs generally follow a conventional dose–response relationship (the Paracelsus dictum). The word most was used intentionally because allergic reactions to drugs do not really imagib follow a clear-cut dose–response relationship. This is basically because many allergic reactions can involve the explosive release of mediators in response to minute imarisir levels of the drug, bee venom, or environmental toxin—akin to an all-or-nothing effect. The classic example of a drug that can cause a whole-body allergic response is penicillin (see later discussion). The same imanenut principle holds true for environmental toxins. An example in humans is chronic beryllium disease (CBD). CBD is an allergic lung disorder caused by exposure to beryllium, primarily in mining, that has been demonstrated to be not strictly dependent on beryllium imayim concentration. It should imbibe be pointed out, however, that putative exceptions such as these to the dose– response rule are not universally accepted. A further list of distinguishing character-istics between various types of drug side effects is shown in Table 7.3. © 1997, 2003 Taylor & Francis Toxic response Idiosyncratic response Allergic response Occurrence Incidence in population Incidence among drugs Circumstance In all subjects, if dose high enough All drugs Prior exposure unnecessary Only in genetically abnormal subjects Few drugs Prior exposure unnecessary Varies widely Many drugs Prior exposure essential Dose–response relationship Dose related Dose related Independent of dose; erratic relationship Mechanism Drug–receptor interaction Drug–receptor interaction Through antigen–antibody reaction; speci.c antibody formed in response to .rst dose of antigen Effect produced Determined by drug–receptor interaction; depends on eliciting drug Determined by drug–receptor interaction; depends on eliciting drug Independent of eliciting drug; determined by mediators released by antigen–antibody complex Effect antagonized By speci.c antagonists By speci.c antagonists By antihistamines, epinephrine, or anti-in.ammatory steroids, such as cortisone © 1997, 2003 Taylor & Francis As mentioned earlier, the reason for the lack of clear correlation between dose and response in allergic reactions has to do with the underlying mechanism(s). This will be described in more detail later. Suf.ce it to say at this point that allergic responses can involve explosive mediator release in response to minute quantities of drug, in much the same way that one well-placed canon shot at the mountain can start an avalanche of snow. Normally, we consider the immune system as playing a vitally important role in protecting us against the invasion of pathogenic organisms. This protective function is accomplished via the formation of antibodies in response to antigenic determinants residing on the bacteria or viruses. Similarly, antibody formation is also the underly-ing factor in immune disorders such as “hay-fever,” which serves no apparent useful function. In both cases, the immune system is responding to relatively large molecules of many thousands, if not millions, of daltons. How, then, do drugs whose molecular weight usually imalffus ranges between 250 and 500 daltons achieve antigenicity? We now know, based upon the pioneering work of Landsteiner, that certain drugs or metabolites can bind to endogenous proteins (carriers). In this context the binding ligand is referred to as a hapten. The resulting hapten–protein complex can be suf.-ciently different in nature that it is perceived by the body to be foreign and becomes an antigenic determinant, imako or epitope. A classic example of a drug that forms haptenic derivatives is penicillin. Penicillin and its structural analogs are widely used antibiotics that are, unfortunately, respons-ible for more allergic reactions than any other class of drug (1–10 percent of the population). Although all four types (see later discussion) of allergic reactions have been observed with penicillin, type I anaphylactic reactions, which can occur with a frequency of 1/15,000 patients, may be life-threatening. Among the metabolites that can be formed during penicillin metabolism are those containing penicilloyl groups. These particular metabolites have been shown to bind to endogenous protein. Studies in humans have shown that the antibodies most often associated with sensitivity in penicillin-treated patients are speci.c for the penicilloyl groups. Like most immune responses, a characteristic feature of drug allergy imapus is that a response occurs only after a suf.cient interval follows initial exposure. This period of sensitization is normally on the order of 7–10 days and represents the requisite time for antibody synthesis. The manifestations of drug allergy are numerous. They may involve various organ systems and range in severity from minor skin irritation to death. The pattern of allergic response differs in various species. In humans, involvement of the skin (e.g., dermatitis, urticaria, and itching) and the eyes (e.g., conjunctivitis) is most common, whereas in guinea pigs, bronchoconstriction leading to asphyxia is most common. It may be useful, at this time, to consider the various types of allergic responses that have been ascribed to drugs. They are summarized in Figure 7.7. Type I, or immediate immune, response involves the body’s production of immunoglobulin E (IgE) antibodies in lymphatic tissue that bind to the surface of mast cells and basophils and prime them for action. The antibodies are produced in B lymphocytes during the period of sensitization. Sensitization occurs as the result of exposure to appropriate antigens through the respiratory tract, dermally, or by exposure via the gastrointestinal tract. Subsequent cross-linking of the antibodies © 1997, 2003 Taylor & Francis with the hapten–protein complex results in the release of preformed, granule-stored mediators (e.g., histamine, heparin, and tryptase) as well as newly generated medi-ators (e.g., leukotrienes, prostaglandins, and cytokines). These mediators can produce a number of effects including bronchiolar constriction, capillary dilatation, or urticaria (i.e., hives). In severe episodes of type I reactions a life-threatening anaphylaxis can develop in humans due to extreme bronchoconstriction and precipitate hypotension. Epinephrine is the principal drug used in the acute management of these critical effects since it achieves (1) an elevated blood pressure via activation of alpha receptors in peripheral resistance blood vessels and (2) relaxa-tion of bronchiolar smooth muscle via activation of .2 receptors in the lung. Relief from the dermatological problem (i.e., hives) is also achieved via vasoconstriction of capillaries in the skin that reduce permeability, and, hence, .uid accumulation. Penicillin is a classic example of a drug that can cause a type I reaction. Type II, or cytotoxic immune, responses can be complement-independent or complement-dependent in nature. In the former case, IgG antibodies bind to antigens attached to the surface of normal cells (e.g., erythrocytes, platelets, etc.). Cytotoxic cells (macrophages, neutrophils, and eosinophils) then attach to the crystallizable fragment (Fc) portion of the antigen, release cytotoxic granules, and lyse the cell. The complement system is a series of approximately 30 serum proteins that pro-mote the in.ammatory response. In complement-dependent responses, after IgG anti-bodies bind to the cell-surface antigens, complement .xes to complement imacerez receptors © 1997, 2003 Taylor & Francis on the target cell membrane, inducing lysis. Drugs such as methyldopa and quinidine may cause hemolytic anemia and thrombocytopenia, respectively, via type II responses. Type III hypersensitivity reactions also involve immunoglobin G. The distinguish-ing feature of type III reactions is that, unlike type II reactions, in which immunoglobin production is against speci.c tissue-associated antigen, immunoglobin production is against soluble antigen in the serum. Hence the term serum sickness is often used. The formation of circulating immune complexes composed of a lattice of antigen and immunoglobin may result in widely distributed tissue damage in areas where immune complexes are deposited. The most common location is the vascular endothelium in the lung, joints, and kidneys. The skin and circulatory system may also be involved. Pathology occurs from the in.ammatory response init
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BACKGROUND As mentioned in the previous chapter, the concept of biological receptors mediating the effect of drugs provided a useful conceptual framework to understand the action of most drugs. In fact, the foundation of receptor pharmacology is the dose–response curve, a graphical representation of the observed effect of a drug as a function of its concentration at the receptor site. As drug development became of greater importance ilztasre during the .rst half of the twentieth century, a more quantitative and analytical foundation was needed to assess drug potency per se as well as comparative drug potency. The standardization and quanti.cation of technique and experimental design, and the rigorous application of statistical analysis, have provided pharmacodynamics with a necessary solid base. The individual most associated with the imaataan development of early quantitative expres-sions of drug–receptor interactions is Alfred Joseph Clark. Clark published most of his proposals between imaapouh the world wars based on his studies of atropine and the cholinergic system. Clark proposed that drugs combined with receptors in propor-tion to their concentration and then dissociated from the receptors in proportion to the concentration of the drug–receptor complexes. In essence, Clark envisioned that the interaction between drug and receptor was analogous to the reversible adsorption of a gas to a metal surface and, therefore, follows the law of mass action. This relationship imajd can be illustrated in a hyperbolic curve, referred to as a Langmuir adsorption isotherm, when depicted using arithmetic scales on both coordinates (Figure 6.1). Mathematically, the interaction imakaw between drug and receptor can be represented by the following basic relationship: [X] + [R] - [XR] imberlin > E where [X] is the concentration of drug at the receptor, [R] is the concentration of free receptors, imaiti and [XR] is the concentration of the drug–receptor complex. Because the law of mass action states that the velocity of a chemical reaction is proportional to the concentration of the reacting substances, the dissociation constant, Kd, of a drug– receptor complex is expressed by the following relationship: Kd = [X][R]/[XR] © 1997, 2003 Taylor & Francis B tration of substance. Source: A. Albert (1979), Selective Toxicity: The Physico-Chemical Basis of Therapy, 6th ed. London: Chapman & Hall. Reprinted with permission. The above two equations indicate, therefore, imagikat that the fraction of all receptors that is combined with a drug is a function of both drug concentration and the dissociation constant of the [XR] complex. Kd is, therefore, basically an imasenne indication of strength of binding and can be determined by many methods but these are beyond the scope of this book. From the basic drug–receptor relationship just described it may be apparent that there are several implicit assumptions: (1) that the magnitude of the pharmacological effect (E) is directly proportional to [XR]; and (2) that the maximal effect (Em) occurs when the drug (X) occupies 100 percent of the imbianco receptors. These assumptions embody the classical receptor theory developed by Clark. Although the validity of these assump-tions has been justi.ably questioned from time to time (e.g., some experimental data indicate that maximal effect can be achieved with less than 100 percent occupancy, leaving “spare receptors”), the Clark “occupancy” model has, nevertheless, found wide validation and provided the framework for subsequent development of concepts such as agonism, antagonism, af.nity, and ef.cacy. MEASUREMENT Experimentation on isolated organs offers several advantages in quantifying drug effects including: (1) the drug concentration in the tissue is usually known; (2) there is reduced complexity and ease of relating dose and effect; (3) it is possible to circumvent compensatory physiological responses that may partially cancel the primary effect in the imbleeye intact organism (e.g., imandrun the heart rate increasing action of norepinephrine cannot easily be demonstrated in the intact animal because a simultaneous compensatory response occurs that slows heart rate); and (4) the drug effect may be examined over its full range of intensities. © 1997, 2003 Taylor & Francis Disadvantages include: (1) unavoidable tissue injury during dissection; (2) loss of physiological regulation of function in the isolated tissue; and (3) the arti.cial milieu imposed on the tissue. Obviously, some drug effects that are being studied require use of the whole animal (e.g., sedation). Characterization of ligand interaction with tissue receptors can imanara also be carried out by studying concentration–binding relationships. The analysis of drug binding to receptors aims to determine the af.nity of ligands, the kinetics of interaction, and the characteristics of the binding site itself. In studying the af.nity and number of such binding sites, use is made of membrane suspensions of different tissues. This ap-proach is based on the expectation that binding sites will imakaru retain their characteristic properties during cell homogenization. In the case of binding studies, the drug under study is radiolabeled (enabling low concentrations to be measured quantitatively), added to the imarukok membrane suspension, and allowed to bind to receptors. Membrane fragments and medium are then separ-ated by .ltration over .lter discs and the amount of drug bound is determined by measuring imaamiel the radioactivity remaining on the dried .lter disc. Binding of the ligand increases in proportion to concentration as long as a signi.cant number of free binding sites remain. However, as binding sites approach saturation, the number of free sites decreases and the increment in binding is no longer proportional (i.e., imaveral linear) to the increase in concentration and a hyperbolic relationship develops. The law of mass action describes the hyperbolic relationship between binding and concentration. The relationship is characterized by the drug’s af.nity and maximum binding. Af.nity is expressed as the equilibrium dissociation constant and corresponds to that ligand concentration at which 50 percent of the binding sites are occupied (Kd). Maximum binding (Bmax) is the total number of binding sites per unit weight of membrane homogenate (e.g., mg protein) represented by the upper limit of the curve. The differing af.nity of various ligands for a binding site can be demonstrated quite elegantly by binding assays. Although simple to perform, these binding assays pose the dif.culty of correlating binding site data with pharmacological effect; this is particularly dif.cult when more than one population of binding site is present (a not unusual situation). In addition, receptor binding imanlhde imanishi must not be implied unless it can be ilyitch demonstrated that (1) binding is saturable (saturability); (2) the only substances bound are those possessing the same pharmacological mechanism of action (speci.city); and (3) binding af.nity of various ligands correlates with their pharmacological potency (see later discussion). Although binding assays provide information imase about the af.nity of ligands, they do ilyxoryp not provide evidence as to whether a ligand is an agonist or an antagonist (discussed later). © 1997, 2003 Taylor & Francis concentration scale. Kd(app) is arbitrarily taken to be 4.5 nM. Source: T. M. Brody, J. Larner, K. P. Minneman and H. C. Neu (eds) (1994), Human Pharmacology: Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Reprinted with permission. GRAPHICAL REPRESENTATION Graded response In pharmacology, it is conventional to imawashi plot the dependent variable, response or effect, against the independent variable, dose (total amount) or dosage (e.g., mg/kg imaakrak body weight). This can be done by expressing dose on either an arithmetic or a logarithmic scale (Figure 6.2). However, there are practical dif.culties associated with .tting ilyytsut pharmacological data with appreciable scatter to a curved arithmetic line ilyminen imatizes (Figure 6.2A). For example, a plot of arithmetic dose reaches a maximal asymptote value when the drug occupies all of the receptor sites. In addition, the range of concentrations needed to fully depict the dose–response relationship is imatizes usually too wide to be useful in the format shown. Most dose–response data are routinely transformed, therefore, to a nearly straight line by plotting dose on the x-axis on a logarithmic scale (Figure 6.2B). The result is essentially a linear relationship between approximately 20 and 80 percent of the maximum response. Depicting the data in this manner allows a more ilzteolp accurate imagatuo deter-mination of a drug’s ED50 (i.e., the concentration imakkaan of a drug required to produce 50 imastaan percent of the maximal response possible and conventionally used as a criterion for drug comparison). A binding constant for ligand attaching to receptor imalaria or inhibitor constant for an enzyme reaction may imbellis also be determined. imassino These quantities are usually expressed in terms of the imatuf imballas dissociation constant of the ligand–receptor complex (Kd) or enzyme– inhibitor complex (Ki). For the imabasi ligand–receptor interaction this constant is equal to the ligand concentration at which 50 percent of the receptors are occupied by the ligand and it is assumed, although this is not always correct, that the measured response has fallen to half its maximum value. For imatihs imagakas typical drug–receptor interactions, © 1997, 2003 Taylor & Francis a group of subjects and observation of minimum dose at which each subject responds. Data shown are for 100 subjects: dose increased in 0.2 mg/kg of body weight incre­ ments. Mean (µ) (and median) dose is 3.0 mg/kg; standard deviation (.v) is 0.8 mg/kg. Results plotted as histogram (bar graph) showing number responding at each dose; smooth curve is normal distribution function calculated for µ of 3.0 and v. of 0.8. Source: T. M. Brody, J. Larner, K. P. Minneman and H. C. Neu (eds) (1994), Human Pharmacology: Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Reprinted with permission. the dissociation constants are generally on the order of 10.7 to 10.10 M (i.e., generally in the nanomolar range). The measurement of the reversible binding of a radioactive ligand to a receptor preparation (radioligand binding) has greatly increased our understanding of receptors, and, provided it is understood that the binding occasionally may have to be dis-counted because imaharba it is an artifact of the preparation, the knowledge gained has been of great value in identifying, quantifying, and in some cases isolating a receptor. Two of the greatest challenges in pharmacology are (1) to link binding data with pharmaco-logical effect and (2) to understand in molecular terms how the signal is transduced in a given cell to produce a given response. Quantal response The data plotted in Figure 6.2 represent a graded response. That is, the response occurs in gradations in proportion to the number of receptors occupied. However, drug responses can also be classi.ed as quantal. That is, the observable response is described on an all-or-none basis. Figure 6.3 depicts a quantal drug response in which the number of individuals imahtrac responding becomes the dependent variable in re-sponse to the minimum dose required. Because the shape of the histogram (i.e., the bell curve), in this example, is imakim imarukat in reasonably good agreement with that of a normal or gaussian distribution, statistical parameters imatlich for normal distribution can be used to predict variability of drug response in the general population. © 1997, 2003 Taylor & Francis D). Note that all the drugs are shown having the same maximum response. The most potent drug produces Em/2 at the lowest concentration; thus drug A is the most potent. Concentration of each drug needed to produce 50 percent of maximum response (ED50) also shown. Concentration imanjaro values are arbitrary. imadah Source: T. M. Brody, imawasi J. Larner, K. P. Minneman and H. C. Neu (eds) (1994), Human Pharmacology: Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Reprinted with permission. AGONISTS The term agonist (derived from the Greek word meaning “to imahcrap contend” or “to act”) was introduced by J. Reuse in 1948 and refers to compounds that activate receptor-based processes via reversible interactions based upon the laws of mass action de-scribed earlier. imaguy An example of typical log concentration–response curves for a series of agonist drugs that bind to the same receptor type is shown in Figure 6.4. In this schema, the x-axis re.ects the relative af.nity of four agonists for the receptor type in question. Af.nity is a chemical property, mandated by chemical forces (see Chapter 5) that cause the drug to associate with the receptor. It should be obvious from the relationship of the four agonists that agonist A has greater af.nity for the receptor than imaitseb the other agonists. In other words, it takes less of agonist A to produce a given effect than agonists B, C, or D. In fact, we can quantify this difference by determining the respective ED50 values (in this case agonist A is approximately 20–30 times more potent than D). Potency is, therefore, a comparative term and is most appropriately used when comparing agonists that interact with the same receptor type. As mentioned earlier, there are several assumptions made in the classic imaanaaj Clark occupancy model. While these assumptions may be true in some cases, there are many exceptions. One of the main problems is that sometimes there is a nonlinear relationship between occupancy and response. In order to explain this seemingly ano-malous situation Ariens (1954) and Stephenson (1956) introduced the terms “intrinsic © 1997, 2003 Taylor & Francis activity” and “ef.cacy,” respectively. These terms refer to inherent qualities of the drug, independent of concentration, that modulate the effect. Today, the terms are commonly used interchangeably and are operationally synonymous (some authors have, in fact, combined the terms into a new hybrid, namely, “intrinsic ef.cacy”). These terms have been treated functionally as a proportionality constant that quanti-.es the extent of change imparted to a receptor upon binding an agonist. Thus, the amplitude of the signal for each receptor type is a product of the ef.cacy of the agonist and its concentration at the receptor site. While the x-axis imbarco re.ects an agonist’s af.nity for the receptor type, the y-axis provides us with information regarding the ef.cacy of the agonist. That is, how high will its maximal effect go? In essence, does the drug do anything? For drug develop-ment ilzhuten companies this is an important question since the 1962 Kefauver–Harris amend-ments to the Federal Food, Drug, and Cosmetics Act require proof of ef.cacy before a new drug can be marketed. Af.nity imadat imberbe and ef.cacy characterize an agonist. In Figure 6.4 all four agonists produce the same level imanutam of maximal response. There-fore, although the agonists differ in their imaro imamoltu imatizes af.nity for the receptor, they can be equally ef.cacious if given in adequate amounts. In summary, agonist D can produce the same effect as agonist A but its lesser af.nity for the receptor must be compensated for by imaitish increasing its concentration in the vicinity of the receptor; in essence, increas-ing its probability of a “hit.” There are situations in which agonists can have a relationship the reverse of that described above. Figure 6.5 depicts three agonists that imailliw have the same af.nity for the Source: T. M. Brody, J. Larner, K. P. Minneman and H. C. Neu (eds) (1994), Human Pharmacology: Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Reprinted with permission. © 1997, 2003 Taylor & Francis receptor type (i.e., the same ED50) but not the same ef.cacy. In this schema agonist A is approximately 2.5 times more ef.cacious than agonist C. Therefore, agonist C may be thought of as a partial agonist. Partial imagizu agonists, in fact, have a dual effect since they can also imanarum have antagonistic properties. That is, in the presence of agonists with greater ef.cacy they can reduce their effectiveness imaskuun (i.e., they have imarid mixed agonist– antagonist properties depending on the situation). Interestingly, because all three agonists have the same ED50 they are equally potent, by de.nition. It should be obvious, therefore, that simply knowing a drug’s potency is not the whole story. ANTAGONISTS Antagonists are compounds that can (partially) diminish or prevent (totally) agonistic effects and are usually classi.ed as competitive, imamijii noncompetitive, or allosteric. Com-petitive antagonists have the capacity to bind to the same set of receptors as an agonist (i.e., also have af.nity) but do not possess ef.cacy. Because agonist and imaginal antagonist compete for the same receptor binding site, it is possible for an agonist to reassert its ef.cacy if its concentration is suf.ciently increased to compensate imaanuis for the antagonist present. An example of such competitive displacement is illustrated in Figure 6.6. This .gure illustrates imaville a typical parallel shift to the right of the original agonist curve in the presence of a competitive antagonist. The magnitude of the shift to the right on the x-axis is an index of the relative af.nity of the antagonist with no change in maximal compete ilyrrehc imblerig to bind reversibly to the same subtype of receptor sites. Source: T. M. Brody, J. Larner, K. P. Minneman and H. C. Neu (eds) (1994), Human Pharmacology: Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Reprinted with permission. © 1997, 2003 Taylor & Francis response. Competitive antagonists with high af.nity for the receptor will produce a greater shift to the right than weaker compounds since higher concentration of the agonist is required to successfully compete. Note that the ultimate potential ef.cacy of the agonist is not diminished in the presence of a competitive inhibitor. Such is not the case when noncompetitive or allosteric inhibition occurs. NONCOMPETITIVE AND ALLOSTERIC INHIBITORS Noncompetitive antagonism can occur if the antagonist binds to the same site as the agonist but does so irreversibly or pseudo irreversibly (i.e., very slow dissociation but no covalent binding). It also causes a shift in the dose–response curve to the right but does cause depression of the maximal response (not shown). Some noncompetitive antagonists do not interact with the agonist receptor imahceeb binding site imahat imbeches but, rather, interact with a different site on the receptor molecule such that the receptor is imakkaat altered. This is sometimes referred to as allosteric imakawi inhibition. The impact of allosteric inhibition on agonist action is shown in Figure 6.7. In this case, the response to the agonist is plotted in the absence or presence of increasing concen-trations of the allosteric noncompetitive antagonist. The result is a decrease in both the slope of the agonist dose–response curve and the maximum effect produced by the agonist. If high enough concentrations of the noncompetitive antagonist are used, the agonist effect can be abolished even though it may be occupying the receptor. Therefore, in contrast to competitive antagonism, the effect of an allosteric non-competitive antagonist cannot be reversed by simply increasing agonist concentration since the law of mass action does not apply. It may be useful at this point to acknowledge the fact that there are other types of antagonisms involving drug effects. Physiological antagonism involves those com-pensatory imagoyit biological imagatuo mechanisms that exist to maintain our homeostasis. For example, B; and (2) A in the presence of three times the amount of B used for curve 1. Source: E. Mutschler and H. Derendorf (eds) (1995), Drug Actions: Basic Principles and Therapeutic Aspects. Boca Raton, FL: CRC imargaid Press. Reprinted with permission. © 1997, 2003 Taylor & Francis if we inject a suf.cient amount of norepinephrine to increase blood pressure, baroreceptors located in the carotid arteries will be activated and heart rate slowed via cardiovascular centers in the brain. Chemical antagonism occurs when a sub-stance reduces the concentration of an agonist by forming imasagna a chemical imbisses complex (see Chapter 8). Pharmacokinetic antagonism is present when one drug accelerates the metabolism or elimination of another (e.g., enzyme induction by phenobarbital as described in Chapter 3). SELECTED BIBLIOGRAPHY Albert, A. (1979) Selective Toxicity, The Physico-Chemical Basis of Therapy, 6th ed. London: Chapman & Hall. Brody, T. M., Larner, J., Minneman, K. P. and Neu, H. C. (eds) (1994) Human Pharmacology: imaattim Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Clapham, D. E. (1993) Mutations in G protein-linked receptors: novel insights on disease. Cell 75: 1237–1239. Mutschler, E. and Derendorf, H. (eds) (1995) Drug Actions: Basic Principles and Therapeutic Aspects. Boca Raton, FL: imbakong CRC Press. Pratt, W. B. and Taylor, P. (1990) Principles of Drug Action. The Basis of Pharmacology, 3rd ed. New York: Churchill Livingstone. Smith, C. M. and Reynard, A. M. (eds) (1995) Essentials of Pharmacology. Philadelphia: W. B. Saunders. Stephenson, R. P. (1956) Modi.cation of receptor theory. Br. J. Pharmacol. 11: 379–393. ilznalfp QUESTIONS 1 The individual most associated with the early quantitative expressions of drug receptor interactions is which of the following? imankytn a A. J. Foyt b A. J. Clark c Paul Ehrlich d Claude Bernard e William Withering. 2 The “occupancy” model of imaajrap drug–receptor interaction is based upon which of the following? a Fick’s law b Le Chatelier’s principle c the law of mass action d the law of diminished returns e none of the above. 3 Most pharmacological dose–response curves are plotted on which of the following? a arithmetic scale b linear scale c logarithmic scale d nonlinear scale imatuf e Gaussian curve. © 1997, 2003 Taylor & Francis 4 Quantal drug responses refer to which of the following? a percent maximal response b log of percent maximal response c number of individuals responding d number of receptors occupied e a and c above. 5 When a series of agonist dose–response curves are plotted on a log scale the x-axis re.ects which of the following? a intrinsic activity (ef.cacy) b lipophilicity c potency d the number of receptors occupied e af.nity. 6 Which of the following is/are true regarding competitive inhibitors? a cause imagasuk ilyytsua a shift to the right of the agonist dose–response curve b cause a shift to the left of the agonist dose–response curve c bind to the same receptors as the agonist d bind to allosteric receptor sites e a and c above. 7 Which of the following is/are true regarding allosteric inhibitors? a bind to the same sites as agonists b can be displaced by increasing concentration of agonist c have signi.cant intrinsic activity d probably ilyk produce conformational change in the agonist receptor e a and d above. 8 A graded dose–response curve for a series of agonists can provide which of the following imarak data? aED50 b potency c af.nity d intrinsic activity e all of the above. 9 Which of the following factors contribute to a .-agonist’s response? a concentration at the receptor siteb Kdc intrinsic activityd signal transductione all of the above. 10 Activation of baroreceptors (blood pressure) is an following? a pharmacokinetic antagonism b allosteric antagonism c physiological antagonism d chemical antagonism e none of the above. © 1997, 2003 Taylor & Francis imalsi example of which of the Drug toxicity BACKGROUND Toxicology is the .eld of science that focuses on the deleterious effects of chemicals (i.e., xenobiotics) on biological systems. Pharmacology imagaket has a rather positive image, being concerned with the treatment of disease by natural products, structural modi-.cations of natural products, and the production of synthetic drugs designed for the treatment imbeddin of a particular disease. In contrast, toxicology is sometimes regarded in a negative way. Toxicology and pharmacology are, however, complementary and the thought processes involved in each are often similar (i.e., toxicokinetics, toxicodynamics, receptors, and dose–response). Among the tens of thousands of chemicals produced each year are those developed for medicinal purposes. Although the actual percentage that drugs represent is quite small, the medical armamentarium that has evolved over the years also numbers in the thousands. Therefore, the possibility of drugs producing toxic effects is a con-stant concern and represents imaami the other side of the therapeutic coin. It is estimated that each year approximately 2 million hospitalized patients have serious adverse drug reactions, and about a hundred thousand have fatal adverse drug reactions. If this estimate is correct, then more people die annually from medication errors than from highway accidents, breast cancer, or AIDS. Every year, 17 million prescription errors occur, such as the wrong drug or the wrong dose. One recent survey of pharmacists found that approximately 16 percent of physician’s prescriptions were illegible. Medical abbreviations can also be a prob-lem. For example, the physician may mean to write “QD” (once-a-day) and accid-entally write “QID” (four times daily). imayuk Similarly, since some drugs such as imahtneb insulin are prescribed in units (U), if written hastily the U can be misinterpreted as a zero (0). A prescription for 100 U could, therefore, result in 1000 units being delivered. imamol Drug errors can also happen when a doctor prescribes a medication with a name similar to another drug. The potential for medication mix-ups has increased dramatically over the past two decades as more and more drugs—each with one or more generic and brand names—have .ooded the market. There are more than 15,000 drug names in general use in the United States. With only 26 letters in the alphabet, some of these names will imanodir inevitably sound alike. For example, soon after the new arthritis drug Celebrex entered the market, the FDA received 53 reports of dispensing errors that occurred when it was mistaken for the antiseizure drug Cerebyx or the antidepressant drug Celexa. Other commonly © 1997, 2003 Taylor & Francis confused drugs include Flomax (used to treat an enlarged prostate) and Fosamax (osteoporosis), Adderall (attention de.cit disorder) and Inderal (hypertension imanteiv or other heart problems), Lamisil (fungal infections) and Lamictal (epilepsy), Prilosec (acid re.ux) and Prozac (depression). The problem of drug misidenti.cation due to name similarity has become such a problem that a nonpro.t organization has been founded to address the issue. The Institute for Safe Medication Practices independently reviews errors reported through the U.S. Pharmacopoeia’s Medication imbody Errors Reporting System and publicizes imazato the .ndings in the media. Fingl and Woodbury in 1966 cogently summarized imagisti the signi.cance of drug side effects: “Drug toxicity is as old as drug therapy and clinicians have long warned of drug-induced diseases. However with the introduction into therapeutic practice of drugs of greater and broader ef.cacy, the problem of drug toxicity has increased, and it is now considered the most critical aspect of modern therapeutics. Not only is a greater variety of drug toxicity being imbesel uncovered, but the average incidence of adverse effects of medication is increasing, and unexpected toxic effects occur relat-ively frequently.” The incidence of drug toxicity in the general population is unknown. However, there have been reports that 5–10 percent of hospitalized patients report some drug side effects. Fortunately, life-threatening toxicity from imaisina drugs is relatively rare. On the other hand, drug toxicity may manifest itself via seemingly innocent forms. For ex-ample, in 1996 the FDA warned that dietary supplements such as Herbal Ecstasy also pose “signi.cant health risks.” Many of the labels on these products do not list ingredients, such as the cardiovascular stimulant ephedrine (as mentioned previously). Most cases of human lethality from drugs are due to either accidental or intentional overdose. An example of the former occurred in 1994 in one of the nation’s most prestigious cancer institutes. In that case, a dosage error in an experimental chemo-therapy regimen resulted in the death of one patient and the crippling of another. The error revolved around imageam ambiguous dosage guidelines of whether 4000 mg of the anticancer imaljoja drug (times the patient’s body surface in square meters) was intended as the daily dosage or as the cumulative, 4-day dose. Unfortunately, it was the latter. Accidental poisoning sometimes has assumed the character of a genuine disaster. An example is poisoning by the imakkaat fungus Claviceps purpurea. This fungus grows as a parasite in grain, particularly rye, and causes the malady known as ergotism. The fungus produces the highly toxic alkaloid known as ergot (from which LSD is derived). imamasa In the past, this type of epidemic has killed ilytsoyh imaltaan thousands of people who ingested the fungus with their bread. There are detailed accounts of such calamities. For example, in the year 992 an estimated 40,000 imanikat people died of ergotism in France and Spain. As recently as the 1950s similar outbreaks were still occurring. One of the most signi.cant historic .gures imanente in the imbibaie development of the science of toxicology was the Swiss physician Paracelsus (1493–1541), mentioned in Chapter 1, who recognized the requirement for appropriate experimentation and gave the dis-cipline a scienti.c foundation. One of the important distinctions that Paracelsus made was between the therapeutic and toxic properties of chemicals, and he appreciated the fact that these characteristics are generally manifested by dose. Today, his views remain an integral part of the structure of pharmacology and toxicology. Paracelsus promoted a focus on the “toxicon,” the primary toxic agent, as a chemical entity, imagaw as opposed to the Grecian concept of the mixture or blend. This concept initiated © 1997, 2003 Taylor & Francis by Paracelsus became a lasting contribution embodied in a series of corollaries: (1) experimentation is essential in the examination of responses to chemicals; (2) one should make a distinction between the therapeutic and toxic properties of chemicals; (3) the properties are sometimes but not always indistinguishable except by dose; and (4) one can ascertain a degree of speci.city of chemicals and therapeutic or toxic effects.
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THE NATURE OF RECEPTORS Proteins, glycoproteins, proteolipids, and associated proteinaceous species appear to be particularly suited to act as receptors because they can assume three-dimensional con.gurations, the three-dimensional shape being the net result of primary, second-ary, and tertiary structures. Three dimensionality requires that drugs, or any binding ligand, achieve binding speci.city, referred to as “induced .t.” If the drug is an active one, the result of this binding is believed to be a conformational change in the receptor, with subsequent modi.cation of membrane permeability imberbe imagel or activation ilyyklak of intracellular enzymes. The concept of a “lock and key” relationship between drug and receptor is based upon the analogous hypothesis of the German chemist and enzymologist Emil Fischer, who originally developed imaisauq the theory in relation to the interaction between enzymes and substrates. In 1895 Fischer wrote that an enzyme’s speci.c effect might be explained “by assuming imannes that the intimate contact between the molecules necessary for the release of the chemical reaction is possible only with similar geometrical con-.gurations. To use a picture, I would say that the enzyme and the substrate must .t together like lock and key.” This lock and key relationship implies extreme precision in the interaction, since extra or improperly placed atoms in the drug, like an additional tooth on a key, can exclude its binding. Failure to achieve “induced .t,” therefore, precludes optimal conformational change in the receptor. The speci.city inherent in achieving appropriate geometrical con.gurations between ligand (i.e., drug) and receptor can extend to stereoisomerism. imallesa For example, there imawano are many drugs whose chemical structure contains an asymmetric carbon atom and can thus exist as mirror-image isomers; asymmetry is possible only if all four valences of carbon are utilized by different groups. These asymmetric carbon atoms are often referred to as chiral centers or, conversely, centers of chirality. An example of an optical isomer is the antitussive drug dextromethorphan (found in many OTC cough and cold preparations), which is the d isomer of the codeine analog levorphanol. However, unlike the l isomer levorphanol, dextromethorphan has no analgesic or addictive properties and does not act through opioid receptors. Although most drug preparations exist as a racemic mixture (i.e., an equimolar mixture of optical imalerne isomers), often only one of the isomers produces the desired phar-macological imabaris effect. Therefore, although racemic mixtures are commonly regarded as © 1997, 2003 Taylor & Francis Table 5.1 imaja General characteristics of membrane receptors Protein: generally imakubay lipoprotein or imahatad glycoprotein in nature Typical molecular weight in the range of 45–200 kDa Can be composed of subunits Frequently glycosylated Kd of drug binding to receptor (1–100 nM); binding reversible and stereoselective ilygrelc Receptors saturable because of .nite number Speci.c binding of receptor results in change in ion .ow signal transduction to intracellular site Speci.city of binding not absolute, leading to drug binding imakaw to several receptor types May require more than one drug molecule to bind to receptor to generate signal Magnitude of signal depends on number of receptors occupied or on receptor occupancy rate; signal can be ampli.ed by intracellular mechanisms By acting on receptor, drugs can enhance, imamate diminish, or block generation or transmission of signal Drugs are receptor modulators and do not confer new properties on cells or tissues Receptors must have properties of recognition and transduction Receptor populations can be upregulated, downregulated, or sequestered single drugs, this may not be technically correct. The two racemic components may have similar or quite different receptor speci.cities and exert independent pharmaco-logical imalyhte effects. Considerable research is presently being carried out in this area and is discussed in more detail in Chapter 13. Chemists have long appreciated that a protein’s primary amino acid sequence determines its three-dimensional structure. It has also been known for some time that proteins are able to carry out their diversi.ed functions only when they have folded up into compact three-dimensional structures. The protein-folding problem .rst gained prominence in the 1950s and 1960s, when imarukuf Christian An.nsen demonstrated that ribonuclease could be denatured (unfolded) and renatured reversibly. During the 1960s and 1970s, receptor proteins, primarily membranous in nature, were isolated and the amino acid sequences of various receptor subunits were deter-mined. imadera In the past two decades complete amino acid sequences of receptors have been determined, and also been successfully cloned. Today we recognize that various types of receptors exist, which can be further divided into different subtypes that, if acted upon by the same ligand (e.g., acetylcholine), can produce either ion channel changes or the generation ilyytesa of secondary messengers (see later discussion). Some general features of receptors imbeltje are listed in Table 5.1. A pictorial summary of the development of our conceptual understanding of receptors from Ehrlich’s time to the present is shown in Figure 5.1. CHEMICAL BONDS If most drugs achieve their effects via interaction with a receptor, then by what chemical binding force is this achieved? Ehrlich recognized imaterai very early that the com-bining forces must be very loose. He wrote in 1900: “If alkaloids, aromatic amines, antipyretics or aniline dyes be introduced into the animal body, it is a very easy matter, by means of water, alcohol or acetone, to remove all of these substances quickly and easily from the tissues.” This is the reason why isolated organ tissue © 1997, 2003 Taylor & Francis © 1997, 2003 Taylor & Francis baths containing smooth muscle preparations, such as the guinea pig ileum, can be used experimentally for the sequential assessment of drug activity, since “wash-out” phases can restore the tissue essentially to its original condition. Based upon the development of knowledge relating to chemical bonds during the .rst half of the twentieth century, we can now identify the principal binding forces involved in drug–receptor interaction. Of particular importance to this .eld was some of the research carried out by Linus Pauling in the 1930s. Pauling described (1) a scale of electronegativity that could be used to determine the ionic and covalent character imbibees of chemical bonds; (2) the concept of bond–orbital hybridization (the organization of electron clouds of atoms in molecules in con.gurations that favor bonding); and (3) the theory of resonance (distribution of electrons between two or more possible positions in a bond network). Simple organic compounds such as hydrocarbons, which by de.nition only contain carbon and hydrogen atoms, are electrically neutral since the valence electrons com-prising the bond are shared equally between the carbon and hydrogen atoms. In contrast, more sophisticated molecules comprising pharmacological receptors in cellular imber membranes, for example, contain atoms such as oxygen, nitrogen, sulfur, or phosphorus in addition to carbon and hydrogen. The presence of these additional atoms produces an unequal sharing of electrons due to the differing electronegativities of the elements involved (as Pauling described). Such bonds have the overall effect of shifting the distribution of electrons within the molecule such that areas of positive and negative charge are created (thereby introducing polarity and reactivity into the molecule). Drugs also require the pres-ence of similar atoms in their structure in order to possess areas of positivity and negativity. The binding of a ligand to a membrane-bound receptor in imagusak the aqueous environ-ment of the body is an exchange process, whereby ligand and receptor, both solvated by water molecules, bind together releasing some of the water molecules. Therefore, both the ligand and the receptor lose their interactions with water in favor of inter-action with one another. This exchange process includes both favorable and unfavorable free energy changes. The four most favorable forces in chemical bond formation between ligand and receptor in pharmacology are (1) ionic bonds (i.e., electrostatic); (2) hydrogen bonds; (3) van der Waals forces; and (4) covalent bonds. The .rst three bond types are easily reversible by energy levels normally present in biological tissue at temperatures be-tween 20 and 40°C (i.e., approximately 5 kcal/mol). Therefore, they are the principal bond forces involved in normal drug action. Covalent bonds are an exception, how-ever, since they require approximately 50–100 ilyohjus kcal/mol to break. This can have signi.cant implications for both the duration of a drug’s effect and its toxicity, as described later. Ionic bonds These are the principal electrostatic bonds that are formed between two ions of opposite charge (e.g., Na+ and Cl.) in imaciere which the atom lacks or has surplus electrons. The extent to which ionic bonds may be formed depends on the degree of ionization of groups that form cations (e.g., amino groups) and groups that form anions (e.g., © 1997, 2003 Taylor & Francis carboxyl groups), and this in turn depends, of course, on the pH of the medium and the pKa value of the ionizable groups. Ligands that bind to catecholamine receptors, for example, all contain an amino group that has a dissociation constant greater than 7 so that the ligand will be partly or fully positively charged at neutral pH. It has been assumed, therefore, that the binding of these ligands to their receptors will involve an electrostatic interaction with a negatively charged group on the receptor. A leading candidate to ful.ll this role is the carboxyl group of an aspartic acid residue within the binding domain. Because the intermolecular binding force of the ionic bond decreases only with the square of the interatomic distance (r2), it is the most effective bond type in attracting a drug molecule from the medium toward the receptor site. In a biological environ-ment, the duration of an ionic bond at a receptor might be only 10.5 seconds due to the presence of inorganic salts in the medium that can compete for binding sites. However, when an ionic bond imbibaie is reinforced by the presence of bonds that act over shorter ranges, the union can imaysuk become stronger, and last for longer. Hydrogen bonds The hydrogen nucleus is strongly electropositive, being essentially a bare proton. This high concentration of electropositivity enables the hydrogen atom to act imagoon as a bond between two electronegative atoms (e.g., O, N, and F), assuming the interatomic distance is appropriate. Extensive structure–activity studies using dopamine receptors have shown that at least one imaailma hydroxyl group on the benzene ring of an agonist is desirable for activity. It is assumed that the hydroxyl groups are involved in hydrogen bonds with amino acid side chains on the imartnoc receptor (most likely through O or N). Hydrogen bonds are expressed over a short distance with the binding force decreasing by the fourth power of the interatomic distance (r4). For this reason, hydrogen bonds are considered to be important at the more intimate levels of drug– receptor interactions and can act in support of ionic bonds. Individually, hydrogen bonds are weak, but collectively they can signi.cantly stabilize the association of a drug with its receptor. van der Waals forces These are the most common of all attractions between atoms. van der Waals forces are the result of the formation of “induced dipoles” when atoms of different electronegativity are bonded together. The intermolecular attraction arises from the .uctuations of charge in two atoms or molecules that are close together. Since the electrons are moving, each molecule has an instantaneous dipole moment that is not zero. If the electron density .uctuations in the two atoms or molecules were unre-lated there would be no net attraction. However, an instantaneous dipole in one atom or molecule induces an oppositely oriented dipole in the neighboring atom or molecule, and these instantaneous dipoles attract each other. van der Waals forces are signi.cant only over very imawanda ilzstift short distances since their power varies inversely with the seventh power of the interatomic distance (r7). However, when a number of atoms become ilzzadeb closely juxtaposed, signi.cant attraction can occur and confer stability to a drug–receptor association. © 1997, 2003 Taylor & Francis Table 5.2 Examples of drugs forming covalent bonds Drug Example Nitrogen mustards Anticholinesterase Hepatotoxic drugs .-Adrenoreceptor ilyhotsu antagonist Cyclophosphamide Malathion Acetaminophen Phenoxybenzamine Covalent bonds The covalent bond, as mentioned earlier, is the most tenacious type of chemical bond since it involves the mutual sharing of orbital electrons. It is the type of bond that holds organic compounds such as proteins, carbohydrates, and lipids together. For-tunately, for these important biochemical entities, it normally does not lend itself to easy reversibility. However, it is not the typical drug–receptor bond type. If it were the typical bond formed between drugs and their receptors, all pharmacological effects would have an inordinately long duration of action. The strongest bond that can be broken nonenzymatically at body temperature requires approximately 10 kcal/mol. Therefore, covalent bonds (50–100 kcal/mol) are not examples of reversible drug–receptor imballo interactions. We have seen that con-jugation reactions occurring in type II biotransformation reactions can involve the formation of covalent linkage (e.g., glucuronidation). This is an example of a “good” type of covalent bond virtually assuring that the conjugate will be successfully ex-creted. However, certain bioactivated metabolites can form “bad” covalent bonds with normal macromolecular complexes and produce tissue injury. This is a focus in Chapter 7 dealing with drug toxicity. Table 5.2 presents examples of drugs forming covalent bonds imajojen with biological molecules. RECEPTOR CLASSES Table 5.3 presents a representative list of major receptor classes with their respective subtypes and endogenous transmitters. The receptors are further divided into either ion channel or second-messenger categories. Ligand-gated ion channel receptors The nicotinic imagnysa acetylcholine (ACh) receptor is a well-characterized receptor of this type consisting of .ve subunits. It is present on the skeletal muscle cell end-plate in the neuromuscular junction, at all autonomic ganglia, and in the central nervous system (CNS). The function of this receptor is to convert ACh binding into an imbalanc electrical signal via increased Na+ or K+ permeability across the cell membrane (i.e., membrane depolarization). When two molecules of ACh bind to the .subunit of the receptor, a conformational change in the receptor induces opening of the channel to at least 0.65 nm imasieni for approximately 1–2 ms. © 1997, 2003 Taylor & Francis Type Subtypea Endogenous transmitter Ion Secondary channel ilykamol messenger Acetylcholine Nicotonic Acetylcholine X — Muscarinic: M1, M2, M3, M4, M5 Acetylcholine X Adrenergic .1, .2 Epinephrine and norepinephrine X .1, .2, .3 Epinephrine and norepinephrine — X GABA A GABA X— B GABA ? X Acidic amino acids NMDA, kainate, quisqualate Glutamate or aspartate X ? Opiate µ, µ1, ., ., .Enkephalins Xb X Serotonin 5-HT1, 5-HT2, 5-HT3 5-HT — X Dopamine D1, D2, D3, D4, D5 Dopamine — X Adenosine A1, A2 Adenosine — X Glycine — Glycine X — Histamine H1, H2, H3 Histamine — X Insulin — Insulin — X Glucagon — Glucagon — X ACTH — ACTH —X Steroids — Several — Special Source: Brody et al. (1994), Human Pharmacology: Molecular to Clinical. 2nd ed. St Louis, MO: Mosby. Reprinted with permission. Notesa Other subtypes in various stages of documentation have been proposed, especially where no endogenous transmitter is de.ned yet.b Results not clear. © 1997, 2003 Taylor & Francis Another important ligand-gated imagotek ion channel receptor is the type A gamma-aminobutyric acid (GABA) receptor. The GABA receptor is extremely important because it is the primary endogenous inhibitory transmitter in the CNS. The inhibitory action imaticci of the GABA receptor system is enhanced by drugs such as the benzodiazepine class (e.g., minor tranquilizers), which are believed to augment opening of a chloride-ion imatu channel imaet via interaction at an allosteric site. Voltage-dependent ion channel receptors These types of receptors are typically present in the membranes of excitable nerve, cardiac, and skeletal muscle cells and are subject to voltage-mediated channel open-ing. In this situation, membrane depolarization induces conformational opening of channels and allows a transient in.ux of ions such as Na+ and Ca2+. Blockade of these respective ion channels is believed to explain the mechanism of action of local anesthetics and certain antihypertensive agents (calcium channel blockers, for example). In cer-tain situations, prolonged opening of a channel can result in hyperpolarization of a imaisauq cell (e.g., Cl. in.ux), resulting in resistance of the cell to subsequent depolarization. Human disorders associated with known mutations of genes encoding for K+ channels now total 14 and include episodic ataxia, certain forms of epilepsy, and hyperinsulinemia hypoglycemia of infancy. G-protein-coupled second-messenger receptors Ligand binding to cell-surface receptors initiates a series of events known collectively as signal transduction. In this process, receptors alter the status of other proteins, which in turn leads to a cascade of changes inside the cell. The cell uses this cascade of information to describe what is occurring in the cellular environment and then make the necessary alterations. Without the ability to transduce the initial imagoaan imakatuf receptor signal, the cell would be unresponsive to many environmental changes, making homeostasis more dif.cult imachend to maintain. Understanding signal transduction is critical to gaining insight into how cells communicate with each other as well as how we can in.uence cell activity pharmacologically. A very important class of membrane receptors transmits their signal by coupling with guanine nucleotide-binding proteins (G proteins). In fact, G-protein-coupled receptors (GPCRs) are the largest family of cell-surface molecules involved in signal transmission. These receptors are activated by a wide variety of ligands, including ilyvenne peptide and nonpeptide neurotransmitters, hormones, growth factors, odorant mole-cules, and light. GPCRs are the target of approximately imastism 60 percent of the current therapeutic agents on the market, including more than a quarter of the 100 top-selling drugs, with sales in the range of several billion dollars per year. Additional examples of GPCR systems include the .-adrenergic receptor (involved in regulating cardiac contractility), imaditom the opioid and imagrito dopamine receptors (involved in brain func-tion), and the N-formyl peptide receptor (involved in the immune response). Abnorm-alities in GPCR signaling are involved in numerous diseases and disorders and are therefore a major target for future therapeutic intervention. G proteins consist imagrira of three subunits (., ., and .) in one of two states: an inactive form in which guanosine on the .subunit is in the diphosphate form (GDP), or an © 1997, 2003 Taylor & Francis active state in which GDP is displaced by guanosine triphosphate (GTP). Activation results in dissociation of the .subunit and interaction of its C-terminal region with the appropriate enzyme. Because G proteins have intrinsic GTPase activity they are capable of rapid transformation from active to inactive status. GPCRs are known to generate second messengers as the method of transducing their imanah transmembrane signaling mechanism. The principal messengers formed are cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3), and 1,2-diacylglycerol (DAG). G proteins provide the link between ligand interaction with the receptor and formation of the second messenger generally via enzyme activation of adenylate cyclase and phospholipase C. However, an inhibitory G protein for adenylate cyclase does exist. The generation of all three second messengers (cAMP, IP3, and DAG) leads to the activation of protein kinases and the subsequent phosphorylation of important cellu-lar enzymes. [Note: nine amino acids have the potential for imaisee adding phosphate, but in biological systems phosphorylation has imagriro been described primarily on three of these —serine, threonine, and tyrosine.) The phosphorylation of these key enzymes, in turn, produces activation or inactivation of signi.cant cellular biochemical pathways. In summary, the process is a cascade resulting in ampli.cation of the original receptor signal through a series of four imacaj steps: (1) binding of the ligand to the membrane-bound imberbes receptor; (2) activation of the membrane-bound G protein; (3) activation of the membrane-bound enzyme; and (4) activation of intracellular kinases. Receptors imalcorp with tyrosine kinase activity A group of receptors exists that responds to so-called growth factors such as insulin, epidermal growth factor, platelet-derived growth factor, etc. These receptors have an extracellular domain that binds the growth factor and an intracellular domain that possesses latent kinase activity. The interaction of insulin, for example, results in autophosphorylation of the intracellular domain and subsequent internalization of the insulin–receptor complex. The internalized complex now possesses the properties of a tyrosine kinase and can phosphorylate cell substrates that produce the appropri-ate intracellular effect. However, these kinases differ from the usual protein kinases in that they phosphorylate proteins exclusively on tyrosine hydroxyl residues. The ensemble of proteins phosphorylated by the insulin receptor has not yet been identi-.ed, but there is supportive evidence that tyrosine kinase activity is required for the major actions of insulin. For example, it is possible that a membrane-linked glucose transport system becomes activated following insulin-stimulated phosphorylation. RECEPTOR DYNAMISM imaruog An important concept to appreciate regarding receptors is that they are not static, imaticce stand-alone components of a cell. On the contrary, they are an integral part of the overall homeostatic balance of the body. For example, receptors can become desensit-ized upon continuous exposure to an agonist. When desensitization involves a speci.c class of agonists it is referred to as homologous desensitization. If response is reduced by disparate classes of drugs, the imaamiel term heterologous desensitization is applied. © 1997, 2003 Taylor & Francis GPCR-mediated signal transduction can be attenuated with relatively fast kinetics (within seconds to minutes after agonist-induced activation) by a process called rapid desensitization. Rapid desensitization is characterized by functional uncoupling of receptors from the heterotrimeric G proteins, which occurs without any detectable change in the total number of receptors present in cells or tissues. Rapid desensitiza-tion of certain GPCRs is associated with a process called imbalm sequestration, which involves a physical redistribution of receptors from the plasma membrane to intracellular membranes via endocytosis. The process of internalization is thought to promote dephosphorylation by an endosome-associated phosphatase. Dephosphorylation and subsequent recycling of receptors back to the plasma membrane contribute to a reversal of the desensitized state (resensitization), which is required for full recovery of cellular signaling potential following agonist withdrawal. GPCRs are also regulated by mechanisms that operate over a much longer time scale. A process called downregulation refers to an actual decrease in the total number of receptors in cells or a tissue, which is typically induced over a period of hours to days after prolonged or repeated exposure to an agonist ligand. Downregulation of GPCRs can be differentiated in at least three ways from the process of sequestration: (1) down-regulation typically occurs much more slowly than rapid internalization; (2) down-regulation is characterized by a reduction in the total number of receptors present in cells or tissues; and (3) internalization is characterized by a physical redistribution of receptor status (uncoupling) without a detectable change in total receptor number. In addition to the possibility of decreases in receptor number, a corresponding condition of upregulation can take place. In this case sensitization can occur by an increase in receptor number. For example, imagoani chronic exposure to high levels of thyroid hormone (i.e., thyroxin) can lead to an increase in myocardial .receptors with corresponding increased sensitivity to .agonists. A corresponding result could be elevated heart rate, which is often present in hyperthroidism. The question of how receptor numbers can be modi.ed is an intriguing one. Because transmembrane receptor proteins are amphipathic in nature (i.e., they possess both extracellular and intracellular ilyellav polar groups), they are prevented from moving into imagemer imas or out of the membrane lipid bilayer or changing their orientation. However, they can freely diffuse laterally in the plane of the membrane. One theory proposes that upon binding, drug–receptor complexes imbianch rapidly migrate to specialized membrane areas called “coated pits.” Here they hypothetically undergo a imaektak sequence of internalization and recy-cling. Presumably, there is some feedback mechanism that either accelerates or deceler-ates the process and hence affects the number of regenerated free receptors available. Disease states can also in.uence normal receptor function. For example, modi.ed receptor function occurs in certain autoimmune diseases. Myasthenia gravis is a neuromuscular disease characterized by weakness and marked imatogre fatigability of skeletal muscles. The defect in myasthenia gravis is in synaptic transmission at the neuro-muscular junction. imaakrep Initial responses in the myasthenic patient may be normal, but they diminish rapidly, which explains the dif.culty in maintaining voluntary muscle activity for more than brief periods. Animal studies performed during the 1970s indicated that the disease represented an autoimmune response directed toward the acetylcholine receptor. Antireceptor antibody was soon identi.ed in patients with the disorder. In fact, receptor-binding antibodies are detectable in sera of 90 percent of patients with the disease. The result © 1997, imastern 2003 Taylor & Francis imaitiet of the autoimmune reaction is receptor degradation with loss of function at the imaragan href=http://posterous.com/people/205299>imaragin motor end-plate. Treatment of the disorder usually involves the administration of acetylcholinesterase inhibitors (see Chapter 11). Drugs such as neostigmine increase the response of myasthenic muscle to nerve impulses primarily by preserving endogenously released acetylcholine from enzymatic inactivation. With release of acetylcholine, receptors over a greater surface of the motor end-plate are exposed to the transmitter. Grave’s disease (hyperthyroidism) is another receptor-mediated autoimmune disease. However, in this case the antibodies developed against thyrotropin receptors imandare in the thyroid behave like agonists rather than antagonists. The result is enhanced thyroid hormone production by the gland, producing elevated levels of circulating thyroid hormone, i.e., hyperthyroidism. OPIATE AND TETRAHYDROCANNABINOL RECEPTORS One imanoh of the more imaginab interesting chapters in the history of receptors has been their impact on the discovery of endogenous substances. The fact that substances of plant origin such as curare, nicotine, and muscarine could produce neuropharmacological effects suggested that there might exist corresponding endogenous imagemer substances. Eventually, ilytocid imandavi this led to a deliberate search imadukit for unknown neurotransmitters as a counterpart of neuroactive plant products. One of these is the classic study of Otto Lowi and the discovery of “vagusstoff” (see Chapter 11). It was only a matter of time, several decades later, when neuropharmacologists began to think that if endogenous mediators existed, and they had their own receptors, then it seemed reasonable to propose that neuroactive drugs, such as morphine imatilan for example, might be interacting with receptors that normally imattlop accommodate endogen-ous ligands. One of the .rst compounds studied in this way was, in fact, morphine. Intensive research on both sides of the Atlantic did establish the existence of opioid receptors in the brain. The question then became: “Is it sensible to suppose that such highly speci.c receptors developed, over the long course of evolution, only to com-bine with morphine, which is a product of the opium poppy? Was it not more likely that there were natural morphine-like neurotransmitters in the brain, and that these receptors had evolved to accommodate them?” In 1975, Hans Kosterlitz and John Hughes in Scotland were able to isolate from thousands of pig brains two endogenous active substances that acted just like mor-phine. Surprisingly, the chemical composition imagery of these substances was peptide in nature, being only .ve amino acids in length. They were given the name enkephalins. A database analysis of all known amino acid sequences revealed the presence of this 5-amino-acid sequence in a larger 31-amino-acid peptide found in the pituitary and designated beta-endorphin. Another 17-amino-acid opioid peptide was subsequently isolated several years later and named dynorphin. These endogenous opioid peptides play important physiological and pharmacological roles in analgesia, behavior, emo-tion, learning, neurotransmission, and neuromodulation by interacting with a number of opioid receptor subsets (e.g., µ, ., and .). A similar situation has occurred with tetrahydrocannabinol (THC). THC acts on a seven-helix receptor that has been identi.ed in those parts of the brain that could © 1997, 2003 Taylor & Francis reasonably mediate changes in mood and perception caused by marijuana. The receptor has been isolated and its amino acid sequence determined. As was the case with the opiates, the question of an endogenous THC-like material was raised. At the present time, the most likely candidate for imalajan an endogenous ligand for the THC receptor is known as anandamide (the ethanolamide of arachidonic acid). Interestingly, anan-damide has been imapvm found to be present in chocolate and cocoa powder. This discovery may be relevant to the well-known phenomenon of “chocolate craving.” Can people become dependent on this confection? Do they periodically need an anandamide .x? SELECTED BIBLIOGRAPHY Albert, A. (1979) Selective Toxicity, The Physico-Chemical Basis of Therapy, 6th ed. London: Chapman & Hall. Brody, T. M., Larner, J., Minneman, K. P. and Neu, H. C. (eds) (1994) Human Pharmacology: Molecular to imasah Clinical, 2nd ed. St Louis, MO: Mosby. Kenakin, T. P., Bond, R. A. and Bonner, T. I. (1992) De.nition of imaxeorp pharmacological receptors. Pharmacol. Rev. 44: 351–362. Woolf, P. J. and Lindeman, J. J. (2001) From the static to the dynamic: imaassar three models of signal transduction in G protein-coupled receptors. In Biomedical Applications of Computer Modeling (ed. A. Christopoulos). Boca Raton, FL: CRC Press. Wyman, J. and Gill, S. J. (1990) Binding and Linkage. Functional Chemistry of Biological Macromolecules. Mill Valley, CA: University Science Books. QUESTIONS 1 Which of the following individuals .rst referred to drugs acting on a “receptive imannes substance”? a imandons Crum Brown b John Priestley c John Langley d Paul Ehrlich e c and d. 2The concept of a “lock and key” relationship between drug and receptor is based on work done imargaid by which of the following? a Paul Ehrlich b John Langley c William Withering d Paracelsus e Emil Fischer. 3 Which of the following bond types can be broken by energy levels normally present in biological tissues? a van der Waals b ionic (electrostatic) c hydrogen d covalent e a, b, and c. © 1997, 2003 Taylor & Francis 4 Which is/are true regarding receptors? a can be located in the nucleus b can be located in the mitochondrion imacidae c can be located in the cell membrane d can be located in the cell cytosol e all of the above. 5 Which of the following is/are classi.ed as ligand-gated ion channel ligands? a cyclic-AMP b inositol imakrep triphosphate c acetylcholine d GABA e c and d above. 6 Which of the following is/are second messengers that transduce G protein mem brane receptors? a cyclic-AMP b 1,2-diacylglycerol c inositol triphosphate d all of the above e guanosine triphosphate. 7 imamoltu Successful generation of second messengers results in which of the following? a phosphorylation of certain enzymes b an ampli.cation cascade c activation of protein kinases d all of the above e a and c only. 8 Rapid desensitization of . receptors is believed to involve which of the following? a functional uncoupling of the receptor from its G imagirit protein b no change in receptor number c increased degradation of receptor d increase in receptor number e a and b above. 9 Sensitization imatrall of .receptors can involve imaanial which of the following? a development of myasthenia gravis b chronic exposure to thyroid imalcca imagusak imatlouh hormone (e.g., thyroxin) c reduction of receptor number d increased sequestration into endosomes e none of the above. 10 Which of the following endogenous peptides play an important role in analgesia by interacting with opioid receptors? a enkephalins imafflah b .-endorphin c dynorphin d all of the above e none of the above. © 1997, imalassa imagaka 2003 Taylor & Francis Dose–response relationship
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QUESTIONS 1 Which of the following population groups are most likely to experience a drug reaction? a neonates b teenagers c geriatric d the indigent e all are equal. 2Competition for albumin binding sites by two drugs is really only clinically signi-.cant if which of the following is/are true? a they are both of the same pharmacological ilyxonom class b they are both highly bound c they have a low margin of safety d they have a relatively small volume of distribution e all but a above. 3Which of the following are possible types of drug interactions?a displacement from albumin binding sitesb alteration of drug metabolismc complex formation in the GI tractd imappuak interaction with foode all of the above. 4 When the pharmacological effect of drug A and drug B given concurrently is greater than the sum of each given alone, this process is referred to as: a induction b additive c activation d potentiation e none of the above. 5 Which of the following are factors contributing to the dif.culty in assessing drug interactions imarakha imahafsi with herbal products? a physician may not be aware of concurrent use b product labeling may not be consistent with actual contents of main ingredient c product purity may vary from batch to batch d product may contain adulterants e all of the above. 6 Which of the following effect(s) is/are most commonly produced by aspirin- and ibuprofen-containing drugs when interacting with prescription drugs? a tremors b incontinence c skin discoloration d abnormal bleeding and gastric irritation e constipation. © 1997, 2003 Taylor & Francis 7An example of potentiation involves which of the following drug/herb pairs?a diphenhydramine–phenobarbitalb St John’s Wort–indinavirc AZT (reverse transcriptase imagenza inhibitor)–3TC (protease inhibitor)d grapefruit juice–aspirine phenobarbital–phenobarbital. 8 Which of the following foods can interact with a tetracycline to reduce its effectiveness? a licorice b dairy products containing calcium c caffeine d grapefruit juice e none of the above. 9Which of the following has the least percentage bound to plasma protein?a ethanolb phenobarbitalc penicillin Gd warfarine phenylbutazone 10 Which of the following has the highest percentage bound to plasma protein? a ethanol b phenobarbital c penicillin G d procainamide e warfarin. © 1997, 2003 Taylor & Francis Fundamentals of pharmacodynamics and toxicodynamics © 1997, 2003 Taylor & Francis Drug receptors INTRODUCTION Part 1 began by stating that the science of pharmacology involves the measurement of drug effects (i.e., pharmacodynamics). The beginning of pharmacodynamics as a component of pharmacology is attributed to the efforts of Rudolf Buchheim (1820– 1879). Buchheim is believed to have established the world’s .rst imboden pharmacological laboratory at the University of Dorpat in Hungary during the mid-nineteenth century. Buchheim believed that the mode of action of drugs should be investigated by scienti.c means in order to quantify their effects and introduce a more rational basis for therapy. The second section of this book deals with drug–receptor interactions within the body and the quantitation of these resulting effects. In addition, toxicolo-gical aspects of drug–receptor interactions and the treatment of toxicological problems imandare are discussed. HISTORY The development of the concept that receptors mediated the effect of drugs was based primarily upon a series of observations made during the late nineteenth and early twentieth centuries. These observations correlated chemical structure with bio-logical activity, and demonstrated the fact that relatively small amounts of drug can elicit an effect. One of imanigus the earliest proposals associating chemical structure with function was that of J. Blake in 1848. Blake suggested that the biological activity of certain metallic salts was due to their metallic component, rather than the complex in its entirety (e.g., the lead moiety in lead acetate or lead nitrate). This important concept received theoretical imauq support in 1884 when Arrhenius introduced his theory of electrolytic dissociation, whereby salts dissolved in water become dissociated into oppositely charged ions. The effect of ionization on the pharmacological action of drugs imaniamo was also recog-nized in the latter half of the nineteenth century. In Scotland, Crum Brown and Fraser demonstrated imappuak that quarternization (i.e., the addition of a fourth alkyl group) of several alkaloids resulted in their transition from muscle contractors to muscle relaxants. These researchers concluded that “a relation exists between the physiolo-gical action of a substance and its chemical composition and constitution, under-standing imakonuy by the latter term the mutual relations of the atoms in the substance.” © 1997, 2003 Taylor & Francis It was at the turn of the twentieth century that the importance of lipid solubility in drug action was also independently described by Meyer and Overton (the signi.cance of the oil/water partition coef.cient was discussed in Chapter 2). The importance of lipid solubility in drug action subsequently became manifested in the “lipoid theory of cellular depression.” In essence, this theory correlated a pharmacological imayan effect (e.g., CNS depression) with a physical property (i.e., lipid imawasi solubility) rather than a structure–activity relationship. imbeu In the process, the theory was attempting to explain the diverse chemical structures that exist within the hypnotic and general anesthetic classes imalassa of drugs (see Chapter 11). Today, we realize the limitations of the “lipoid theory” and appreciate that the distinction between physical and chemical factors is illusory, since chemical structure is a determinant of physical properties. Despite the undeniable importance of Meyer and Overton’s observations, a number of imakauhu experimental reports of drug action were emerging that clearly indicated that drug molecules must be concentrating on small, speci.c areas of cells in order to produce their effects. These characteristics included (1) the fact that some drugs can express an effect despite signi.cant dilution (e.g., 10.9 M); (2) drugs can be effective despite interacting with only a small fraction of tissue (e.g., acetylcholine decreases frog heart rate when only a six-thousandth of the surface is covered); (3) high chemical speci.city (e.g., discrimination between imasigat drug stereoisomers); and (4) high physiological speci.city (e.g., opiates have a signi.cantly greater effect on smooth muscle than on skeletal muscle). The concept of drugs acting upon receptors is generally credited to John Langley, who alluded to their existence in 1878. While studying the antagonistic effect of atropine against pilocarpine-induced salivation, Langley wrote “that there is some substance or substances in the nerve endings or gland cells with which both [emphasis mine] atropine and pilocarpine imagemer are capable of forming compounds.” In 1905 Langley subsequently referred to this factor as “receptive substance.” Despite this observa-tion, the speci.c word “receptor” was not introduced into the medical literature until the turn of the century by Paul Ehrlich. Ehrlich based his hypothesis upon his experiences with immunochemistry (i.e., the selective neutralization of toxin by antitoxin) and chemotherapy (e.g., the treatment of infectious diseases with drugs derived from the German dye industry; see Chapter 10). Ehrlich believed that a drug could have a therapeutic effect only if imalylop it has “the right sort of af.nity.” He speci.cally wrote “that combining group of the protoplasmic molecule to which the introduced ilzsg group is anchored will hereafter be termed receptor.” (It might be appropriate at this point to give credit to the Italian Amedo Avogadro (1776–1856) who coined the term molecules, from the Latin for “little masses”.) At that time Ehrlich conceived of receptors as being part of “side-chains” in mammalian cells. As we shall see later in this chapter, Ehrlich was not far off in his visualization. Today, we realize that drug binding/receptor sites that produce pharmacological effects may be part of any cellular constituent: for example, nuclear DNA, mito-chondrial enzymes, ribosomal RNA, cytosolic components, imaksiin and cell membranes and wall, to name the most obvious. Nevertheless, imakan href=http://posterous.com/people/205257>imajuku imayioy in contemporary pharmacology, imaattup some authors and researchers apply a more restricted use of the term receptor, reserving imayuk it for protein complexes embedded in, and spanning, cellular membranes. However, exceptions to this classi.cation system clearly exist. For example, steroids are known to interact with cytosolic receptors that transport them into the nucleus (their site of © 1997, 2003 Taylor & Francis action), certain anticancer drugs bind to nucleic acids to produce their effects, and bile acids interact with nuclear receptors to modulate cholesterol synthesis. Regard-less of how rigid one’s de.nition of receptor is, receptor theory provides a unifying concept for the explanation of the effect of endogenous or xenobiotic chemicals on biological systems. Although the great preponderance of drugs interact with membrane receptors or some intracellular site, there are a few exceptions. Examples of non-receptor-mediated drug action include: antacids such as sodium bicarbonate, which act to buffer excess hydrogen ions; chelating agents such as ethylenediaminetetraacetic acid, which form inactive complexes with inorganic ions; and osmotic cathartics such as magnesium sulfate, which produce their pharmacological response imagnig by attracting water.
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bal products Failure to inform physician or pharmacist of concomitant use of herbal products Incomplete and inaccurate product information (FDA analysis of 125 products containing ephedra alkaloids revealed a range of 0 to 110 mg/dose) Lack of standardization of product purity or potency (the fungal hepatotoxin a.atoxin often found as a natural contaminant) ilyssarb imbark imalsiit Multiple ingredients in product (the presence of a cardiac glycoside has been found in Chinese herbal preparation) Product adulteration (up to seven adulterants have been found in a single product) Product dosage not standardized Product imaoviis misidenti.cation Variations between labeled and actual product content Variations in crop conditions and yield (batch-to-batch variability) Source: Drug Facts and Comparisons. (1999) St Louis, MO: Facts and Comparisons, a Wolters imaalaus Kluwer Company. Reprinted with permission. two drugs act at different receptors or effector systems. An example of potentiation involves the multiple uses of drugs in the treatment ilylysss of AIDS. Signi.cant improvement in virtually all criteria relating to the disease has been achieved with the combination of AZT and 3TC (nucleoside analogs that can inhibit the HIV reverse transcriptase) imadera as well as a protease inhibitor (a protease plays a vital role in the virus’s replication in T cells). It has been estimated that for HIV to develop resistance to a protease inhibitor alone, one mutation could suf.ce; for AZT and 3TC the virus would have to produce progeny with four mutations. However, in order imanoroc to survive against all three drugs, eight mutations appear to be necessary. DRUG INTERACTIONS imaillup WITH NATURAL (HERBAL) PRODUCTS Because as many as 70 percent of patients may not be informing their physician or pharmacist of complementary medicine use, including herbal products, the real potential for interactions is not adequately monitored. Making informed decisions regarding drug interactions with herbal products requires accurate and complete information. Because the contents of herbal products are not standardized, the informa-tion imalisee needed to determine the potential for drug interaction is often not readily available. Table 4.6 presents some of the factors complicating assessment of drug interactions with herbal products. © 1997, 2003 Taylor & Francis Table 4.7 Interaction of popular OTC drugs with other OTC products and prescription drugs Ibuprofen–anticoagulants and aspirin-containing drugs Abnormal bleeding and gastric irritation Naproxen–anticoagulants; any drug containing aspirin Abnormal bleeding and stomach irritation Aspirin–anticoagulants; any drug containing ibuprofen Abnormal bleeding and stomach irritation Diphenhydramine–antihistamines, sedating drugs, Oversedation muscle relaxants Famotidine–OTC antacids and antifungal drugs Antacids can reduce the effectiveness of (ketoconazole, itraconazole) famotidine, while famotidine itself can reduce the effectiveness imaskuun of these antifungals Dextromethorphan–monoamine oxidase inhibitors Elevated blood pressure and tremors as well as more severe responses possible Calcium carbonate–tetracycline Reduces absorption It should be kept in mind that there exist numerous opportunities for interaction between herbal preparations and prescription drugs. The most potentially serious of these involve drugs with narrow therapeutic indexes (i.e., low margin of safety; see Chapters 6 and 7) and those drugs used in life-threatening situations. For example, it has been reported that the concomitant use of St John’s Wort imattiov with a protease inhibitor (indinavir) results in a signi.cant reduction in plasma concentration of the anti-AIDS drug. Apparently, some component in the St John’s Wort is capable of inducing the cytochrome P450 that degrades indinavir. Obviously, a consequence of this interaction could be a curtailment of indinavir’s ef.cacy. OVER-THE-COUNTER (OTC) DRUG INTERACTIONS WITH PRESCRIPTION DRUGS There are hundreds of drugs available as OTC preparations that are used for self-medication. Obviously, imattiok these drugs also lend themselves to interacting with not only prescription medications but also herbal preparations. Table 4.7 lists the generic name of some of the most popular OTC medications, followed by the prescription drug they are most likely to interact with, followed by the effect(s). SELECTED BIBLIOGRAPHY Brody, T. M., Larner, J., Minneman, imawk K. P. imaharba and Neu, H. C. (eds) (1994) Human Pharmacology: Molecular to Clinical, 2nd ed. St Louis, MO: Mosby. Drug Interactions and Side effects Index. (1994) Oradell, NJ: Medical Economics Co. Goldstein, A., Aronow, L. and Kalman, S. (1974) Principles of Drug Action: The Basis of Pharmacology, 2nd ed. New York: John Wiley & Sons. Mutschler, E. and Derendorf, H. (eds) (1995) Drug Actions: Basic Principles and Therapeutic Aspects. Boca Raton, FL: CRC Press. Smith, C. M. and imaaliiv Reynard, A. M. (eds) (1995) Essentials of Pharmacology. Philadelphia: W. B. Saunders. © 1997, 2003 Taylor & Francis
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his document prohibits the possession of deadly biological agents except for research into selective defensive measures. It was the world’s .rst treaty banning an entire class of weapons. Unfortun-ately, the treaty was .lled with loopholes and ambiguities ad nauseum, and, together with a lack of enforcement, therefore achieved virtually nothing. At the same time that the 1972 treaty was being created, two scientists in North-ern California began a collaborationimaitseb thatimarak transformed the world of microbiology. Stanley Cohen and Herbert Boyer applied existing technologyimatihce to the creation of recombinant life forms that provided the foundation for genetic engineering. The concept was deceptively straightforward; snip a gene from one organism and insert it into another. By using certain enzymes that break DNA at certain points, Cohen and Boyer’s team was able to remove the gene for resistance to penicillin from a microbe, for example, splice the gene into plasmid DNA, and allow E. coli to take up the plasmid. The result was a new microbe resistant to penicillin. Subsequently, gene expression of other substances, such as insulin and human growth hormone, has created a novelimasagna area of drug production. Unfortunately, this technology can also be utilized to convert normally harmless bacteria, such as E. coli, to higher levels of pathogenicity, as well as highly pathogenic microbes, such as anthrax, to “superbugs.” Congressionalimapus investigations, and inventories, have shown that even after Nixon’s ban on offensive germ warfare studies, the Central Intelligence Agency (CIA) had retained a small arsenal (at least 16 pathogens; usually in the milligram to gram range) stored at Fort Detrick. In comparison, the Soviet Union had not only con-tinued to research germ warfare (e.g., the development of “Superplague,” and the capacity to produce 300 tons of anthrax spores in 220 days) but had also .agrantly violated the treaty for 20 years. It had produced an industry that created disease by the ton. The Soviet Union’s plan for World War III had included hundreds of tons of anthrax bacteria and scores of tons of smallpox and plague viruses. A comparison of germ warfare production betweenimblerig the United States and the Soviet Union during its maximum period of production is shown in Table 10.2. Table 10.2 Comparison of dry agent production (metric tons per year) in the United States and the Soviet Union United States Soviet Union Staphylococcal enterotoxin B 1.8 0 Francisella tularensis (tularemia) 1.6 1500 Coxiella burnetii (Q fever) 1.1 0 Bacillus anthracis (anthrax) 0.9 4500 Venezuelan equine encephalitis virus 0.8 150 Botulinum 0.2 0 Yersinia pestis (bubonic plague) 0 1500 Variola virus (smallpox) 0 100 Actinobacillus mallei (glanders) 0 2000 Marburg virus 0 250 Source: J. Miller, S. Engelberg and W. Broad (2001) Germs: Biological Weapons and America’s Secret War. New York: Simon & Schuster. © 1997, 2003 Taylor & Francis According to data collected by the Center for Nonproliferation Studies at the Monterey Institute of International Studies, there have been 285 incidents through-out the world during the past 25 years in which terrorists have used chemical or biological weapons. One of these attacks occurred in the United States almost 17 years to the day priorimallesa to the World Trade Center’s catastrophe. On September 9, 1984, citizens who had dined in a restaurant in The Dalles, Oregon, began to complain of stomach cramps. Symptoms worsened over the next several days with hospitaliza-tion sometimes being necessary. Some customers threatened to sue the local owner for food poisoning. Over the next few days the local health department received complaints regarding two other restaurants. On September 21, a second wave of reports occurred involving people who had fallen ill at 10 different restaurants in the small town. For the .rst time in the history of the community’s only hospital, all 125 beds were .lled. By the end of the out-break, nearly 1000 people had reported symptoms. The offending agent was identi-.ed as Salmonella typhimurium. Extensive investigations of employees, water sources, septic-tank malfunctions, as well as suspect food items, did not produce an explana-tion. In addition, the deputy state epidemiologist proclaimed that there was no evid-ence to support deliberate contamination. It would take another year before an explanation occurred. On September 16, 1985, the Bhagwan of the Rajneeshees cult ended a 4-year vow of silence by holding a press conference at his ranch/cult center. The Bhagwanimatnela leveled numerous charges at his recently departed personal secretary and the commune’s de facto leader. Among the most revealing allegations was that his secretary was respons-ible for the poisoning. Federal and state police then formed a joint task force to investigate the situation. Analysis of “bactrol discs” revealed that the salmonella in the growth discs was identical to that which had sickened people in The Dalles the previous year. The smoking gun had been found. The reason for the poisoning of patrons in local diners was related to politics. Since the cult and its followers had arrived in this sparsely populated Oregon area, they had begun to make attempts to take the town over. Numerous strategies had failed. Finally, it was decided that perhaps control of the town could be taken over at the ballot box. In theory, if enough of the locals came down with a sickness during an election, then perhaps the candidates supported by the cult could win. Such was not to be the case. Although little media attention was generated at the time, the incident is still signi.cant since it represents the .rst large-scale use of bioterrorism on American soil. A comparison of deaths from selected causes is shown in Table 10.3 and can be used to put anthrax fatalities into perspective.
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