406 Chapter 7 Drug Metabolism 2.Glucuronic Acid Conjugation 456 3.Sulfate Conjugation. 460 4.Amino Acid Conjugation. 462 5.Glutathione Conjugation 464 6.Water Conjugation............... 466 7.Acetyl Conjugation........ 466 8.Fatty Acid and Cholesterol Conjugation. 468 9.Methyl Conjugation 469 D.Hard and Soft Drugs;Antedrugs 471 7.5 General References 473 7.5 Problems..… 475 7.7 References. 479 ☐7.1 Introduction When a foreign organism enters the body,the immune system produces antibodies to interact with and destroy it.Small molecules,however,do not stimulate antibody production.So how has the human body evolved to protect itself against low molecular weight environmental pollutants?The principal mechanism is the use of nonspecific enzymes that transform the foreign compounds(often highly nonpolar molecules)into polar molecules that are excreted by the normal bodily processes.Although this mechanism to rid the body of xenobiotics (molecules foreign to the organism)is highly desirable,especially when one considers all of the foreign materials to which we are exposed every day,it can cause problems when the foreign agent is a drug that we want to enter and be retained in the body sufficiently long to be effective. The enzymatic biotransformations of drugs is known as drug metabolism.Because many drugs have structures similar to those of endogenous compounds,drugs may get metabolized by specific enzymes for the related natural substrates as well as by nonspecific enzymes. The principal site of drug metabolism is the liver,but the kidneys,lungs,and GI tract also are important metabolic sites.When a drug is taken orally (the most common route of administration),it is usually absorbed through the mucous membrane of the small intestine or from the stomach.Once out of the GI tract it is carried by the bloodstream to the liver where it is usually first metabolized.Metabolism by liver enzymes prior to the drug reaching the sys- temic circulation is called the presystemic or first-pass effect.which may result in complete deactivation of the drug.If a large fraction of the drug is metabolized,then larger or multiple doses of the drug will be required to get the desired effect.Another undesirable effect of drug metabolism is that occasionally the metabolites of a drug may be toxic.even though the drug is not. The first-pass effect sometimes can be avoided by changing the route of drug admin- istration.The sublingual route (the drug is placed under the tongue)bypasses the liver. After absorption through the buccal cavity,the drug enters the systemie circulation.This is the route employed with nitroglycerin (7.1.Nitrostat),a drug used for the treatment of angina pectoris that is converted by mitochondrial aldehyde dehydrogenase to nitrite ion. which is then reduced to nitric oxide a second messenger molecule that dilates blood ves- sels in the heart.The recal roue in the form of a solid suppository or in solution as an
Section 7.1 Introduction enema.leads to absorption through the colon mucosa.Ergotamine (7.2.Ergomar).a drug 407 for migraine headaches,is administered this way (who would have guessed) (iv.)injection introduces the drug directly into the systemic circulation and is used when a rapid therapeutic response is desired.The effects are almost immediate when drugs are administered by this route,because the total blood circulation time in humans is 15-20 sec. Intramuscular (i.m.)injection is used when large volumes of drugs need to be administered. if slow absorption is desirable,or if the drug is unstable in the gastric acid of the stomach. A subcutaneous (s.c.)injection delivers the drug through the loose connective tissue of the subcutaneous layer of the skin.Another method of administration.particularly for gaseous or highly volatile drugs such as general anesthetics,is by pulmonary absorption through the respiratory tract.The asthma drug isoproterenol (7.3.Isuprel)is metabolized in the intestines and liver.but administration by aerosol inhalation is effective at getting the drug directly to the bronchi.Topical applicarion of the drug to the skin or a mucous membrane is used for local effects;few drugs readily penetrate the intact skin.Not all drugs can be administered by these alternate routes,so their structures may have to be altered to minimize the first-pass effect or to permit them to be administered by one of these alternate routes.These struc- tural modification approaches in drug design to avoid the first-pass effect are discussed in Chapter 8.Even if the first-pass effect is avoided,there are many enzymes in tissues other than the liver that are capable of catalyzing drug metabolism reactions.Once a drug has reached its site of action and elicited the desired response,it usually is desirable for the drug to be metabolized and eliminated at a reasonable rate.Otherwise,it may remain in the body and produce the effect longer than desired or it could accumulate and become toxie to the cells. CH HO NHI ONO2 ONO2 HO ONO2 HO H nitroglycerin ergotamine isoproterenol 7.1 7.2 7.3 Drug metabolism studies are essential for evaluating the potential safety and efficacy of drugs.Consequently,prior to approval of a drug for human use,an understanding of the metabolie pathways and disposition of the drug in humans and in preclinical animal species is required.The animal species used for metabolism studies are often those in which the toxicological evaluations are conducted.Additional toxicological studies have to be carried out on metabolites found in humans that were not observed in the animal metabolism studies. Metabolism studies also can be a useful lead modification approach.For example,after many years on the drug market,terfenadine(7.4.R=CH3:Seldane)was removed because it was found to cause life-threatening cardiac arrhythmias when coadministered with inhibitors of hepatic cytochrome P450.such as erythromycin and ketoconazole.2 The active metabolite
408 Chapter 7 Drug Metaboliam of terfenadine,fexofenadine (7.4.RCOOH:Allegra),however,produces no arhythmias. and it has replaced terfenadine on the market.31 CH HO OH terfenadine HCI(R=CH3) fexofenadine HCI(R COOH) 7.4 Once the metabolic products are known,it is possible to design a compound that is inactive when administered,but which utilizes the metabolic enzymes to convert it into the active form. These compounds are known as prodrugs,and are discussed in Chapter 8.In this chapter we consider the various reactions that are involved in the biotransformations of drugs.Because only very small quantities of drugs generally are required to elicit the appropriate response, it may be difficult to detect all of the metabolic products.To increase the sensitivity of the detection process,drug candidates are typically radioactively labeled.Radioactive compounds are useful for studying all aspects of absorption,distribution,metabolism,and excretion (ADME).4 Metabolite studies often can be done directly by tandem mass spectrometry/mass spectrometry techniques.51 In the next two sections we look briefly at how radiolabeling is carried out,how metabolites are detected,and how their structures are elucidated. 7，.2 Synthesis of Radioactive Compounds Because of the sensitivity of detection of particles of radioactive decay,a common approach used for detection,quantification,and profiling of metabolites in whole-animal studies is the incorporation of a radioactive label,typically a weak B-emitter such as 14Cl6]or3H.7 into the drug molecule.When this approach is used,it does not matter how few metabolites are produced or how small the quantities of metabolites,even in the presence of a large number of endogenous compounds.Only the radioactively labeled compounds are isolated from the urine and the feces of the animals,and the structures of these metabolites are elucidated(see Section 7.3).If one of the carbon atoms of a drug is metabolized to carbon dioxide.as is the case of erythromycin(7.5.Erythrocin),14C labeling of the carbon atom that becomes CO2(the NMe2 methyl groups of erythromyein are oxidized to CO2),makes it possible to measure the rate of metabolism of the compound by measuring the rate of exhaled To incorporate a radioactive label into a compound,a synthesis must be designed so that a commercially available,radioactively labeled compound or reagent can be used in one of the steps.It is highly preferably to incorporate the radioactive moiety in a step at or near the end of the synthesis because once the radioactivity is introduced.the scale of the reaction is generally diminished and special precautions and procedures regarding radiation safety and disposal of radioactive waste must be followed.Often the radioactive synthesis is quite different fromor longer than the synthesis of the unlabeled compound in order touse availableradioactive material.It is preferable to preparebeed hensrated in the drug.the site ofnrtion must be su that loss of therim by exchange with the medium does notoccurevenfer an early metabolic step
Secon 7.2 Synthesis of Radioactive Compounds Generally,only one radioactive label is incorporated in a drug because drug metabolism 409 typicalyadstoastrucurewthiamiooemoeyobk farenough inasynthesis,however.it is possible to synthesizeadrug with several om radioactively labeled.Radioactive labeling at multipeiofaoceoudethe identification of more fragments of the drug and consequently the elucidation of metabolite structures and the fate of the molecule invivo. Me Me HO OH OH Me Me O.HOZ OMe Me Me Me -OH Me erythromycin 7.5 Industrially,the radioactive drug is synthesized with high specific radioactivity (a measure of the amount of radioactivity per mole of compound),often>57 mCi/mmol of14C(the theo- retical maximum is 64 mCi/mmol).When needed,the specific radioactivity is diluted with non-[14C]-labeled drug for use in metabolism studies.Typically,commercially available radio- active compounds have relatively low specific radioactivities.This means that may be only one in 106 or fewer molecules actually contains the radioactive tag:the remainder of the molecules are unlabeled and are carriers of the relatively few radioactive molecules.In the case of 4C there will be no difference in the reactivity of the labeled and unlabeled molecules. so the statistical amount of radioactivity in the products formed is the same as that in the starting materials.The specific radioactivity of the metabolites formed during metabolism, then,should be identical to the specific radioactivity of the drug.In the case of tritiated drugs. however,if a carbon-hydrogen bond is broken,the radiolabel will be lost as tritiated water, and satisfactory recovery of total radioactivity in animal studies cannot occur.Also,a kineric isorope effect will occur on those molecules that are tritiated.This will lead to metabolite formation with a lower specific radioactivity than that of the drug.As a result,quantitation of the various metabolic pathways,where some involve C-H bond cleavage and others do not,may require knowledge of the tritium isotope effect.This,then,is another reason why it is preferable to use [4C]-labeling of a drug for metabolism studies rather than tritium labeling. If the drug is a natural product or derivative of a natural product,the easiest procedure for incorporation of a radioactive label could be a biosynthetie approach.namely,to grow the organism that produces the natural product in the presence of a radioactive precursor.and let Nature incorporate the radioactivity into the molecule.Because of the volume of media generally involved and,therefore,the large amount of radioactive precursor required,this could be a very expensive approach:however,generally the expense is compensated by the ease of the method and the attractive yield of product obtained. An example ofadrug class that could use this approach is the penicillins.which are biosyn- thesized by Penicillium fungi from valine,cysteine,and various carboxylic acids(Scheme7.1). Valine is commercially available with a c label at the carboxylate carbon or it may be
Chapter 7 Drug Metabolism 410 +HS P所 COOH NH Penicillim +COOH Scheme 7.1Biosynthesis of penicillins (iPr)2EtN H NO 98% Cbz-Cl NaHCO 91% 00 85% 1.MsCI/EN Ac2O 2.NaNs pyridine 3.H2/Pd-C 68% CH 93% linezolid 7.7 Scheme 7.2Chemical synthesis of linezolid obtained uniformly labeled:that is,all of the carbon atoms are labeled to some small extent with 4C(albeit very few molecules would contain all of the carbon atoms labeled in the same molecule).It also can be purchased with a tritium label at the 2-and 3-positions or at the 3-and 4-positions.Cysteine is available uniformly labeled in 14C or with a35s label. Penicillin G could be produced if phenylacetic acid(available with a 4C label at either the 1-or 2-position)were inoculated into the Penicillium growth medium. If the drug is not a natural product (the more common case)a chemical synthesis must be carried out.For example,the synthesis of the first in a new class of antibacterial drugs. (7.Zyvx)sshowninScheme72The last step in the synthesis.acetytion of heprmymne can becedouth Clacetic anhydride toinraedoacive labethe acetyl group of 7.7.The oxoido earbonyl carbon lso coul have been abenCC-Clomke.6 but radioactive Cbz-Clsommly availablesoit would have had to be synthesized fromChosgene and benzyl alcohol