COMMON PHARMACOLOGY
Common Pharmacology
Drug and Active Principle
Until the end of the 19th century, medicines were natural organic or inorganic products, mostly dried, but also fresh, plants or plant parts. These might contain substances possessing healing (therapeutic) properties or substances exerting a toxic effect. In order to secure a supply of medically useful products not merely at the time of harvest but year-round, plants were preserved by drying or soaking them in vegetable oils or alcohol. Drying the plant or a vegetable or animal product yielded a drug (from French “drogue” – dried herb). Colloquially, this term nowadays often refers to chemical substances with high potential for physical dependence and abuse. Used scientifically, this term implies nothing about the quality of action, if any. In its original, wider sense, drug could refer equally well to the dried leaves of peppermint, dried lime blossoms, dried flowers and leaves of the female cannabis plant (hashish, marijuana), or the dried milky exudate obtained by slashing the unripe seed capsules of Papaver somniferum (raw opium). Nowadays, the term is applied quite generally to a chemical substance that is used for pharmacotherapy. Soaking plants parts in alcohol (ethanol) creates a tincture. In this process, pharmacologically active constituents of the plant are extracted by the alcohol. Tinctures do not contain the complete spectrum of substances that exist in the plant or crude drug, only those that are soluble in alcohol. In the case of opium tincture, these ingredients are alkaloids (i.e., basic substances of plant origin) including: morphine, codeine, narcotine = noscapine, papaverine, narceine, and others. Using a natural product or extract to treat a disease thus usually entails the administration of a number of substances possibly possessing very different activities. Moreover, the dose of an individual constituent contained within a given amount of the natural product is subject to large variations, depending upon the product‘s geographical origin (biotope), time of harvesting, or conditions and length of storage. For the same reasons, the relative proportion of individual constituents may vary considerably. Starting with the extraction of morphine from opium in 1804 by F. W. Sertrner (1783–1841), the active principles of many other natural products were subsequently isolated in chemically pure form by pharmaceutical laboratories.
The aims of isolating active principles are:
1. Identification of the active ingredient( s).
2. Analysis of the biological effects (pharmacodynamics) of individual ingredients
and of their fate in the body (pharmacokinetics).
3. Ensuring a precise and constant dosage in the therapeutic use of chemically
pure constituents.
4. The possibility of chemical synthesis, which would afford independence from
limited natural supplies and create conditions for the analysis of structure-activity
relationships.
Finally, derivatives of the original constituent may be synthesized in an effort to optimize pharmacological properties. Thus, derivatives of the original constituent with improved therapeutic usefulness may be developed.
Drug Development
This process starts with the synthesis of novel chemical compounds. Substances with complex structures may be obtained from various sources, e.g., plants (cardiac glycosides), animal tissues (heparin), microbial cultures (penicillin G), or human cells (urokinase), or by means of gene technology (human insulin). As more insight is gained into structure- activity relationships, the search for new agents becomes more clearly focused.
Preclinical testing yields information on the biological effects of new substances. Initial screening may employ biochemical-pharmacological investigations or experiments on cell cultures, isolated cells, and isolated organs. Since these models invariably fall short of replicating complex biological processes in the intact organism, any potential drug must be tested in the whole animal. Only animal experiments can reveal whether the desired effects will actually occur at dosages that produce little or no toxicity. Toxicological investigations serve to evaluate the potential for: (1) toxicity associated with acute or chronic administration; (2) genetic damage (genotoxicity, mutagenicity); (3) production of tumors (onco- or carcinogenicity); and (4) causation of birth defects (teratogenicity). In animals, compounds under investigation also have to be studied with respect to their absorption, distribution, metabolism, and elimination (pharmacokinetics). Even at the level of preclinical testing, only a very small fraction of new compounds will prove potentially fit for use in humans. Pharmaceutical technology provides the methods for drug formulation.
Clinical testing starts with Phase I studies on healthy subjects and seeks to determine whether effects observed in animal experiments also occur in humans. Dose-response relationships are determined. In Phase II, potential drugs are first tested on selected patients for therapeutic efficacy in those disease states for which they are intended. Should a beneficial action be evident and the incidence of adverse effects be acceptably small, Phase III is entered, involving a larger group of patients in whom the new drug will be compared with standard treatments in terms of therapeutic outcome. As a form of human experimentation, these clinical trials are subject to review and approval by institutional ethics committees according to international codes of conduct (Declarations of Helsinki,
Dosage Forms for Oral, Ocular, and Nasal Applications
A medicinal agent becomes a medication only after formulation suitable for therapeutic use (i.e., in an appropriate dosage form). The dosage form takes into account the intended mode of use and also ensures ease of handling (e.g., stability, precision of dosing) by patients and physicians. Pharmaceutical technology is concerned with the design of suitable product formulations and quality control. Liquid preparations (A) may take the form of solutions, suspensions (a sol or mixture consisting of small water- insoluble solid drug particles dispersed in water), or emulsions (dispersion of minute droplets of a liquid agent or a drug solution in another fluid, e.g., oil in water). Since storage will cause sedimentation of suspensions and separation of emulsions, solutions are generally preferred. In the case of poorly watersoluble substances, solution is often ccomplished by adding ethanol (or other solvents); thus, there are both aqueous and alcoholic solutions.
These solutions are available to patients in specially designed drop bottles, enabling single doses to be measured exactly in terms of a defined number of drops, the size of which depends on the area of the drip opening at the bottle mouth and on the viscosity and surface tension of the solution. The advantage of a drop solution is that the dose, that is, the number of drops, can be precisely adjusted to the patient‘s need. Its disadvantage lies in the difficulty that some patients, disabled by disease or age, will experience in measuring a prescribed number of drops. When the drugs are dissolved in a larger volume — as in the case of syrups or mixtures — the single dose is measured with a measuring spoon. Dosing may also be done with the aid of a tablespoon or teaspoon (approx. 15 and 5 ml, respectively). However, due to the wide variation in the size of commercially available spoons, dosing will not be very precise. (Standardized medicinal teaspoons and tablespoons are available.) Eye drops and nose drops (A) are designed for application to the mucosal surfaces of the eye (conjunctival sac) and nasal cavity, respectively. In order to prolong contact time, nasal drops are formulated as solutions of increased viscosity. Solid dosage forms include tablets, coated tablets, and capsules (B).
Tablets have a disk-like shape, produced by mechanical compression of active substance, filler (e.g., lactose, calcium sulfate), binder, and auxiliary material (excipients). The filler provides bulk enough to make the tablet easy to handle and swallow. It is important to consider that the individual dose of many drugs lies in the range of a few milligrams or less. In order to convey the idea of a 10-mg weight, two squares are marked below, the paper mass of each weighing 10 mg. Disintegration of the tablet can be hastened by the use of dried starch, which swells on contact with water, or of NaHCO3, which releases CO2 gas on contact with gastric acid. Auxiliary materials are important with regard to tablet production, shelf life, palatability, and identifiability (color). Effervescent tablets (compressed effervescent powders) do not represent a solid dosage form, because they are dissolved in water immediately prior to ingestion and are, thus, actually, liquid preparations.
The coated tablet contains a drug within a core that is covered by a shell, e.g., a wax coating, that serves to: (1) protect perishable drugs from decomposing; (2) mask a disagreeable taste or odor; (3) facilitate passage on swallowing; or (4) permit color coding. Capsules usually consist of an oblong casing — generally made of gelatin
— that contains the drug in powder or granulated form. In the case of the matrix-type tablet, the drug is embedded in an inert meshwork from which it is released by diffusion upon being moistened. In contrast to solutions, which permit direct absorption of drug (A, track 3), the use of solid dosage forms initially requires tablets to break up and capsules to open (disintegration) before the drug can be dissolved (dissolution) and pass through the gastrointestinal mucosal lining (absorption). Because disintegration of the tablet and dissolution of the drug take time, absorption will occur mainly in the intestine (A, track 2). In the case of a solution, absorption starts in the stomach (A, track 3). For acid-labile drugs, a coating of wax or of a cellulose acetate polymer is used to prevent disintegration of solid dosage forms in the stomach. Accordingly, disintegration and dissolution will take place in the duodenum at normal speed (A, track 1) and drug liberation per se is not retarded. The liberation of drug, hence the site and time-course of absorption, are subject to modification by appropriate production methods for matrix-type tablets, coated tablets, and capsules. In the case of the matrix tablet, the drug is incorporated into a lattice from which it can be slowly leached out by gastrointestin fluids. As the matrix tablet undergoes enteral transit, drug liberation and absorption proceed en route (A, track 4). In the case of coated tablets, coat thickness can be designed such that release and absorption of drug occur either in the proximal (A, track 1) or distal (A, track 5) bowel. Thus, by matching dissolution time with small-bowel transit time, drug release can be timed to occur in the colon. Drug liberation and, hence, absorption can also be spread out when the drug is presented in the form of a granulate consisting of pellets coated with a
waxy film of graded thickness. Depending on film thickness, gradual dissolution occurs during enteral transit, releasing drug at variable rates for absorption. The principle illustrated for a capsule can also be applied to tablets. In this case, either drug pellets coated with films of various thicknesses are compressed into a tablet or the drug is incorporated into a matrix-type tablet. Contrary to timed-release capsules, slow-release tablets have the advantage of being dividable ad libitum; thus, fractions of the dose contained within the entire tablet may be administered. This kind of retarded drug release is employed when a rapid rise in blood level of drug is undesirable, or when absorption is being slowed in order to prolong the action of drugs that have a short sojourn in the body.
Dosage Forms for Parenteral (1), Pulmonary (2), Rectal or Vaginal (3), and Cutaneous Application Drugs need not always be administered orally (i.e., by swallowing), but may also be given parenterally. This route usually refers to an injection, although enteral absorption is also bypassed when drugs are inhaled or applied to the skin. For intravenous, intramuscular, or subcutaneous injections, drugs are often given as solutions and, less frequently, in crystalline suspension for intramuscular, subcutaneous, or intraarticular injection. An injectable solution must be free of infectious agents, pyrogens, or suspended matter. It should have the same osmotic pressure and pH as body fluids in order to avoid tissue damage at the site of injection. Solutions for injection are preserved in airtight glass or plastic sealed containers. From ampules for multiple or single use, the solution is aspirated via a needle into a syringe. The cartridge ampule is fitted into a special injector that enables its contents to be emptied via a needle. An infusion refers to a solution being administered over an extended period of time. Solutions for infusion must meet the same standards as solutions for injection. Drugs can be sprayed in aerosol form onto mucosal surfaces of body cavities accessible from the outside (e.g., the respiratory tract. An aerosol is a dispersion of liquid or solid particles in a gas, such as air. An aerosol results when a drug solution or micronized powder is reduced to a spray on being driven through the nozzle of a pressurized container. Mucosal application of drug via the rectal or vaginal route is achieved by means of suppositories and vaginal tablets, respectively. On rectal application, absorption into the systemic circulation may be intended. With vaginal tablets, the effect is generally confined to the site of application. Usually the drug is incorporated into a fat that solidifies at room temperature, but melts in the rectum or vagina. The resulting oily film spreads over the mucosa and enables the drug to pass into the mucosa. Powders, ointments, and pastes (p. 16) are applied to the skin surface. In many cases, these do not contain drugs but are used for skin protection or care. However, drugs may be added if a topical action on the outer skin or, more rarely, a systemic effect is intended. Transdermal drug delivery systems are pasted to the epidermis. They contain a reservoir from which drugs may diffuse and be absorbed through the skin. They offer the advantage that a drug depot is attached noninvasively to the body, enabling the drug to be administered in a manner similar to an infusion. Drugs amenable to this type of delivery must: (1) be capable of penetrating the cutaneous barrier; (2) be effective in very small doses (restricted capacity of reservoir); and (3) possess a wide therapeutic margin (dosage not adjustable).
Drug Administration by Inhalation
Inhalation in the form of an aerosol , a gas, or a mist permits drugs to be applied to the bronchial mucosa and, to a lesser extent, to the alveolar membranes. This route is chosen for drugs intended to affect bronchial smooth muscle or the consistency of bronchial mucus. Furthermore, gaseous or volatile agents can be administered by inhalation with the goal of alveolar absorption and systemic effects. Aerosols are formed when a drug solution or micronized powder is converted into a mist or dust, respectively. In conventional sprays (e.g., nebulizer), the air blast required for aerosol formation is generated by the stroke of a pump. Alternatively, the drug is delivered from a solution or powder packaged in a pressurized canister equipped with a valve through which a metered dose is discharged. During use, the inhaler (spray dispenser) is held directly in front of the mouth and actuated at the start of inspiration.
The effectiveness of delivery depends on the position of the device in front of the mouth, the size of aerosol particles, and the coordination between opening of the spray valve and inspiration. The size of aerosol particles determines the speed at which they are swept along by inhaled air, hence the depth of penetration into the respiratory tract. Particles > 100 µm in diameter are trapped in the oropharyngeal cavity; those having diameters between 10 and 60µm will be deposited on the epithelium of the bronchial tract. Particles < 2 µm in diameter can reach the alveoli, but they will be largely exhaled because of their
low tendency to impact on the alveolar epithelium. Drug deposited on the mucous lining of the bronchial epithelium is partly absorbed and partly transported with bronchial mucus towards the larynx. Bronchial mucus travels upwards due to the orally directed undulatory beat of the epithelial cilia. Physiologically, this mucociliary transport functions to remove inspired dust particles. Thus, only a portion of the drug aerosol (~ 10 %) gains access to the respiratory tract and just a fraction of this amount penetrates the mucosa, whereas the remainder of the aerosol undergoes mucociliary transport to the laryngopharynx and is swallowed.
From Application to Distribution in the Body As a rule, drugs reach their target organs via the blood. Therefore, they must first enter the blood, usually the venous limb of the circulation. There are several possible sites of entry. The drug may be injected or infused intravenously, in which case the drug is introduced directly into the bloodstream. In subcutaneous or intramuscular injection, the drug has to diffuse
from its site of application into the blood. Because these procedures entail injury to the outer skin, strict requirements must be met concerning technique. For that reason, the oral route (i.e., simple application by mouth) involving subsequent uptake of drug across the gastrointestinal mucosa into the blood is chosen much more frequently. The disadvantage of this route is that the drug must pass through the liver on its way into the general circulation. This fact assumes practical significance with any drug that may be rapidly transformed or possibly inactivated in the liver (first-pass hepatic elimination. Even with rectal administration, at least a fraction of the drug enters the general circulation via the portal vein, because only veins draining the short terminal segment of the rectum communicate directly with the inferior vena cava. Hepatic passage is circumvented when absorption occurs buccally or sublingually, because venous blood from the oral cavity drains directly into the superior vena cava. The same would apply to administration by inhalation. However, with this route, a local effect is usually intended; a systemic action is intended only in exceptional cases. Under certain conditions, drug can also be applied percutaneously in the form of a transdermal delivery system. In this case, drug is slowly released from the reservoir, and then penetrates the epidermis and subepidermal connective tissue where it enters blood capillaries. Only a very few drugs can be applied transdermally. The feasibility of this route is determined by both the physicochemical properties of the drug and the therapeutic requirements (acute vs. long-term effect). Speed of absorption is determined by the route and method of application. It is fastest with intravenous injection, less fast which intramuscular injection, and slowest with subcutaneous injection. When the drug is applied to the oral mucosa (buccal, sublingual route), plasma levels rise faster than with conven ional oral administration because the drug preparation is deposited at its actual site of absorption and very high concentrations in saliva occur upon the dissolution of a single dose. Thus, uptake across the oral epithelium is accelerated. The same does not hold true for poorly water-soluble or poorly absorbable drugs. Such agents should be given orally, because both the volume of fluid for dissolution and the absorbing surface are much larger in the small intestine than in the oral cavity.
Bioavailability is defined as the fraction of a given drug dose that reaches the circulation in unchanged form and becomes available for systemic distribution. The larger the presystemic elimination, the smaller is the bioavailability of an orally administered drug.
Potential Targets of Drug Action Drugs are designed to exert a selective influence on vital processes in order to alleviate or eliminate symptoms of disease. The smallest basic unit of an organism is the cell. The outer cell membrane, or plasmalemma, effectively demarcates the cell from its surroundings, thus permitting a large degree of internal autonomy.
Embedded in the plasmalemma are transport proteins that serve to mediate controlled metabolic exchange with the cellular environment. These include energy-consuming pumps (e.g., Na, K-ATPase,), carriers (e.g., for Na/glucose-cotransport), and ion channels e.g., for sodium or calcium (1). Functional coordination between single cells is a prerequisite for viability of the organism, hence also for the survival of individual cells. Cell functions are regulated by means of messenger substances for the transfer of information. Included among these are “transmitters” released from nerves, which the cell is able to recognize with the help of specialized membrane binding sites or receptors. Hormones secreted by endocrine glands into the blood, then into the extracellular fluid, represent another class of chemical signals. Finally, signalling substances can originate from neighboring cells, e.g., prostaglandins and cytokines. The effect of a drug frequently results from interference with cellular function. Receptors for the recognition of endogenous transmitters are obvious sites of drug action (receptor agonists and antagonists). Altered activity of transport systems affects cell function (e.g., cardiac glycosides, loop diuretics, calcium-antagonists). Drugs may also directly interfere with intracellular metabolic processes, for instance by inhibiting (phosphodiesterase inhibitors) or activating (organic nitrates, ) an enzyme (2). In contrast to drugs acting from the outside on cell membrane constituents, agents acting in the cell’s interior need to penetrate the cell membrane. The cell membrane basically consists of a hospholipid bilayer (80Е = 8 nm in thickness) in which are embedded proteins (integral membrane proteins, such as receptors and transport molecules). Phospholipid molecules contain two long-chain fatty acids in ester linkage with two of the three hydroxyl groups of glycerol. Bound to the third hydroxyl group is phosphoric acid, which, in turn, carries a further residue, e.g., choline, (phosphatidylcholine = lecithin), the amino acid serine (phosphatidylserine) or the cyclic polyhydric alcohol inositol (phosphatidylinositol). In terms of solubility, phospholipids are amphiphilic: the tail region containing the apolar fatty acid chains is lipophilic, the remainder – the polar head – is hydrophilic. By virtue of these properties, phospholipids aggregate spontaneously into a bilayer in an aqueous medium, their polar heads directed outwards into the aqueous medium, the fatty acid chains facing each other and projecting into the inside of the membrane (3). The hydrophobic interior of the phospholipid membrane constitutes a diffusion barrier virtually impermeable for charged particles. Apolar particles, however, penetrate the membrane easily. This is of major importance with respect to the absorption, distribution, and elimination of drugs.
External Barriers of the Body
Prior to its uptake into the blood (i.e., during absorption), a drug has to overcome barriers that demarcate the body from its surroundings, i.e., separate the internal milieu from the external milieu. These boundaries are formed by the skin and mucous membranes. When absorption takes place in the gut (enteral absorption), the intestinal
epithelium is the barrier. This singlelayered epithelium is made up of enterocytes and mucus-producing goblet cells. On their luminal side, these cells are joined together by zonulae occludentes (indicated by black dots in the inset, bottom left). A zonula occludens or tight junction is a region in which the phospholipid membranes of two cells establish close contact and become joined via integral membrane proteins (semicircular inset, left center). The region of fusion surrounds each cell like a ring, so that neighboring cells are welded together in a continuous belt. In this manner, an unbroken phospholipid layer is formed (yellow area in the schematic drawing, bottom left) and acts as a continuous barrier between the two spaces separated by the cell layer – in the case of the gut, the intestinal lumen (dark blue) and the interstitial space (light blue). The efficiency with which such a barrier restricts exchange of substances can be increased by arranging these occluding junctions in multiple arrays, as for instance in the endothelium of cerebral blood vessels. The connecting proteins (connexins) furthermore serve to restrict mixing of other functional membrane proteins (ion pumps, ion channels) that occupy specific areas of the cell membrane.
This phospholipid bilayer represents the intestinal mucosa-blood barrier that a drug must cross during its enteral absorption. Eligible drugs are those whose physicochemical properties allow permeation through the lipophilic membrane interior (yellow) or that are subject to a special carrier transport mechanism.
Absorption of such drugs proceeds rapidly, because the absorbing surface is greatly enlarged due to the formation of the epithelial brush border (submicroscopic foldings of the plasmalemma). The absorbability of a drug is characterized by the absorption quotient, that is, the amount absorbed divided by the amount in the gut available
for absorption. In the respiratory tract, cilia-bearing epithelial cells are also joined on the luminal side by zonulae occludentes, so that the bronchial space and the interstitium are separated by a continuous phospholipid barrier. With sublingual or buccal application, a drug encounters the non-keratinized, multilayered squamous pithelium of the oral mucosa. Here, the cells establish punctate contacts with each
other in the form of desmosomes (not shown); however, these do not seal the intercellular clefts. Instead, the cells have the property of sequestering phospholipid-
containing membrane fragments that assemble into layers within the extracellular space (semicircular inset, center right). In this manner, a continuous phospholipid barrier arises also inside squamous epithelia, although at an extracellular location, unlike that of intestinal epithelia. A similar barrier principle operates in the multilayered keratinized squamous epithelium of the outer skin. The presence of a continuous phospholipid layer means that squamous epithelia will permit passage
of lipophilic drugs only, i.e., agents capable of diffusing through phospholipid membranes, with the epithelial thickness determining the extent and speed
of absorption.
Membrane Permeation
An ability to penetrate lipid bilayers is a prerequisite for the absorption of drugs, their entry into cells or cellular organelles, and passage across the bloodbrain barrier. Due to their amphiphilic nature, phospholipids form bilayers possessing a hydrophilic surface and a hydrophobic interior. Substances may traverse this membrane in three different ways.
Diffusion (A). Lipophilic substances (red dots) may enter the membrane from the extracellular space (area shown in ochre), accumulate in the membrane, and exit into the cytosol (blue area). Direction and speed of permeation depend on the relative concentrations in the fluid phases and the membrane. The steeper the gradient (concentration difference), the more drug will be diffusing per unit of time (Fick’s Law). The lipid membrane represents an almost insurmountable obstacle for hydrophilic substances (blue triangles). Transport (B). Some drugs may penetrate membrane barriers with the help of transport systems (carriers), irrespective of their physicochemical properties, especially lipophilicity. As a prerequisite, the drug must have affinity for the carrier (blue triangle matching recess on “transport system”) and, when bound to the latter, be capable of being ferried across the membrane. Membrane passage via transport mechanisms is subject to competitive inhibition by another substance possessing similar affinity for the carrier. Substances lacking in affinity (blue circles) are not transported.
Drugs utilize carriers for physiological substances, e.g., L-dopa uptake by L-amino acid carrier across the blood-intestine and blood-brain barriers, and uptake of aminoglycosides
by the carrier transporting basic polypeptides through the luminal membrane of kidney tubular cells. Only drugs bearing sufficient resemblance to the physiological substrateof a carrier will exhibit affinity for it. Finally, membrane penetration may occur in the form of small membrane- covered vesicles. Two different systems are considered.
Transcytosis (vesicular transport, C). Wheew vesicles are pinched off, substances dissolved in the extracellular fluid are engulfed, and then ferried through the cytoplasm, vesicles (phagosomes) undergo fusion with lysosomes to form phagolysosomes, and the transported substance is metabolized. Alternatively, the vesicle may fuse with the opposite cell membrane (cytopempsis). Receptor-mediated endocytosis (C). The drug first binds to membrane surface receptors (1, 2) whose cytosolic domains contact special proteins (adaptins, 3). Drug-receptor complexes migrate laterally in the membrane and aggregate
with other complexes by a clathrin-dependent process (4). The affected membrane region invaginates and eventually pinches off to form a detached vesicle (5). The clathrin coat is
shed immediately (6), followed by the adaptins (7). The remaining vesicle then fuses with an “early” endosome (8), whereupon proton concentration rises inside the vesicle. The drug-receptor complex dissociates and the receptor returns into the cell membrane. The
“early” endosome delivers its contents to predetermined destinations, e.g., the Golgi complex, the cell nucleus, lysosomes, or the opposite cell membrane (transcytosis). Unlike simple endocytosis, receptor-mediated endocytosis is contingent on affinity for specific receptors and operates independently of concentration gradients.
Biotransformation of Drugs Many drugs undergo chemical modification in the body (biotransformation). Most frequently, this process entails a loss of biological activity and an increase in hydrophilicity (water solubility), thereby promoting elimination via the renal route . Since rapid drug elimination improves accuracy in titrating the therapeutic concentration, drugs are often designed with built-in weak links. Ester bonds are such links, being subject to hydrolysis by the ubiquitous esterases. Hydrolytic cleavages, along
with oxidations, reductions, alkylations, and dealkylations, constitute Phase I reactions
of drug metabolism. These reactions subsume all metabolic processes apt to alter drug molecules chemically and take place chiefly in the liver. In Phase II (synthetic) reactions, conjugation products of either the drug itself or its Phase I metabolites are formed, for
instance, with glucuronic or sulfuric acid. The special case of the endogenous transmitter acetylcholine illustrates well the high velocity of ester hydrolysis. Acetylcholine is broken down at its sites of release and action by acetylcholinesterase so rapidly as to negate its therapeutic use. Hydrolysis of other esters catalyzed by various esterases is slower, though relatively fast in comparison with other biotransformations. The local anesthetic, procaine, is a case in point; it exerts its action at the site of application while being largely devoid of undesirable effects at other locations because it is inactivated by hydrolysis during absorption from its site of application. Ester hydrolysis does not invariably lead to inactive metabolites, as exemplified by acetylsalicylic acid. The cleavage product, salicylic acid, retains pharmacological activity. In certain cases, drugs are administered in the form of
esters in order to facilitate absorption (enalapril _ enalaprilate; testosterone undecanoate _ testosterone) or to reduce irritation of the gastrointestinal mucosa (erythromycin succinate _ erythromycin). In these cases, the ester itself is not active, but the cleavage product is. Thus, an inactive precursor or prodrug is applied, formation of the active molecule occurring only after hydrolysis in the blood. Some drugs possessing amide bonds, such as rilocaine, and of course, peptides, can be hydrolyzed by peptidases and inactivated in this manner. Peptidases are also of pharmacological interest because they are responsible
for the formation of highly reactive cleavage products (fibrin) and potent mediators (angiotensin II; bradykinin, enkephalin,) from biologically inactive peptides. Peptidases exhibit some substrate selectivity and can be selectively inhibited, as exemplified by the formation of angiotensin II, whose actions inter alia include vasoconstriction. Angiotensin II is formed from angiotensin I by cleavage of the C-terminal dipeptide histidylleucine.
Hydrolysis is catalyzed by “angiotensin- converting enzyme” (ACE). Peptide analogues such as captopril block this enzyme. Angiotensin II is degraded by angiotensinase A, which clips off the N-terminal asparagine residue. The product, angiotensin III, lacks vasoconstrictor activity.
Enterohepatic Cycle (A) After an orally ingested drug has been absorbed from the gut, it is transported via the portal blood to the liver, where it can be conjugated to glucuronic or sulfuric acid (shown in B for salicylic acid and deacetylated bisacodyl, respectively) or to other organic acids. At the pH of body fluids, these acids are predominantly ionized; the negative charge confers high polarity upon the conjugated drug molecule and, hence, low membrane penetrability. The conjugated products may pass from hepatocyte into biliary fluid and from there back into the intestine. O-glucuronides can be cleaved by bacterial в-glucuronidases in the colon, enabling the liberated drug molecule to be reabsorbed. The enterohepatic cycle acts to trap drugs in the body. However, conjugated products
enter not only the bile but also the blood.
Glucuronides with a molecular weight (MW) > 300 preferentially pass into the blood, while those with MW > 300 enter the bile to a larger extent. Glucuronides circulating in the blood undergo glomerular filtration in the kidney
and are excreted in urine because their decreased lipophilicity prevents tubular reabsorption. Drugs that are subject to enterohepatic cycling are, therefore, excreted
slowly. Pertinent examples include digitoxin and acidic nonsteroidal anti-inflammatory agents. Conjugations (B) The most important of phase II conjugation reactions is glucuronidation. This reaction does not proceed spontaneously, but requires the activated form of glucuronic acid, namely glucuronic acid uridine diphosphate. Microsomal glucuronyl transferases link the activated glucuronic acid with an acceptor molecule. When the latter is a phenol or alcohol, an ether glucuronide will be formed. In the case of carboxyl-bearing molecules, an ester glucuronide is the result. All of these are O-glucuronides. Amines may form N-glucuronides that, unlike O-glucuronides, are resistant to bacterial в-glucuronidases. Soluble cytoplasmic sulfotransferases conjugate activated sulfate (3’-phosphoadenine-5’-phosphosulfate) with alcohols and phenols. The conjugates
are acids, as in the case of glucuronides.
In this respect, they differ from conjugates formed by acetyltransferases from activated acetate (acetylcoenzyme A) and an alcohol or a phenol. Acyltransferases are involved in the conjugation of the amino acids glycine
or glutamine with carboxylic acids. In these cases, an amide bond is formed between the carboxyl groups of the acceptor and the amino group of the donor molecule (e.g., formation of salicyluric acid from salicylic acid and glycine). The acidic group of glycine or glutamine remains free.
The Kidney as Excretory Organ Most drugs are eliminated in urine either chemically unchanged or as metabolites. The kidney permits elimination because the vascular wall structure in the region of the glomerular capillaries (B) allows unimpeded passage of blood
solutes having molecular weights (MW) < 5000. Filtration diminishes progressively as MW increases from 5000 to 70000 and ceases at MW > 70000. With few exceptions, therapeutically used drugs and their metabolites have much smaller molecular weights and can, therefore, undergo glomerular filtration, i.e., pass from blood into primary urine. Separating the capillary endothelium from the tubular epithelium, the basal membrane consists of charged glycoproteins and acts as a filtration barrier for high-molecular-weight substances. The relative density of this barrier depends on the electrical charge of molecules that attempt to permeate it. Apart from glomerular filtration (B), drugs present in blood may pass into urine by active secretion. Certain cations and anions are secreted by the epithelium of the proximal tubules into the tubular fluid via special, energyconsuming transport systems. These transport systems have a limited capacity. When several substrates are present simultaneously, competition for the carrier may occur .
During passage down the renal tubule, urinary volume shrinks more than 100-fold; accordingly, there is a corresponding concentration of filtered drug or drug metabolites (A). The resulting concentration gradient between urine and interstitial fluid is preserved in the case of drugs incapable of permeating the tubular epithelium. However, with lipophilic drugs the concentration gradient will favor reabsorption of the filtered molecules. In this case, reabsorption is not based on an active process but results instead from passive diffusion. Accordingly, for protonated substances, the extent of reabsorption is
dependent upon urinary pH or the degree of dissociation. The degree of dissociation varies as a function of the urinary pH and the pKa, which represents the pH value at which half of the substance exists in protonated (or unprotonated) form. This relationship is graphically
illustrated (D) with the example of a protonated amine having a pKa of 7.0. In this case, at urinary pH 7.0, 50 % of the amine will be present in the protonated, hydrophilic, membrane-impermeant form (blue dots), whereas the other half, representing the uncharged amine (orange dots), can leave the tubular lumen in accordance with the resulting concentration gradient. If the pKa of an amine is higher (pKa = 7.5) or lower (pKa = 6.5), a correspondingly smaller or larger proportion of the amine will be present in the uncharged, reabsorbable form. Lowering or raising urinary pH by half a pH unit would result in analogous changes for an amine having a pKa of 7.0. The same considerations hold for acidic molecules, with the important difference that alkalinization of the urine (increased pH) will promote the deprotonization of -COOH groups and thus impede reabsorption. Intentional alteration in urinary pH can be used in intoxications with proton-acceptor substances in order to hasten elimination of the toxin (alkalinization _ phenobarbital; acidification _amphetamine).
Drug Concentration in the Body as a Function of Time. First-Order (Exponential) Rate Processes
Processes such as drug absorption and elimination display exponential characteristics. As regards the former, this follows from the simple fact that the amount of drug being moved per unit of time depends on the concentration difference (gradient) between two body compartments (Fick’s Law). In drug absorption from the alimentary tract, the intestinal contents and blood would represent the compartments containing an initially high and low concentration, respectively. In drug elimination via the kidney, excretion often depends on glomerular filtration, i.e., the filtered amount of drug present in primary urine. As the blood concentration falls, the amount of drug filtered per unit of time diminishes. The resulting exponential decline is illustrated in (A). The exponential time course implies constancy of the interval during which the concentration decreases by one-half. This interval represents the half-life (t1/2) and is related to the elimination rate constant k by the equation t1/2 = ln 2/k. The two parameters, together with the initial concentration co, describe a first-order (exponential) rate process. The constancy of the process permits
calculation of the plasma volume that would be cleared of drug, if the remaining drug were not to assume a homogeneous distribution in the total volume (a conditioot met in reality).
This notional plasma volume freed of drug per unit of time is termed the clearance. Depending on whether plasma concentration falls as a result of urinary excretion or metabolic alteration, clearance is considered to be renal or hepatic. Renal and hepatic clearances add up to total clearance (Cltot) in the case of drugs that are eliminated unchanged via the kidney and biotransformed in the liver. Cltot represents the sum of all processes contributing to elimination; it is related to the half-life (t1/2) and the apparent volume of distribution Vapp by the equation: Vapp t1/2 = In 2 x –––– Cltot The smaller the volume of distribution or the larger the total clearance, the shorter is the half-life.
In the case of drugs renally eliminated in unchanged form, the half-life ofelimination can be calculated from the cumulative excretion in urine; the final total amount eliminated corresponds to the amount absorbed. Hepatic elimination obeys exponential kinetics because metabolizing enzymes operate in the quasilinear region of their concentration-activity curve; hence the amount of drug metabolized per unit of time diminishes with decreasing blood concentration. The best-known exception to exponential kinetics is the limination of alcohol (ethanol), which obeys a linear time course (zero-order kinetics), at least at blood concentrations > 0.02 %. It does so because the rate-limiting enzyme, alcohol dehydrogenase, achieves half-saturation at very low substrate concentrations, i.e., at about 80 mg/L (0.008 %). Thus, reaction velocity reaches a plateau at blood ethanol concentrations of about 0.02 %, and the amount of drug eliminated per unit of time remains constant at concentrations above this level.
Drug receptors and pharmacodynamics
The therapeutic and toxic effects of drugs result from their interactions with molecules in the patient. In most instances, drugs act by associating with specific macromolecules in ways that alter their biochemical or biophysical activity. This idea, now almost a century old, is embodied in the terms receptive substances and receptor: the component of a cell or organism that ‘ interacts with a drug and initiates the chain of biochemical events leading to the drug’s observed effects.
Initially, the existence of receptors was inferred from observations of the chemical and physiologic specificity of drug effects. Thus, Ehrlich noted that certain synthetic organic agents had characteristic antiparasitic effects while other agents did not, although their chemical structures differed only slightly.
In addition to its usefulness for explaining biology, the receptor concept has immensely important practical consequences for the development of drugs and for making therapeutic decisions in clinical practice. These consequences—explained more fully in later sections of this chapter—form the basis for understanding the actions and clinical uses of drugs described in every chapter of this book. They may be briefly summarized as follows:
1). Receptors largely determine the quantitative relations between dose or concentration of drug and pharmacologic effects. The receptor’s affinity for binding a drug determines the concentration of drug required to form a significant number of drug-receptor complexes, and the total number of receptors often limits the maximal effect a drug may produce.
2). Receptors are responsible for selectivity of drug action. The molecular size, shape, and electrical charge of a drug determine whether—and with what avidity —it will bind to a particular receptor among the vast array of chemically different binding sites available in a cell, animal, or patient. Accordingly, changes in the chemical structure of a drug can dramatically increase or decrease the new drug’s affinities for different classes of receptors, with resulting alterations in therapeutic and toxic effects.
3). Receptors mediate the actions of pharmacologic antagonists. Many drugs and endogenous chemical signals, such as hormones, regulate the function of receptor macromolecules as agonists; they change the function of a macromolecule as a more or less direct result of binding to it.
Pure pharmacologic antagonists, however, bind to receptors without directly altering the receptors’ function. Thus, the effect of a pure antagonist on a cell or in a patient depends entirely upon its preventing the binding and blocking the biologic actions of agonists molecules. Some of the most useful drugs in clinical medicine are pharmacologic antagonists.
MACROMOLECULAR NATURE OF DRUG RECEPTORS
It is still true that the chemical structures and even the existence of receptors for most clinically useful drugs can only be inferred from the chemical structures of the drugs themselves. By noting which chemical groups are required for specific pharmacologic effects of chemical congeners of a drug, pharmacologists imagine the complementary shape and distribution of electrical charge of the receptor site. In recent years, however, investigators have begun to characterize drug receptors in biochemical terms. Most of these receptors turn out to be proteins, presumably because the polypeptide structure provides the necessary diversity and specificity of shape and charge.
The best-characterized drug receptors are regulatory proteins, which mediate the actions of endogenous chemical signals such as neurotransmitters, autacoids, and hormones. This class of receptors mediates effects of many of the most useful therapeutic agents, which either mimic actions of endogenous agonists or, acting as antagonists, prevent responses to endogenous chemical signals. Although the physiologist or endocrinologist may think of these regulatory proteins as the only class of receptors, virtually any kind of protein molecule may serve as a receptor for a drug. Other classes of proteins that have been clearly
identified as drug receptors include enzymes, which may be inhibited (or, less commonly, activated) by binding a drug (eg, dihydrofolate reductase, the receptor for the antineoplastic drug methotrexate); transport proteins (eg, Na+K+-ATPase, the membrane receptor for cardioactive digitalis glycosides); and structural proteins (eg, tubulin, the receptor for colchicine, an anti-inflammatory agent).
Of the membrane-bound proteins that serve as receptors for neurohormones and drugs, the nicotinic cholinergic receptor is the best-characterized. This receptor is a pentamer composed of 5 peptide subunits. All 5 peptides span the membrane’s lipid bilayer, but only one or 2 peptides bind acetylcholine, the neurotransmitter. The binding of acetylcholine triggers opening of a transmembrane channel or pore through which sodium ions penetrate from the extracellular fluid into the cell, an event that initiates an excitatory postsynaptic potential in the nerve or muscle cells that are targets for nicotinic stimulation. The function of the remaining subunits of the nicotinic receptor is the subject of intensive investigation. These subunits somehow transduce the binding of a cholinergic ligand into opening the ion channel and probably constitute the channel itself. Thus, the nicotinic receptor, a single oligomeric protein molecule, performs 2 distinct functions as a receptor: 1) specific recognition of a drug or regulatory ligand (binding of acetylcholine), and 2) initiation of a biochemical event (opening of the sodium channel) that leads to the characteristic response of the cell (an excitatory postsynaptic potential).
RELATION BETWEEN DRUG CONCENTRATION AND RESPONSE
The relation between dose of a drug and the clinically observed response may be quite complex. In carefully controlled in vitro systems, however, the relation between concentration of a drug and its effect is often simple and can be described with mathematical precision. We will analyze this simple, idealized relation first because it underlies virtually all of the more complex relations between dose and effect that occur when drugs are given to patients.
Concentration-effect curves and receptor binding of agonists
Even in intact animals or patients, responses to low doses of a drug usually increase in direct proportion to dose. As doses increase, however, the incremental response diminishes; finally, doses may be reached at which no further increase in response can be achieved. In idealized or in vitro systems, the relation between drug concentration and effect is described by a hyperbolic curve.
Partial agonists
Based on the maximal pharmacologic response that occurs when all receptors are occupied, agonists can be divided into 2 classes: Partial agonists produce a lower maximal response, at full receptor occupancy, than do full agonists. As compared to full agonists, partial agonists produce concentration-effect curves that resemble curves observed with full agonists in the presence of a noncompetitive antagonist that irreversibly blocks receptor sites. Nonetheless, radioligand-binding experiments have demonstrated that partial agonists may occupy all receptor sites at concentrations that will fail to produce a maximal response comparable to that seen with full agonists. In addition, the failure of partial agonists to produce a “full” maximal response is not due to decreased affinity for binding to receptors. Such drugs compete, frequently with high affinity, for the full complement of receptors. Indeed, the partial agonists’ ability to occupy the total receptor population is indicated by the fact that partial agonists competitively inhibit the responses produced by full agonists.
The precise molecular mechanism that accounts for blunted maximal responses to partial agonists is not known. It is simplest to imagine that the partial agonist produces an effect on receptors that is intermediate between the effect produced by a full agonist and that produced by a competitive antagonist. The full agonist changes receptor conformation in a way that initiates subsequent* pharmacologic effects of receptor occupancy, while the “pure” competitive antagonist produces no such change in receptor conformation; in this view, the partial agonist changes receptor conformation, but not to the extent necessary to result in full efficacy of the occupied receptor.
To express this idea, pharmacologists refer to the efficacy (or ”maximal efficacy”) of a drug as a way of indicating the relation between pharmacologic response and occupancy of receptor sites. Efficacy for a full agonist is considered to be 1.0, while the efficacy of a pure antagonist is zero. Partial agonists have efficacious between zero and 1.0. Many drugs used as competitive antagonists are in fact weak partial agonists.
Drug receptors and pharmacodynamics
Agonist-receptor interactions presumably result in full or ”tight” coupling of full agonists to response, in less tight coupling of partial agonists, and in ‘ ‘uncoupling” of pure antagonists. However, it is possible for even full agonists to become ‘ ‘uncoupled” from responses as the result of changes in coupling processes that take place distal to the receptor.
High efficiency of receptor-effector coupling may also be interpreted as the result of spare receptors. Receptors are said to be “spare” for a given pharmacologic response when the maximal response can be elicited by an agonist at a concentration that does not result in occupancy of the full complement of available receptors. Experimentally, spare receptors may be demonstrated by using noncompetitive (irreversible) antagonists to prevent binding of agonist to a proportion of available receptors and showing that high concentrations of agonist can still produce an undiminished maximal response.
The spare receptors are not qualitatively different from nonspare ones. They are not “hidden” or unavailable, and they can be coupled to response. This will happen if the concentration or amount of a cellular component other than the receptor limits the coupling of receptor occupancy to response.
Not all of the mechanisms of pharmacologic antagonism involve interactions of drugs or endogenous ligands at a single type of receptor. Indeed, chemical antagonists need not involve a receptor at all. Thus, one drug may antagonize the actions of a second drug by binding to and inactivating the second drug. For example, protamine, a protein that is positively charged at physiologic pH, is used clinically to counteract the effects of heparin, an anticoagulant that is negatively charged; in this case, one drug antagonizes the other simply by binding it and making it unavailable for interactions with proteins involved in formation of a blood clot.
The clinician often uses drugs that take advantage of physiologic antagonism between endogenous regulatory pathways. Many physiologic functions are controlled by opposing regulatory pathways. For example, several catabolic actions of the glucocorticoid hormones lead to increased blood sugar, an effect that is physiologically opposed by insulin. Although glucocorticoids and insulin act on quite distinct receptor-effector systems, the clinician must sometimes administer insulin to oppose the hyperglycemic effects of glucocorticoid hormones, whether the latter are elevated by endogenous synthesis (eg, an inoperable tumor of the adrenal cortex) or as a result of glucocorticoid therapy.
In general, use of a drug as a physiologic antagonist produces effects that are less specific and less easy to control than are the effects of a receptor-specific antagonist. Thus, for example, to treat bradycardia caused by increased vagal tone associated with the acute pain of a myocardial infarction, the physician could use isoproterenol, a beta-adrenergic agonist that increases heart rate by mimicking sympathetic stimulation of the heart. However, use of this physiologic antagonist would be less rational—and potentially more dangerous—than would use of a receptor-specific antagonist such as atropine (a competitive antagonist at the muscarinic receptors through which vagal stimuli slow heart rate).
The existence of a specific drug receptor is usually inferred from studying the structure-activity relationship of a group of structurally similar congeners of the drug that mimic or antagonize its effects.
Evidence that a particular drug acts via 2 or more distinct receptors usually presents an important therapeutic opportunity, because it suggests the possibility of developing new drugs that will exhibit enhanced selectivity for one receptor over the other. Such opportunities have been extensively exploited with the receptors for histamine, acetylcholine, and norepineph-rine. Thus, for example, beta-adrenergic antagonists can block cardioacceleration produced by norepinephrine without preventing catecholamine regulation of arteriolar constriction, which is mediated by alpha-adrenergic receptors. Similarly, alpha-adrenergic agonists can induce vasoconstriction without stimulating beta receptors in the heart.
The quantal dose-effect curve is often characterized by stating the median effective dose (ED50), the dose at which 50% of individuals exhibit the specified quantal effect. Similarly, the dose required to produce a particular toxic effect in 50% of animals is called the median toxic dose (TD50). If the toxic effect is death of the animal, a median lethal dose (LD50) may be experimentally defined. Such values provide a convenient way of comparing the potencies of drugs in experimental and clinical settings. Thus, if the ED50s of 2 drugs for producing a specified quantal effect are 5 and 500 mg, respectively, then the first drug can be said to be 100 times more potent than the second for that particular effect. Similarly, one can obtain a valuable index of the selectivity of a drug’s action by comparing its ED50s for 2 different quantal effects in a population (eg, cough suppression versus sedation for opiate drugs; increase in heart rate versus increased vasoconstriction for adrenergic amines; anti-inflammatory effects versus sodium retention for corticosteroids; etc).
Quantal dose-effect curves may also be used to generate information regarding the margin of safety to be expected from a particular drug used to produce a specified effect. One measure, which relates the dose of a drug required to produce a desired effect to that which produces an undesired effect, is the therapeutic index. In animal studies, the therapeutic index is usually defined as the ratio of the TD50 to the ED50 for some therapeutically relevant effect. The clinical usefulness of a drug usually relates to a much more conservative definition of therapeutic index and critically depends upon the severity of the disease under treatment. Thus, for the treatment of headache the physician might require a very large therapeutic index, defined as the ratio of the dose required to cause serious toxicity in a very small percentage of subjects (TD 0.001) to the dose required to ameliorate headache in a very large proportion of subjects (ED 99). For treatment of a lethal disease, such as Hodgkin’s lymphoma, an acceptable therapeutic index might be defined less stringently.
Variation in drug responsiveness
Individuals may vary considerably in their responsiveness to a drug; indeed, a single individual may respond differently to the same drug at different times during the course of treatment. Occasionally, individuals exhibit an unusual or idiosyncratic drug response, one that is infrequently observed in most patients. These idiosyncratic responses are usually caused by genetic differences in metabolism of the drug or by immunologic mechanisms, including allergic reactions.
Quantitative variations in drug response are in general more common and more clinically important. An individual patient is hyporeactive or hyperreactive to a drug in that the intensity of effect of a given dose of drug is diminished or increased in comparison to the effect seen in most individuals. The term hypersensitivity usually refers to allergic or other immunologic responses to drugs. With some drugs, the intensity of response to a given dose may change during the course of therapy; in these cases, responsiveness usually decreases as a consequence of continued drug administration, producing a state of relative tolerance to the drug’s effects. When responsiveness diminishes rapidly after administration of a drug, the response is said to be subject to tachyphylaxis.
The general clinical implications of individual variability in drug responsiveness are clear: The physician must be prepared to change either the dose of drug or the choice of drug, depending upon the response observed in the patient. Even before administering the first dose of a drug, the physician should consider factors that may help in predicting the direction and extent of possible variation in responsiveness. These include the propensity of a particular drug to produce tolerance or tachyphylaxis as well as the effects of age, sex, body size, disease state, and simultaneous administration of other drugs.
Four general mechanisms may contribute to variation in drug responsiveness among patients or within an individual patient at different times. The classification described below is necessarily artificial in that most variation in clinical responsiveness is caused by more than one mechanism. Nonetheless, the classification may be useful because certain mechanisms of variation are best dealt with according to different therapeutic strategies:
Alteration in concentration of drug that reaches the receptor.
Patients may differ in the rate of absorption of a drug, in distributing it through body compartments, or in clearing the drug from the blood. Any of these pharmacokinetic differences may alter the concentration of drug that reaches relevant receptors and thus alter clinical response. These pharmacokinetic differences can often be predicted on the basis of age, weight, sex, disease state, or liver and kidney function of the patient, and such predictions may be used to guide quantitative decisions regarding an initial dosing regimen. Repeated measurements of drug concentrations in blood during the course of treatment are often helpful in dealing with the variability of clinical response caused by pharmacokinetic differences among individuals.Variation in concentration of an endogenous receptor ligand.
This mechanism contributes greatly to variability in responses to pharmacologic antagonists. Thus, propranolol, a beta-adrenergic antagonist, will markedly slow the heart rate of a patient whose endogenous catecholamines are elevated (as in heart failure or pheochromocytoma) but will not affect the resting heart rate of a well-trained marathon runner. A partial agonist may exhibit even more dramatically different responses: Saralasin, a weak partial agonist at angiotensin II receptors, lowers blood pressure in patients with hypertension caused by increased angiotensin II production and raises blood pressure in patients who produce low amounts of angiotensin.
In assessing clinical response to antagonist drugs, the physician must always make a judgment about the probable stimulation of receptors by endogenous agonists. Thus, unsatisfactory response to a dose of a competitive antagonist might be due to greatly elevated endogenous agonist, and a larger dose of antagonist would be appropriate. Alternatively, the unsatisfactory response might be due to low rates of stimulation by agonist; this would suggest that the diagnosis is wrong and that a different mode of therapy, rather than more drug, is indicated.
Alterations iumber or function of receptors.
Experimental studies have documented changes in drug responsiveness caused by increases or decreases in the number of receptor sites or by alterations in the efficiency of coupling of receptors to distal effector mechanisms. Although such changes have not been rigorously documented in human beings, it is likely that they account for much of the individual variability in response to some drugs, particularly those that act at receptors for hormones, biogenic amines, and neurotransmitters. In some cases, the change in receptor number is caused by other hormones; for example, thyroid hormones increase both the number of beta-adrenergic receptors in rat heart muscle and the cardiac sensitivity to catecholamines. Similar changes probably contribute to the tachycardia of thyrotoxicosis in patients and may account for the usefulness of propranolol, a beta-adrenergic antagonist, in ameliorating symptoms of this disease.
In other cases, the agonist ligand itself induces a decrease in the number (“down regulation”) or coupling efficiency of its receptors. Receptor-specific de-sensitization mechanisms presumably act physiologically to allow cells to adapt to changes in rates of stimulation by hormones and neurotransmitters in their environment. These mechanisms may contribute to tachyphylaxis or tolerance to the effects of some drugs, particularly the biogenic amines and their congeners. Recent investigations suggest, in addition, that similar adaptive mechanisms may be responsible for so-called “overshoot” phenomena that follow withdrawal of certain drugs (propranolol, opiates, some antihypertensive agents, etc). Thus, for example, an antagonist may actually raise the number of receptors in a cell by preventing down regulation caused by endogenous agonist; when the antagonist is withdrawn, the elevated receptor number allows an exaggerated response to physiologic concentrations of agonist.
Therapeutic strategies required to deal with receptor-specific changes in drug responsiveness vary according to the clinical situation. In some cases, the dose of an agonist must be increased to achieve a continuing satisfactory response, while in other cases different or additional drugs should be administered. Cessation of treatment with certain drugs should be gradual and carefully monitored.
Changes in components of response distal to receptor.
Although a drug initiates its actions by binding to receptors, the response observed in a patient depends on the functional integrity of biochemical processes in the responding cell and physiologic regulation by interacting organ systems. Clinically, changes in these postreceptor processes represent the largest and most important class of mechanisms that cause variation in responsiveness to drug therapy.
Before initiating therapy with a drug, the physician should be aware of patient characteristics that may limit the clinical response. These characteristics include the age, sex, and general health of the patient and—most importantly—the severity and pathophysiologic mechanism of the disease. Once treatment is begun, the most important potential cause of failure to achieve a satisfactory response is that the diagnosis is wrong or physiologically incomplete. Thus, congestive heart failure will not respond satisfactorily to agents that increase myocardial contractility if the underlying pathologic mechanism is unrecognized stenosis of the mitral valve rather than myocardial insufficiency. Conversely, drug therapy will always be most successful when it is accurately directed at the pathophysiologic mechanism responsible for the disease.
When the diagnosis is correct and the drug is appropriate, treatment may still not produce an optimal result. An unsatisfactory therapeutic response can often be traced to compensatory mechanisms in the patient that respond to and oppose the beneficial effects of the drug. Compensatory increases in sympathetic nervous tone and fluid retention by the kidney, for example, can contribute to tolerance to antihy-pertensive effects of a vasodilator drug. In such cases, additional drugs may be required to achieve a useful therapeutic response.
Clinical selectivity: beneficial versus toxic effects of drugs
Although we classify drugs according to their principal actions, it is clear that no drug causes only a single, specific effect. Why is this so? It is exceedingly unlikely that any kind of drug molecule will bind to only a single molecular species of receptor, if only because the number of potential receptors in a patient is astronomically large. (Consider that the human genome codes for approximately 104 different peptide gene products and that the chemical complexity of each of these peptides is sufficient to provide many different potential binding sites.) Even if the chemical structure of a drug allowed it to bind to only one kind of receptor, the biochemical processes controlled by such receptors would take place in multiple cell types and would be coupled to many other biochemical functions; as a result, the patient and the physician would probably perceive more than one drug effect.
Accordingly, drugs are only selective—rather than specific—in their actions, because they bind to one or a few types of receptor more tightly than to others, and because these receptors control discrete processes that result in distinct effects. As we have seen, selectivity can be measured by comparing binding affinities of a drug to different receptors or by comparing ED50s for different effects of a drug in vivo. In drug development and in clinical medicine, selectivity is usually considered by separating effects into 2 categories: beneficial or therapeutic effects versus toxic effects. Pharmaceutical advertisements and physicians occasionally use the term side effect, implying that the effect in question is insignificant or occurs via a pathway that is to one side of the principal action of the drug; such implications are frequently erroneous.
It is important to recognize that the designation of a particular drug effect as either therapeutic or toxic is a value judgment and not a statement about the phar-macologic mechanism underlying the effect. As a value judgment, such a designation depends on the clinical context in which the drug is used.
is only because of their selectivity that drugs are useful in clinical medicine. Thus, it is important, both in the management of patients and in the development and evaluation of new drugs, to analyze ways in which beneficial and toxic effects of drugs may be related, in order to increase selectivity and usefulness of drug therapy. Fig 2-10 depicts 3 possible relations between the therapeutic and toxic effects of a drug based on analysis of the receptor-effector mechanisms involved.
Beneficial and toxic effects mediated by the same receptor-effector mechanism. Much of the serious drug toxicity in clinical practice represents a direct pharmacologic extension of the therapeutic actions of the drug. In some of these cases (bleeding caused by anticoagulant therapy; hypoglycemic coma due to insulin), toxicity may be avoided by judicious management of the dose of drug administered, guided by careful monitoring of effect (measurements of blood coagulation or serum glucose) and aided by ancillary measures (avoiding tissue trauma that may lead to hemorrhage; regulation of carbohydrate intake). In still other cases, the toxicity may be avoided by not administering the drug at all, if the therapeutic indication is weak or if other therapy is available (eg, sedative-hypnotics ordinarily should not be used to treat patients whose complaints of insomnia are due to underlying mental depression).
In certain situations, a drug is clearly necessary and beneficial but produces unacceptable toxicity when given in doses that produce optimal benefit. In such situations, it may be necessary to add another drug to the treatment regimen. For example, sympatholytic agent octadinum (guanethidine) lowers blood pressure in essential hypertension by inhibiting cardiovascular stimulation by sympathetic nerves; as an inevitable consequence, patients will suffer from symptoms of postural hypotension if the dose of drug is large enough. (Note that postural hypotension has been called a ”side effect” of guanethidine, although in fact it is a direct effect, closely related to the drug’s principal therapeutic action.) Appropriate management of such a problem takes advantage of the fact that blood pressure is regulated by changes in blood volume and tone of arterial smooth muscle in addition to the sympathetic nerves. Thus, concomitant administration of diuretics and vasodilators may allow the dose of guanethidine to be lowered, with relief of postural hypotension and continued control of blood pressure.
Beneficial and toxic effects mediated by identical receptors but in different tissues or by different effector pathways.
Examples of drugs in this category include digitalis glycosides, which may be used to augment cardiac contractility but also produce cardiac arrhythmias, gastrointestinal effects, and changes in vision (all probably mediated by inhibition of Na+K+-ATPase in cell membranes); methotrexate, used to treat leukemia and other neoplastic diseases, which also kills normal cells in bone marrow and gastrointestinal mucosa (all mediated by inhibition of the enzyme dihydrofolate reductase); and congeners of glucocorticoid hormones, used to treat asthma or inflammatory disorders, which also can produce protein catabolism, psychosis, and other toxicities (all thought to be mediated by similar or identical glucocorticoid receptors). In addition to these and other well-documented examples, it is likely that “side effects” of many drugs are mediated by receptors identical to those that produce the recognized beneficial effect.
Three therapeutic strategies are used to avoid or mitigate this sort of toxicity. First, the drug should always be administered at the lowest dose that produces acceptable benefit, recognizing that complete abolition of signs or symptoms of the disease may not be achieved. Second (as described above for guanethidine), adjunctive drugs that act through different receptor mechanisms and produce different toxicity’s may allow lowering the dose of the first drug, thus limiting its toxicity (eg, use of other immunosuppressive agents added to glucocorticoids in treating inflammatory disorders). Third, selectivity of the drug’s actions may be increased by manipulating the concentrations of drug available to receptors in different parts of the body. Such “anatomic” selectivity may be achieved, for example, by aerosol administration of a glucocorticoid to bronchi or by selective arterial infusion of an antimetabolite into an organ containing tumor cells.
Beneficial and toxic effects mediated by different types of receptors: Therapeutic advantages resulting from new chemical entities with improved receptor selectivity were mentioned earlier in this chapter and are described in detail in later chapters. Such drugs include the alpha- and beta-adrenergic agonists and antagonists, the H1 and H2 antihistamines, nicotinic and muscarinic blocking agents, and receptor-selective steroid hormones. All of these receptors are grouped in functional families, each responsive to a small class of endogenous agonists. The receptors—and their associated therapeutic uses— were discovered by analyzing effects of the physiologic chemical signals—catehecholamines, histamine, acetylcholine, and corticosteroids.
A number of other drugs were discovered in a similar way, although they may not act at receptors for known hormones or neurotransmitters. These drugs were discovered by exploiting toxic or side effects of other agents, observed in a different clinical context. Examples include quinidine, the sulfonylureas, thiazide diuretics, tricyclic antidepressants, monoamine oxidase inhibitors, and phenothiazine antipsychotics among many others.
It is likely that some of these drugs will eventually be shown to act via receptors for endogenous agonists, as was recently established for morphine, a potent analgesic agent. Morphine has been shown to act on receptors physiologically stimulated by the opioid peptides. Pharmacologists now subclassify the opioid receptors, in a fashion reminiscent of earlier studies of adrenergic and cholinergic reactions.
Thus, the propensity of drugs to bind to different classes of receptor sites is not only a potentially vexing problem in treating patients – it also presents a continuing challenge to pharmacology and an opportunity for developing new and more useful drugs.
Pharmacokinetics: absorption, distribution and excretion
When a clinician prescribes a drug and the patient takes it, their main concern is with the effect on the patient’s disease. Several processes are going forward from the time a dose is administered until the appearance of any therapeutic effect. These pharmacokinetic processes, defined above, determine how rapidly and in what concentration and for how long the drug will appear at the target organ. Input, distribution, and loss—are the major pharmacokinetic variables. In most cases, input will consist of absorption from the most convenient site that meets the requirements for speed and completeness of absorption. For most drugs, oral administration is appropriate, and measurable concentrations of the drug in the blood result. The pattern of the concentration-time curve in the blood is a function of the input, distribution, and loss factors. In this chapter, we will examine the quantitative aspects of these relationships.
A fundamental hypothesis of pharmacokinetics is that a relationship exists between a pharmacologic or toxic effect of a drug and the concentration of the drug in a readily accessible site of the body (eg, blood).
This hypothesis has been documented for many drugs, although for some drugs no clear relationship has been found between pharmacologic effect and plasma or blood concentrations. In most cases, the concentration of drug in the general circulation will be related to its concentration at the site of action. The drug will then elicit a number of pharmacologic effects at the site of action. These pharmacologic effects may include toxic effects in addition to the desired clinical effect. The clinician then must balance the toxic potential of a particular dose of a drug with its efficacy to determine the utility of that agent in that clinical situation. Pharmacokinetics plays its role in the dose efficacy scheme by providing the quantitative relationship between drug efficacy and drug dose, with the aid of measurements of drug concentrations in various biologic fluids.
The importance of pharmacokinetics in patient care rests upon the improvement in drug efficacy that can be attained when the measurement of drug levels in the general circulation is added to traditional methods of predicting the dose of the drug. Knowledge of the relationship between efficacy and drug concentration measurements allows the clinician to take into account the various pathologic and physiologic features of a particular patient that make him or her different from the normal individual in responding to a dose of the drug.
Several pathologic and physiologic processes dictate dosage adjustment in individual patients (eg, heart failure, renal failure). They do so by modifying specific pharmacokinetic parameters. The 2 basic variables are clearance, the measure of the ability of the body to eliminate the drug, and volume of distribution, the measure of the apparent space in the body available to contain the drug.
Volume of distribution relates the amount of drug in the body to the concentration of drug (C) in blood or plasma. Volume of distribution is defined in terms of blood or plasma concentrations, depending upon the fluid measured, and reflects the apparent space available in both the general circulation and the tissues of distribution. The plasma volume of a normal 70-kg man is
Depending on the pKa of the drug, the degree of plasma protein binding, the partition coefficient of the drug in the fatty tissues, and the degree of binding to other tissues within the body, volume of distribution may vary widely. For a drug extensively bound to plasma proteins but not to tissue proteins, most of the drug in the body will be retained in the blood, and the volume of distribution will have a lower limit of approximately
A further definition of clearance is useful in understanding the effects of physiologic and pathologic variables on drug elimination, particularly with respect to a specific organ. The rate of elimination of a drug by a single organ can be defined in terms of the blood flow entering and exiting from the organ and the concentration of drug in the blood. The rate of presentation of drug to the organ is the product of blood flow and entering drug concentration, while the rate of exit of drug from the organ is the product of blood flow and exiting drug concentration. The difference between these rates at steady state is the rate of drug elimination.
Half-life is a useful kinetic parameter in that it indicates the time required to attain steady state or to decay from steady-state conditions after a change (ie, starting or stopping) in a particular rate of drug administration (the dosing regimen). However, as an indicator of either drug elimination or distribution, it has little value. Early studies of drug pharmacokinetics in diseased subjects were compromised by reliance on drug half-life as the sole measure of alterations in drug disposition. Disease states can affect both of the physiologically related parameters, volume of distribution and clearance; thus, the derived parameter, h/3, will not necessarily reflect the expected change in drug elimination.
Bioavailability
Bioavailability is defined as the fraction of unchanged drug reaching the systemic circulation following administration by any route. For an intravenous dose of the drug, bioavailability is equal to unity. For a drug administered orally, bioavailability may be less than unity for several reasons. The drug may be incompletely absorbed. It may be metabolized in the gut, the gut wall, the portal blood, or the liver prior to entry into the systemic circulation. It may undergo enterohepatic cycling with incomplete reabsorption following elimination into the bile. Biotransformation of some drugs in the liver following oral administration is an important factor in the pharmacokinetic profile, as discussed below.
Absorption, bioavailability and routes of administration
In addition to the definition given above, bioavailability is often used to indicate the rate at which an administered dose reaches the general circulation. In general, the relative order of peak times following the administration of different dosage forms of the drug thus corresponds to the rates of availability of the drug from the various dosage forms. The extent of availability may be measured by using either drug concentration in the blood or drug amounts in the urine. The area under the blood concentration-time curve (area under the curve, AUC) for a drug is a common measure of the extent of availability. For most drugs, drug clearance is linear (a constant function of concentration), and the relative areas under the curve or the total amounts of unchanged drug excreted in the urine quantitatively describe the relative availability of the drug from the different dosage forms. However, even ionlinear cases, where clearance is dose-dependent, the relative areas under the curve will yield a measurement of the rank order of availability from different dosage forms or from different sites of administration.
In many cases, the duration of pharmacologic effect is a function of the length of time the blood concentration curve is above the minimum effective concentration, and the intensity of the effect is usually a function of the height of the blood level curve above the minimum effective concentration.
Extraction ratio and the first-pass effect
For most drugs, disposition or loss from the biologic system is independent of input, where disposition is defined as what happens to the active drug after it reaches a site in the circulation where drug concentration measurements can be made. Although disposition processes may be independent of input, the inverse is not necessarily true, since disposition can markedly affect the extent of availability. Drug absorbed from the stomach and the intestine must pass through the liver before reaching a site in the circulation that can be sampled for measurement. Thus, if a drug is metabolized in the liver or excreted in bile, some of the active drug absorbed from the gastrointestinal tract will be inactivated by hepatic processes before the drug can reach the general circulation and be distributed to its sites of action. If the metabolizing or biliary excreting capacity of the liver is great, the effect on the extent of availability will be substantial (first-pass effect). Thus, if the hepatic clearance for a drug is large the extent of availability for this drug will be low when it is given by a route that yields first-pass metabolic effects. This decrease in availability is a function of the physiologic site from which absorption takes place, and no amount of dosage form modification can improve the fractional availability. Of course, therapeutic blood levels may still be reached by this route of administration if larger doses are given. However, in this case, the levels of the drug metabolites will be increased significantly over those that would occur following intravenous administration, especially if the drug has a large volume of distribution. Therefore, the toxicity potential and elimination kinetics of the metabolites must be thoroughly understood before a decision to administer a large oral dose is made.
Drugs with high extraction ratios will show marked intersubject variability in bioavailability because of variations in hepatic function or blood flow or both. Consider a drug with an extraction ratio of 0.95 that falls to 0.90 as a result of hepatic impairment. In this instance, the bioavailability of the drug will double, from 0.05 to 0.10. These relationships can explain the marked variability in plasma or blood drug concentrations that occurs among individuals given similar doses of a highly extracted drug. Small variations in hepatic extraction between individuals will result in large differences in availability and plasma drug concentrations . These considerations are also pertinent for hepatic disease states accompanied by significant intrahepatic or extrahepatic circulatory shunting and in the presence of surgically created anastomoses between the portal system and the systemic venous circulation. For drugs that are highly extracted by the liver, shunting of blood past hepatic sites of elimination will result in substantial increases in drug availability, whereas for drugs that are poorly extracted by the liver (for which the difference in equations and between entering and exiting drug concentration is small), shunting of blood past the liver will cause little change in drug availability. Drugs that are poorly extracted by the liver are chlordiazepoxide, diazepam, digitoxin, phenytoin, theophylline, tolbutamide, and warfarin.
The first-pass effect can be avoided to a great extent by use of sublingual tablets and to some extent by use of rectal suppositories. The capillaries in the lower and mid sections of the rectum drain into the inferior and middle hemorrhoidal veins, which in turn drain into the inferior vena cava, thus bypassing the liver. However, suppositories tend to move upward in the rectum into a region where veins that lead to the liver, such as the superior hemorrhoidal, predominate. In addition, there are extensive anastomoses between the superior and middle hemorrhoidal veins; thus, only about 50% of a rectal dose can be assumed to bypass the liver. The lungs represent a good temporary clearing site for a number of drugs, especially basic compounds, as a result of partition into lipid tissues. The lungs also provide a filtering function for particulate matter that may be given by intravenous injection. The lung may serve as a site of first-pass loss by excretion and possible metabolism for drugs administered by nongastrointestinal (“parenteral”) routes.
THE USE OF PHARMACOKINETICS IN DESIGNING A DOSAGE REGIMEN
The “dosage” of a drug represents a decision about 4 variables: 1) the amount of drug to be administered at one time; 2) the route of administration; 3) the interval between doses; and 4) the period of time over which drug administration is to be continued. The choice of the route of administration and the implications of this choice upon the extent and rate of drug availability were discussed in the previous section. Most patterns of administration fall into 2 classes, both of which may be described using pharmacokinetic principles: 1) continuous input by intravenous infusion (or any route that delivers drug at a constant rate), and 2) a series of intermittent drug doses, usually of equal size and given at approximately equally spaced intervals.
Maintenance dose
In most clinical situations, drugs are administered in such a way as to maintain a steady state of drug in the body. Thus, calculation of the appropriate maintenance dose is a primary goal. Dosing rate is also defined as the product of the extent of availability (F) and the dose divided by the dosing interval. Thus, if the clinician can specify the desired plasma drug concentration and knows the clearance and availability for that drug in a particular patient, the appropriate dosing rate can be calculated.
Loading dose
When the time to reach steady state is appreciable, as it is for drugs with long half-lives, it may be desirable to administer a loading dose that promptly raises the concentration of drug in plasma to the projected steady-state value. In theory, only the amount of the loading dose need be computed, not the rate of its administration; to a first approximation, this is so. The amount of drug required to achieve a given steady-state concentration in the plasma is the amount that must be in the body when the desired steady state is reached. (For intermittent dosage schemes, the amount is that at the average concentration). The volume of distribution (Vd) is the proportionality factor that relates the total amount of drug in the body to the concentration in the plasma. However, in some cases the distribution phase may not be ignored, particularly in connection with the calculation of loading doses. If the rate of absorption is rapid relative to distribution (this is always true for intravenous bolus administration), the concentration of drug in plasma that results from an appropriate loading dose can initially be considerably higher than desired. Severe toxicity may occur, albeit transiently. This may be particularly important, for example, in the administration of antiarrhythmic drugs, where an almost immediate toxic response is obtained when plasma concentrations exceed a particular level. Thus, while the estimation of the amount of a loading dose may be quite correct, the rate of administration can sometimes be crucial in preventing excessive drug concentrations, and slow administration of an intravenous drug (over minutes rather than seconds) is almost always wise. For intravenous doses of theophylline, initial injections should be given over a 20-minute period to avoid the possibility of high plasma levels during the distribution phase.
THE EFFECT OF DISEASE ON PHARMACOKINETIC PROCESSES
Disease states may modify all of the variables listed in Table 3-1. The ability to predict or understand how pathologic conditions may modify drug kinetics requires an understanding of the interrelationship between the variables. Clearance is the most important parameter in the design of drug dosage regimens. As shown in equation (5), clearance of an eliminating organ may be defined in terms of blood flow to the organ and the extraction ratio.
When the capability for elimination is of the same order of magnitude as the blood flow, clearance is dependent upon the blood flow as well as on the intrinsic clearance and plasma protein binding. Enzyme induction or hepatic disease may change the rate of imipramine metabolism in an isolated hepatic microsomal enzyme system, but no change in clearance is found in the whole animal with similar hepatic changes. This is explained by the fact that imipramine is a high-extraction-ratio drug and clearance is limited by blood flow rate, so that changes in dim due to enzyme induction or liver disease have no effect on clearance. Also, although imipramine is highly protein-bound, changes in protein binding due to disease or competitive binding should have no effect on clearance even though volume of distribution is changed. In the latter case, a change in volume of distribution with no change in clearance will result in a change in half-life, although the elimination mechanisms have not been altered.
The differences between clearance and half-life are important in defining the underlying mechanisms for the effect of a disease state on drug disposition. For example, the half-life of diazepam increases with age. One explanation for this change is that the ability of the liver to metabolize this drug decreases as a function of age.
In many reports hepatic disease has been shown to reduce drug clearance and prolong half-life. However, for many other drugs known to be eliminated by hepatic processes, no changes in clearance or half-life have beeoted with hepatic disease. This reflects the fact that hepatic disease does not always affect the hepatic intrinsic clearance. This may be due to the multiplicity of liver metabolizing enzymes available to degrade drugs and other exogenous compounds. There is no reliable marker of hepatic drug-metabolizing function that can be used to predict changes in liver clearance in a manner analogous to the changes in drug renal clearance that can be predicted as a function of creatinine clearance.
Generally, hepatic impairment would be expected to reduce clearance and prolong half-life or to cause no change in drug elimination. However, there is some evidence that hepatic disease can also increase clearance and shorten half-life. For example, the clearance of tolbutamide may increase and its half-life decrease with no change in volume of distribution in individuals with acute viral hepatitis during the acute phase of illness in comparison to the recovery period. Tolbutamide is a low-extraction-ratio drug, and its hepatic clearance may be described by equation. The explanation for the observations appears to be an increase in the unbound fraction of drug in plasma (fu) in the absence of a change in dim. The half-life is changed, since total clearance is changed without a change in volume of distribution. For many drugs, volume of distribution would be expected to increase as the free fraction of drug in plasma increases. However, the volume of distribution for tolbutamide is quite small (11 L/70 kg), and the majority of the distribution space is related to blood volume, which is independent of fu.
Pharmacokinetic changes in renal disease may also be explained in terms of clearance concepts. However, since the net renal excretion of a drug is determined by filtration, active secretion, and reabsorption, the treatment of renal clearance is more complicated than that described above.
The secretion of drug in the kidney will depend on the relative binding of drug to the active transport carriers in relation to the binding to plasma proteins, the degree of saturation of these carriers, transfer of the drug across the tubular membrane, and the rate of delivery of the drug to the secretory site. With a model that combines these factors, the influence of changes in protein binding, blood flow, and number of functioning nephrons may be predicted and explained in a manner analogous to the examples given above for hepatic elimination.
ADJUSTMENT OF THE DOSAGE REGIMEN FOR THE INDIVIDUAL PATIENT
Attainment of an appropriate maintenance dosage regimen often requires adjustment for that patient. It is reasonable to assume that the usual recommended dose of a drug has been determined from appropriate experience with many “typical” cases. If a particular patient being treated is not expected to differ from the “typical” one with respect to sensitivity to the drug, one may adjust the usual maintenance dosage regimen on the basis of the patient’s clearance of the drug. This requires computation or measurement of clearance for the patient (and bioavailability, if it will differ from usual). At the corrected dosing rate, the patient’s mean steady-state concentration of drug will be identical to that achieved in the presumably successful treatment of a “typical” patient.
When an intermittent regimen is adjusted for an individual patient, one may choose to alter the dose given per dosing interval, the dosing interval, or both. As a rule, adjustment of the amount of each dose, not the interval, is preferred. Adjustments of dosage are usually made to compensate for smaller-than-usual clearances. If the amount of each dose is reduced to compensate for reduced clearance, then fluctuations about the mean steady-state concentration will be smaller. For most drugs, a more constant concentration-time profile is not only acceptable but desirable.
Estimates of clearance must often be adjusted when patients have alterations in renal function. The quantities required for this adjustment are the fraction of normal renal function remaining and the fraction of drug usually excreted unchanged in the urine. The latter parameter appears in Table 3-1; the former can be estimated as the ratio of the patient’s creatinine clearance to a normal creatinine clearance (100-120 mL/min/70 kg). If creatinine clearance has not been measured, it may be estimated from measurements of the concentration of creatinine in serum, using one of several different equations and nomograms.
Pharmacokinetics: drug biotransformation
Humans are daily exposed to a wide variety of foreign compounds called xenobiotics—substances absorbed across the lungs or skin or, more commonly, ingested either unintentionally as compounds present in food and drink or deliberately as drugs for therapeutic or “recreational” purposes. Exposure to environmental xenobiotics may be inadvertent and accidental and may even be inescapable. Some xenobiotics are innocuous, but many can provoke biologic responses both pharmacologic and toxic iature. These biologic responses often depend on conversion of the absorbed or ingested substance into an active metabolite. The discussion that follows is applicable to xenobiotics in general as well as to drugs and to some extent to endogenous compounds.
parent drug and may even be inactive. However, some biotransformation products have enhanced activity or toxic properties, including mutagenicity, teratogenicity, and carcinogenicity. This observation undermines the once popular theory that drug-biotransforming enzymes evolved as a biochemical defense mechanism for the detoxification of environmental xenobiotics. It is noteworthy that the synthesis of endogenous substrates such as steroid hormones, cholesterol, and bile acids involves many enzyme-catalyzed pathways associated with the metabolism of xenobiotics. The same is true of the formation and excretion of endogenous metabolic products such as bilirubin, the end catabolite of heme. Finally, drug-metabolizing enzymes have been exploited through the design of pharmacologically inactive pro-drugs that are converted in vivo to the pharmacologically active species.
WHY IS DRUG BIOTRANSFORMATION NECESSARY? Renal excretion plays a pivotal role in terminating the biologic activity of a few drugs, particularly those that have small molecular volumes or possess polar characteristics such as functional groups fully ionized at physiologic pH. Most drugs do not possess such physicochemical properties. Pharmacologically active organic molecules tend to be highly lipophilic and remain un-ionized or only partially ionized at physiologic pH. They are often strongly bound to plasma proteins. Such substances are not readily filtered at the glomerulus. The lipophilic nature of renal tubular membranes also facilitates the reabsorption of hydrophobic compounds following their glomerular filtration. Consequently, most drugs would have a prolonged duration of action if termination of their action depended solely on renal excretion. An alternative process that may lead to the termination or alteration of biologic activity is metabolism. In general, lipophilic xenobiotics are transformed to more polar and hence more readily excretable products. The role metabolism may play in the inactivation of lipid-soluble drugs can be quite dramatic. For example, lipophilic barbiturates such as thiopental and phenobarbital would have half-lives greater than 100 years if it were not for their metabolic conversion to more water-soluble compounds.
THE ROLE OF BIOTRANSFORMATION IN DRUG DISPOSITION. Most metabolic biotransformations occur at some point between absorption of the drug into the general circulation and its renal elimination. A few transformations occur in the intestinal lumen or intestinal wall. In general, all of these reactions can be assigned to one of 2 major categories, called phase I and phase II reactions.
Phase I reactions usually convert the parent drug to a more polar metabolite by introducing or unmasking a functional group (-OH, -NH2, -SH). Often these metabolites are inactive, although in some instances activity is only modified.
If phase I metabolites are sufficiently polar, they may be readily excreted. However, many phase 1 products are not eliminated rapidly and undergo a subsequent reaction in which an endogenous substrate such as glucuronic acid, sulfuric acid, acetic acid, or an amino acid combines with the newly established functional group to form a highly polar conjugate. Such conjugation or synthetic reactions are the hallmarks of phase II metabolism. A great variety of drugs undergo these sequential biotransformation reactions, although in some instances the parent drug may already possess a functional group that may form a conjugate directly. For example, the hydrazide moiety of isoniazid is known to form an N-acetyl conjugates – in a phase II reaction – that is a substrate for a phase I type reaction, namely, hydrolysis to isonicotinic acid. Thus, phase II reactions may actually precede phase I reactions.
WHERE DO DRUG BIOTRANSFORMATIONS OCCUR? Although every tissue has some ability t( metabolize drugs, the liver is the principal organ o drug metabolism. Other tissues that display consider able activity include the gastrointestinal tract, the lungs, the skin, and the kidneys. Following oral ad ministration, many drugs (eg, isoproterenol, meperi dine. pentazocine, morphine) are absorbed intact from the small intestine and transported first via the portal system to the liver, where they undergo extensive metabolism. This process has been called a first-pass effect. Some orally administered drugs (eg, clonazepam, chlorpromazine) are more extensively metabolized in the intestine than in the liver. Thus, intestinal metabolism may contribute to the overall first-pass effect. First-pass effects may so greatly limit the bioavailability of orally administered drugs that alternative routes of administration must be employed to achieve therapeutically effective blood levels. The lower gut harbors intestinal microorganisms that are capable of many biotransformation reactions. In addition, drugs may be metabolized by gastric acid (eg, penicillin), digestive enzymes (eg, polypeptides such as insulin), or by enzymes in the wall of the intestine (eg, sympathomimetic catecholamines).
Although drug biotransformation in vivo can occur by spontaneous, noncatalyzed chemical reactions, the vast majority are catalyzed by specific cellular enzymes. At the subcellular level, these enzymes may be located in the endoplasmic reticulum, mitochondria, cytosol, lysosomes, or even the nuclear envelope or plasma membrane.
MICROSOMAL MIXED FUNCTION OXIDASE SYSTEM
Many drug-metabolizing enzymes are located in the lipophilic membranes of the endoplasmic reticulum of the liver and other tissues. When these lamellar membranes are isolated by homogenization and fractionation of the cell, they re-form into vesicles called microsomes. Microsomes retain most of the morphologic and functional characteristics of the intact membranes, including the rough and smooth surface features of the rough (ribosome-studded) and smooth (no ribosomes) endoplasmic reticulum. Whereas the rough microsomes tend to be dedicated to protein synthesis, the smooth microsomes are relatively rich in enzymes responsible for oxidative drug metabolism. In particular, they contain the important class of enzymes known as the mixed function oxidases (MFO), or monooxygenases. The activity of this enzyme system requires both a reducing agent (NADPH) and molecular oxygen; in a typical reaction, one molecule of oxygen is consumed (reduced) per substrate molecule, with one oxygen atom appearing in the product and the other in the form of water.
In this oxidation-reduction process, 2 microsomal enzymes play a key role. The first of these is a flavo-protein, NADPH-cytochrome P-450 reductase. One mol of this enzyme (molecular weight ~ 80,000) contains 1 mol each of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Because cytochrome c can serve as an electron acceptor, the enzyme is often referred to as NADPH-cytochrome c reductase. The second microsomal enzyme is a hemoprotein called cytochrome P-450 and serves as the terminal oxidase. The name cytochrome P-450 is derived from the spectral properties of this hemoprotein. In its reduced (ferrous) form, it binds carbon monoxide to give a ferrocarbonyl adduct that absorbs maximally in the visible region of the electromagnetic spectrum at 450 nm. As with other naturally occurring heme-containing proteins, the iron present in this molecule is complexed with protoporphyrin IX. Over half of the heme synthesized in the liver is committed to hepatic cytochrome P-450 formation. The relative abundance of cytochrome P-450, as compared to that of the reductase in the liver, contributes to making cytochrome P-450 heme reduction the rate-limiting step in hepatic drug oxidations.
Microsomal drug oxidations require cytochrome P-450, cytochrome P-450 reductase, NADPH, and molecular oxygen. Briefly, oxidized (Fe+) cytochrome P-450 combines with a drug substrate to form a binary complex (step 1). NADPH donates an electron to the flavoprotein reductase, which in turn reduces the oxidized cytochrome P-450-drug complex (step 2). A second electron is introduced from NADPH via the same flavoprotein reductase, which serves to reduce molecular oxygen and to form an “activated oxygen “-cytochrome P-450-substrate complex (step 3). This complex in turn transfers ‘ ‘activated” oxygen to the drug substrate to form the oxidized product (step 4).
The potent oxidizing properties of this activated oxygen permit oxidation of a large number of substrates. Substrate specificity is very low for this enzyme complex. High solubility in lipids is the only common structural feature of the wide variety of structurally unrelated drugs and chemicals that serve as substrates in this system.
Enzyme induction
An interesting feature of some of these chemically dissimilar drug substrates is their ability, on repeated administration, to ‘ ‘induce” cytochrome P-450 by enhancing the rate of its synthesis or reducing its rate of degradation. Induction results in an acceleration of metabolism and usually in a decrease in the phar-macologic action of the inducer and also ofcoadminis-tered drugs. However, in the case of drugs meta-bolically transformed to reactive intermediates, enzyme induction may exacerbate drug-mediated tissue toxicity.
Various substrates appear to induce forms of cytochrome P-450 having different molecular weights and exhibiting different substrate specificities and im-munochemical and spectral characteristics. The 2 isozymes that have been most extensively studied are: 1). cytochrome P-450b, or LMz (for liver microsomal form 2), which is induced by treatment with phenobar-bital; and 2). cytochrome P-448 (cytochrome Pi-450, or P-450c, or LIVLi), which is induced by polycyclic aromatic hydrocarbons, of which 3-methylcholanthrene is a prototype. Environmental pollutants are capable of inducing cytochrome P-450. For example, exposure to benzo(a)pyrene, present in tobacco smoke, charcoal-broiled meat, and other organic pyrolysis products is known to induce cytochrome P-448 and to alter the rates of drug metabolism in both experimental animals and in humans. Other environmental chemicals known to induce specific cytochrome P-450 isozymes include the polychlorinated biphenyls (PCBs), which are used widely in industry as insulating materials and plasticizers, and 2,3,7,8-tetrachlorodibenzo-beta-dioxon (dioxin, TCDD), a trace by-product of the chemical synthesis of the defoliant 2,4,5-trichlorophenol.
Enzyme inhibition
Other drug substrates may inhibit cytochrome P-450 enzyme activity. A well-known inhibitor is proadifen. This compound binds avidly to the cytochrome molecule and thereby competitively inhibits the metabolism of potential substrates. Cimetidine is a popular therapeutic agent that has been found to impair the in vivo metabolism of other drugs by the same mechanism. Some substrates irreversibly inhibit cytochrome P-450 via covalent interaction of a metabolically generated reactive intermediate that may react with either the apoprotein or the heme moiety of the cytochrome. A growing list of such inhibitors includes the steroids ethinylestradiol, norethindrone, and spironolactone; the anesthetic agent fluroxene; the barbiturates secobarbital and allobarbital; the analgesic sedatives allylisopropylacetylurea, diethylpentenamide, and ethchlorvynol; the solvent carbon disulfide; and propylthiouracil.
PHASE II REACTIONS
Parent drugs or their phase I metabolites that contain suitable chemical groups often undergo coupling or conjugation reactions with an endogenous substance to yield drug conjugates (table 1). In general, conjugates are polar molecules that are readily excreted and often inactive. Conjugate formation involves high-energy intermediates and specific transfer enzymes. Such enzymes (transferases) may be located in microsomes or in the cytosol. They catalyze the coupling of an activated endogenous substance (such as the uridine 5′-diphosphate [UDP] derivative of glucuronic acid) with a drug (or endogenous compound), or of an activated drug (such as the S-CoA derivative of benzoic acid) with an endogenous substrate. Because the endogenous substrates originate in the diet, nutrition plays a critical role in the regulation of drug conjugations.
Drug conjugations were once believed to represent terminal inactivation events and as such have been viewed as “true detoxification” reactions. However, this concept must be modified, since it is now known that certain conjugation reactions (0-sulfation of N-hydroxyacetylaminofluorene and N-acetylation of isoniazid) may lead to the formation of reactive species responsible for the hepatotoxicity of the drug.
Table 1. Phase II reactions.
|
Type of conjugation
|
Endogenous reactant
|
Transferase (location)
|
Types of substrates
|
Examples
|
|
Glucuronidation
|
UDP glucuronic acid.
|
UDP-glucuronyl trans-ferase (microsomes).
|
Phenols, alcohols, carboxylic acids, hydroxylamines, sul-fonamides.
|
Nitrophenol, morphine, acetaminophen, diazepam, N-hydroxy-dapsone, sulfathiazole, meproba-mate.
|
|
Acetylation
|
Acetyl-CoA.
|
N-Acetyl transferase (cytosol).
|
Amines.
|
Sulfonamides, isoniazid, clonazepam, dapsone, mescaline.
|
|
Glutathione conjugation
|
Glutathione.
|
GSH-S-transferase (cytosol, microsomes).
|
Epoxides, arene oxides, nitro groups, hydroxylamines.
|
Ethacrynic acid, bromobenzene.
|
|
Glycine conjugation
|
Glycine.
|
Acyl-CoA glycine trans-ferase (mito-chondria).
|
Acyl-CoA derivatives of carboxy-lic acids.
|
Salicylic acid, benzoic acid, nico- tinic acid, cinnamic acid, cholic acid, deoxycholic acid.
|
|
Sulfate conjugation
|
PhosphoadenosyI phosphosulfate.
|
Sulfotransferase (cytosol).
|
Phenols, alcohols, aromatic amines.
|
Estrone, aniline, phenol, 3-hy-droxycoumarin, acetaminophen, methyldopa.
|
|
Methyla-tion
|
S-Adenosyl-methionine.
|
Transmethylases (cytosol).
|
Catecholamines, phenols, amines, histamine.
|
Dopamine, epinephrine, pyridine, histamine, thiouracil.
|
CLINICAL RELEVANCE OF DRUG METABOLISM
The dose and the frequency of administration required to achieve effective therapeutic blood and tissue levels vary in different patients because of individual differences in drug distribution and rates of drug metabolism and elimination. These differences are determined by genetic factors and nongenetic variables such as age, sex, liver size, liver function, circadian rhythm, body temperature, and nutritional and environmental factors such as concomitant exposure to inducers or inhibitors of drug metabolism. The discussion that follows will summarize the most important variables relating to drug metabolism that are of clinical relevance.
Individual Differences
Individual differences in metabolic rate depend on the nature of the drug itself. Thus, within the same population, steady-state plasma levels may reflect a 30-fold variation in the metabolism of one drug only a 2-fold variation in the metabolism of another.
Genetic factors that influence enzyme levels account for some of these differences. Succinylcholine, for example, is metabolized only half as rapidly in persons with genetically determined defects in pseudocholines-terase as iormals. Analogous pharmacogenetic differences are seen in the acetylation ofisoniazid and the hydroxylation of warfarin. Similarly, genetically determined defects in the oxidative metabolism of de-brisoquine, phenacetin, guanoxan, sparteine, and phenformin have been recently reported. The defects are apparently transmitted as autosomal recessive traits and may be expressed at any one of the multiple metabolic transformations that a chemical might undergo in vivo. Environmental factors also contribute to individual variations in drug metabolism. Cigarette smokers metabolize some drugs more rapidly thaonsmokers because of enzyme induction (see p 37). Industrial workers exposed to some pesticides metabolize certain drugs more rapidly thaonexposed individuals. Such differences make it difficult to determine effective and safe doses of drugs that have narrow therapeutic indices.
Age and sex
Increased susceptibility to the pharmacologic or toxic activity of drugs has been reported in very young and old patients as compared to young adults. Although this may reflect differences in absorption, distribution, and elimination, differences in drug metabolism cannot be ruled out—a possibility supported by studies in other mammalian species indicating that drugs are metabolized at reduced rates during the pre-pubertal period and senescence. Slower metabolism could be due to reduced activity of metabolic enzymes or reduced availability of essential endogenous cofac-tors. Similar trends have been observed in humans, but incontrovertible evidence is yet to be obtained.
The activities are divided by age …
Sex-dependent variations in drug metabolism have been well documented in rats but not in other rodents. Young adult male rats metabolize drugs much faster than mature female rats or prepubertal male rats. These differences in drug metabolism have been clearly associated with androgenic hormones. A few clinical reports suggest that similar sex-dependent differences in drug metabolism also exist in humans for benzodiazepines, estrogens, salicylates.
Drug-drug interactions during metabolism
Many substrates, by virtue of their relatively high lipophilicity, are retained not only at the active site of the enzyme but remaionspecifically bound to the lipid membrane of the endoplasmic reticulum. In this state, they may induce microsomal enzymes; depending on the residual drug levels at the active site, they also may competitively inhibit metabolism of a simultaneously administered drug. Such drugs include various sedative-hypnotics, tranquilizers, anticonvulsants, and insecticides (see table 2). Patients who routinely ingest barbiturates, other sedative-hypnotics, or tranquilizers may require considerably higher doses of warfarin or dicumarol, when being treated with these oral anticoagulants, to maintain a prolonged prothrombin time. On the other hand, discontinuation of the sedative may result in reduced metabolism of the anticoagulant and bleeding – a toxic effect of the enhanced plasma levels of the anticoagulant. Similar interactions have been observed in individuals receiving various combination drug regimens such as tranquilizers or sedatives with contraceptive agents, sedatives with anticonvulsant drugs, and even alcohol with hypoglycemic drugs (tolbutamide).
It must also be noted that an inducer may enhance not only the metabolism of other drugs but also its own metabolism. Thus, continued use of a drug may result in one form of tolerance – progressively reduced effectiveness due to enhancement of its own metabolism.
Conversely, simultaneous administration of 2 or more drugs may result in impaired elimination of the more slowly metabolized drug and prolongation or potentiation of its pharmacologic effects (table 3). Both competitive substrate inhibition and irreversible substrate-mediated enzyme inactivation may augment plasma drug levels and lead to toxic effects from drugs with narrow therapeutic indices. For example, it has been shown that dicumarol inhibits the metabolism of the anticonvulsant phenytoin and leads to the expression of side effects such as ataxia and drowsiness. Similarly, allopurinol both prolongs the duration and enhances the chemotherapeutic action of mercaptopurine by competitive inhibition of xanthine oxidase.
Consequently, to avoid bone marrow toxicity, the dose of mercaptopurine is usually reduced in patients receiving allopurinol. Cimetidine, a drug used in the treatment of peptic ulcer, has been shown to potentiate the pharmacologic actions of anticoagulants and sedatives. The metabolism of Chlordiazepoxide has been shown to be inhibited by 63% after a single dose of cimetidine; such effects are reversed within 48 hours after withdrawal of cimetidine. For such interactions to occur, drug metabolism must follow zero-order kinetics. Elimination of most drugs proceeds, however, by exponential (first-order) kinetics, thus greatly reducing the probability of such metabolically dependent interactions.
Impairment of metabolism may also result if a simultaneously administered drug irreversibly inactivates a common metabolizing enzyme, as is the case with secobarbital or novonal (diethylpentenamide) overdoses. These compounds, in the course of their metabolism by cytochrome P-450, inactivate the enzyme and result in impairment of their own metabolism and that of other cosubstrates.
Interactions between drugs and endogenous compounds
Various drugs require conjugation with endogenous substrates such as glutathione, glucuronic acid, and sulfuric acid for their inactivation. Consequently, different drugs may compete for the same endogenous substrates, and the faster-reacting drug may effectively deplete endogenous substrate levels and impair the metabolism of the slower-reacting drug. If the latter has a steep dose-response curve or a narrow margin of safety, potentiation of its pharmacologic and toxic effects may result.
Diseases affecting drug metabolism
Acute or chronic diseases that affect liver architecture or function markedly affect hepatic metabolism of some drugs. Such conditions include fat accumulation, alcoholic hepatitis, active or inactive alcoholic cirrhosis, hemochromatosis, chronic active hepatitis, biliary cirrhosis, and acute viral or drug hepatitis. Depending on their severity, these conditions impair hepatic drug-metabolizing enzymes, particularly microsomal oxidases, and thereby markedly affect drug elimination. For example, the half-lives of Chlordiazepoxide and diazepam in patients with liver cirrhosis or acute viral hepatitis are greatly increased, with a corresponding prolongation of their effects. Consequently, these drugs may cause coma in patients with liver disease when given in ordinary doses.
Liver cancer has been reported to impair hepatic drug metabolism in humans. For example, aminopyrine metabolism is slower in patients with malignant hepatic tumors than iormal controls. These patients also exhibit markedly diminished aminopyrine clearance rates. Studies with biopsy specimens of livers from patients with hepatocellular carcinoma also indicate impaired ability to oxidatively metabolize drugs in vitro. This is associated with a correspondingly reduced cytochrome P-450 content.
Cardiac disease, by limiting blood flow to the liver, may impair disposition of those drugs whose metabolism is flow-limited. These drugs are so readily metabolized by the liver that hepatic clearance is essentially equal to liver blood flow. Pulmonary disease may affect drug metabolism as indicated by the impaired hydrolysis of procainamide and procaine in patients with chronic respiratory insufficiency and the increased half-life of antipyrine in patients with lung cancer. Impairment of enzyme activity or defective formation of enzymes associated with heavy metal poisoning or porphyria also results in reduction of hepatic drug metabolism. For example, lead poisoning has been shown to increase the half-life of antipyrine in humans.
Although the effects of endocrine dysfunction on drug metabolism have been well explored in experimental animal models, corresponding data for humans with endocrine disorders are scanty. Thyroid dysfunction has been associated with altered metabolism of some drugs and of some endogenous compounds as well. Hypothyroidism increases the half-life of antipyrine, digoxin, methamizole, andpractolol, as hyperthyroidism has the opposite effect. A few clinical studies in diabetic patients indicate no apparent impairment of drug metabolism, as reflected by the half-lives of antipyrine, tolbutamide, and phenylbutazone. In contrast, the metabolism of several drugs is impaired in male rats treated with diabetogenic agents such as alloxan or streptozocin. These alterations are abolished by administration of insulin, which has no direct influence on hepatic drug-metabolizing enzymes. Malfunctions of the pituitary, adrenal cortex, and gonads markedly impair hepatic drug metabolism in rats. On the basis of these findings, it may be supposed that such disorders could significantly affect drug metabolism in humans. However, until sufficient evidence is obtained from clinical studies in patients, such extrapolations must be considered tentative.
Rapidly metabolized drugs whose hepatic clearance is blood flow-limited: Alprenolol, Lidocaine, Meperidine, Morphine, Pentazocine, Propoxyphene, Propranolol, Verapamil, Amitriptyline, Chlormethiazole, Desipramine, Imipramine, Isoniazid, Labetalol.
METABOLISM OF DRUGS TO TOXIC PRODUCTS
Metabolism of drugs and other foreign chemicals may not always be an innocuous biochemical event leading to detoxification and elimination of the compound. Indeed, several compounds have been shown to be metabolically transformed to reactive intermediates that are toxic to various organs. Such toxic reactions may not be apparent at low levels of exposure to parent compounds when alternative detoxification mechanisms are not yet overwhelmed or compromised and the availability of endogenous detoxifying cosubstrates (glutathione, glucuronic acid, sulfate) is not limited. However, when these possibilities are exhausted, the toxic pathway may prevail resulting in overt organ toxicity or carcinogenesis. The number of specific examples of such drug-induced toxicity is expanding rapidly. An example is acetaminophen (paracetamol-induced hepatotoxicity. This analgesic antipyretic drug is quite safe in therapeutic doses (1.2 g/daily). It normally undergoes glucuronidation and sulfation to the corresponding conjugates, which together comprise 95% of the total excreted metabo-lites. The alternative cytochrome P-450-dependent glutathione (GSH) conjugation pathway accounts for the remaining 5%. When acetaminophen intake far exceeds therapeutic doses, the glueuronidation and sulfation pathways are saturated, and the cytochrome P-450-dependent pathway becomes increasingly important. Little or no hepatotoxicity results as long as glutathione is available for conjugation. However, with time, hepatic glutathione is depleted faster than it can be regenerated, and accumulation of a reactive and toxic metabolite occurs. In the absence of intracellular nucleophiles such as glutathione, this reactive metabolite (thought to be an N-hydroxylated product or an N-acetylbenzoiminoquinone) reacts with nucleophilic groups present on cellular macro-molecules such as protein, resulting in hepatotoxicity.
The chemical and toxicologic characterization of the electrophilic nature of the reactive acetaminophen metabolite has led to the development of effective antidotes—cysteamine and acetylcysteine (Mucomyst). Administration of acetylcysteine (the safer of the 2) within 24 hours following acetaminophen over-dosage has been shown to protect victims from fulminant hepatotoxicity and death.
Similar mechanistic interpretations can be invoked to explain the nephrotoxicity of phenacetin and the hepatotoxicity of aflatoxin and of benzo(a)pyrene, a pyrolytic product of organic matter present in cigarette tar and smoke and in smoked foods.
1. http://www.medicinethroughtime.co.uk/Medicine_worksheets/videos.htm
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9. http://www.apchute.com/moa.htm
