Organic Chemistry Lecture 02 PDF

PRODRUG Initially, the term prodrug referred to a pharmacologically inactive compound that is transformed by the human body into an active substance by either chemical or metabolic means. This included both compounds that are designed to undergo a transformation to yield an active substance an...

PRODRUG Initially, the term prodrug referred to a pharmacologically inactive compound that is transformed by the human body into an active substance by either chemical or metabolic means. This included both compounds that are designed to undergo a transformation to yield an active substance and those that were discovered by chance. Drug latenation refers to drugs specifically designed to require bioactivation. The type of prodrug to be produced depends on the specific type of the drug's action that requires improvement and the type of functionality that is present in the active drug. Generally, prodrug approaches are done to improve patient acceptability of the agent (i.e., reduce pain associated with administration), alter absorption, alter distribution, alter metabolism, or alter elimination. The chemical nature of the prodrugs that can be prepared is limited by the chemical nature of the active species. Recently, the terms hard drugs and soft drugs were introduced. Hard drugs are compounds that are designed to contain the structural characteristics necessary for pharmacological activity but in a form that is not susceptible to metabolic or chemical transformation. In this way, the production of any toxic metabolite is avoided, and there is increased efficiency of action. Since the drug is not inactivated by metabolism, it may be less readily eliminated. Soft drugs are active compounds that after exerting their desired pharmacological effect arc designed to undergo metabolic inactivation to give a nontoxic product. Thus soft drugs are considered to be the opposite of prodrugs. BASIC CONCEPTS A prodrug by definition is inactive and must be converted into an active species within the biological system. There are a variety of mechanisms by which this conversion maybe accomplished. Generally, the conversion to an active form is most often carried out by metabolizing enzymes within the body. Conversion to an active form may be accomplished by chemical means (e.g., hydrolysis or decarboxylation). Prodrugs can be grouped into carrier-linked prodrugs and bioprecursor prodrugs. Carrier-linked prodrugs are drugs that have been attached through a metabolically labile linkage to another molecule, the so-called promoiety, which is not necessary for activity but may impart some desirable property to the drug, such as increased lipid or water solubility or site-directed delivery. 1  Several advantages may be gained by generating a prodrug: increased absorption, alleviation of pain at the site of injection if the agent is given parenterally, elimination of an unpleasant taste associated with the drug, decreased toxicity, decreased metabolic inactivation, increased chemical stability, and prolonged or shortened action, whichever is desired in a particular agent. Administration of a drug parenterally may cause pain at the site of injection, especially if the drug begins to precipitate out of solution and damage the surrounding tissue. This situation can be remedied by preparing a drug with increased solubility in the administered solvent. Since chloramphenicol has low water solubility, the succinate ester was prepared to increase the water solubility of the agent and facilitate parenteral administration. The succinate ester used is inactive as an antibacterial agent, so it must be converted to chloramphenicol for this agent to be effective. This occurs in the plasma to give the active drug and succinate. The ester hydrolysis reaction can be catalyzed by esterases present enlarge amounts in the plasma. The ability to prepare ester-type prodrugs depends, of course, on the presence of either a hydroxyl group or a carboxyl moiety in the drug molecule. OH Cl OH Cl NH Cl NH – Cl O + O – N HO O + O O N O O C – + O O Na chloramphenicol O O chloramphenicol succinate + HO – Na O O sodium 3-carboxypropanoate The promoiety should be easily and completely removed after it has served its function and should be nontoxic, as in the case with succinate. The selection of the appropriate promoiety depends on which properties are required for the agent. If it is desirable to increase water solubility, then a promoiety containing an ionizable function or numerous polar functional groups is used. If on the other hand, the goal is to increase lipid solubility or decrease water solubility, a nonpolar promoiety is appropriate. A slight variation on the currier-linked prodrug approach is seen with mutual prodrugs in which the carrier also has activity. The antineoplastic agent estramustine, 2 which is used in the treatment of prostatic cancer, provides an example of such an approach. Estramustine is composed of a phosphorylated steroid (17-estradiol) linked to a normustard [HN(CH2CH2Cl)2] through a carbamate linkage. The steroid portion of the molecule helps to concentrate the drug in the prostate, where hydrolysis occurs to give the normustard and the normustard then acts as an alkylating agent and elicits a cytotoxic effect. The 17-estradiol has an antiandrogenic effect on the prostate and thereby, slows the growth of the cancer cells. Since both the steroid and the mustard possess activity, estramustine is termed a mutual prodrug. Note that phosphorylation of the estradiol can be used to increase the water solubility, which also constitutes a prodrug modification. Both types of esters (carbamates and phosphates) are hydrolyzed by chemical or enzymatic means. OH OH H H H H O HO H H Cl 17-estradiol N O Cl Cl estramustine NH + CO2 + PO 4 Cl In contrast to carrier-linked prodrugs, bioprecursor prodrugs contain no promoiety but rather rely on metabolism to introduce the functionality necessary to create an active species, e.g., the NSAID sulindac is inactive as the sulfoxide and must be reduced metabolically to the active sulfide. Sulindac is administered orally, absorbed in the small intestine, and subsequently reduced to the active species. Administration of the inactive form has the benefit of reducing the gastrointestinal (GI) irritation associated with the sulfide. This example also illustrates one of the problems associated with this approach, namely, participation of alternate metabolic paths that may inactivate the compound. In this case, after absorption of sulindac, irreversible metabolic oxidation of the sulfoxide to the sulfone can also occur to give an inactive compound. 3  Although seen less frequently, some prodrugs rely on chemical mechanisms for conversion of the prodrug to its active form. For example, hetacillin is a prodrug form of ampicillin in which the amide nitrogen and -amino functionalities have been allowed to react with acetone to give an imidazolidinone ring system. This decreases the basicity of the -amino group and reduces protonation in the small intestine so that the agent is more lipophilic. In this manner, the absorption of the drug from the small intestine is increased after oral dosing, and chemical hydrolysis after absorption regenerates ampicillin. In such an approach, the added moiety, or promoiety, in this case acetone, must be nontoxic and easily removed after it has performed its function. PRODRUGS OF FUNCTIONAL GROUPS There are a variety of different types of prodrugs. The major types of prodrugs (grouped according to functional group) and bioprecursor drugs (grouped according to type of metabolic activation), however, are discussed briefly below. Carboxylic Acids and Alcohols Prodrugs of agents that contain carboxylic acid or alcohol functionalities can often be prepared by conversion to an ester. This is the most common type of prodrug because of the ease with which the ester can be hydrolyzed to give the active drug. 4 Hydrolysis is normally accomplished by esterase enzymes present in plasma and other tissues that are capable of hydrolyzing a wide variety of ester linkages. Types of esterases that prodrugs may use are: ester hydrolase, lipase, cholesterol esterase, acetylcholinesterase, carboxypeptidase and cholinesterase. Activation of ester prodrugs In addition to these agents, microflora present within the gut produce a wide variety of enzymes that can hydrolyze esters. Chemical hydrolysis of the ester function may also occur to some extent. An additional factor that has contributed to the popularity of esters as prodrugs is the ease with which they can be formed, if the drug molecule contains either an alcohol or carboxylic acid functionality, an ester prodrug may be synthesized easily. The carboxylic or alcohol pro-moiety can be chosen to provide a wide range of lipophilic or hydrophilic properties to the drug, depending on what is desired. Manipulation of the scene and electronic properties of the pro-moiety allows control of the rate and extent of hydrolysis. This can be an important consideration when the active drug must be revealed at the correct point in its movement through the biological system. When it is desired to decrease water solubility, a nonpolar alcohol or carboxylic acid is chosen as the prodrug moiety. Decreasing the hydrophilicity of the compound may yield a number of benefits, including increased absorption, decreased dissolution in the aqueous environment of the stomach, and a longer duration of action. An example of increased absorption by the addition of a nonpolar carboxylic acid is seen with dipivefrin HCI. This is a prodrug form of epinephrine in which the catechol hydroxyl groups have been used in the formation of an ester linkage with pivalic acid. The agent is used in the treatment of open-angle glaucoma. The increased lipophilicity relative to epinephrine allows the agent, when applied to move across the membrane of the eye easily and achieve higher intraocular concentrations. Hydrolysis of the ester functions then occurs in the cornea, conjunctiva, and aqueous humor to generate the 5 active form, epinephrine. Using pivalic acid as the promoiety increases the steric bulk around the ester bond, which slows the ester hydrolysis relative to less bulky groups, yet still allows this reaction to proceed after the drug has crossed the membrane harriers of the eye. In addition to this benefit, the catechol system is somewhat susceptible to oxidation, and protecting the catechol as the diester prevents this oxidation and the resulting drug inactivation. Decreasing the water solubility of a drug by the formation of a prodrug may have additional benefits beyond simply increasing absorption. A number of agents have an unpleasant taste when given orally. This results when the drug begins to dissolve in the mouth and then is capable of interacting with taste receptors. This can present a significant problem, especially in pediatric patients and may lead to low compliance. A prodrug with reduced water solubility does not dissolve to any appreciable extent in the mouth and, therefore, does not interact with taste receptors. This approach has been used in the case of the antibacterial chloramphenicol, which produces a bitter taste when given as the parent drug. The hydrophobic palmitate ester does not dissolve to any appreciable extent in the mouth, so there is little chance for interaction with taste receptors. The ester moiety is subsequently hydrolyzed in the GI tract and the agent is absorbed as chloramphenicol. Other examples of ester prodrugs to overcome the unpleasant taste are: N-Acetyl sulfisoxazole, N-Acetyl sulfamethoxypyridazine, erythmmycin estolate, clindamycin palmitate, and troleandomycin. Not all carboxylic esters are easily hydrolyzed in vivo. Steric inhibition around the ester in some cases prevents the prodrug from being hydrolyzed. This is seen in the -lactams, in which it is often desirable to increase the hydrophobicity of the agent to improve absorption or prevent dissolution in the stomach where acid-catalyzed 6 decomposition may occur. Simple esters of the carboxylic acid moiety, however, are not hydrolyzed in vivo to the active carboxylate. A solution to this problem was to use the so-called double ester approach, in which an additional ester or carbonate function is incorporated into the R2 substituent further removed from the heterocyclic nucleus. Hydrolysis of such a function occurred readily, and the moiety was selected that chemical hydrolysis of the second ester occurred quickly. This is seen in the cephalosporin cefpodoxime proxetil, where a carbonate function was used. The carbonate is also susceptible to the action of esterase enzymes, and the unstable product undergoes further reaction to give the active carboxylate. This approach is frequently used to improve absorption or prevent dissolution in the stomach and the subsequent acid-catalyzed decomposition of aminopenicillins and second- and third-generation cephalosporins (cefpodoxime proxetil has been classified as both a second- and a third-generation agent) so that these agents can be administered orally. To increase the hydrophilicity of an agent, several different types of ester prodrugs have been used, including succinates, phosphates, and sulfonates. All are ionized at physiological pH and, therefore, increase the water solubility of the agents, making them more suitable for parenteral or oral administration when high water solubility is desirable. 7  Succinate esters containing an ionizable carboxylate are useful when rapid in vivo hydrolysis of the ester functionality is required. The rapid hydrolysis is related to the intramolecular attack of the carboxylate on the ester linkage, which does not require the participation of enzymes. As a result, these agents may be somewhat unstable in solution and should be dissolved immediately prior to administration. 8  Phosphate esters of alcohols offer another method of increasing the water solubility of an agent. The phosphates are completely ionized at physiological pH and generally hydrolyzed rapidly in vivo by phosphatase enzymes. Ionization of the phosphate function imparts high stability to these derivatives in solution, and solutions for administration can be stored for long periods of time without hydrolysis of the phosphate. Such an approach has been used to produce clindamycin phosphate, which produces less pain at the injection site than clindamycin itself. Pain after parenteral administration is associated with local irritation caused by low aqueous solubility or highly acidic or basic solutions. With clindamycin phosphate, the reduction in pain is attributed to the increased water solubility of the agent. Amines 9  Derivatization of amines to give amides has not been widely used us a prodrug strategy because of the high chemical stability of the amide linkage and the lack of amidase enzymes necessary for hydrolysis. There have been efforts at incorporating amines into peptide linkages in which the peptide serves to increase cellular uptake by use alanine amino acid transporter. The amino acids are then cleaved by specific peptidase enzymes. A more common approach has been to use Mannich bases as a prodrug form of the amines. Mannich bases result from the reaction of two amines with an aldehyde or ketone. As seen with hetacillin, the effect of forming the Mannich base is to tower the basicity of the amine and, thereby, increase lipophilicity and absorption. When nitrogen is present in an amide linkage, it is sometimes desirable to use the amide nitrogen as one of the amines necessary to form a Mannich base. This approach was used with the antibiotic tetracycline—the amide nitrogen was allowed to react with formaldehyde and pyrrolidine to give the Mannich base rolitetracycline. In this case, addition of the basic pyrrolidine nitrogen introduces an additional ionizable functionality and increases the water solubility of the parent drug. The Mannich base hydrolyzes completely and rapidly in aqueous media to give the active tetracycline. Azo linkage Amines have occasionally been incorporated into an azo linkage to produce a prodrug. In fact, it was an azo dye, prontosil that led to the discovery of the sulfonamides as the first antibacterials to be used to treat systemic infections. Although prontosil itself was inactive in vitro, it was active in vivo and was converted by azo reductase enzymes in the gut to sulfanilamide, the active species. 10  Although prontosil is no longer used as an antibacterial, this type of linkage appears in sulfasalazine, which is used in the treatment of ulcerative colitis. The azo linkage is broken in the gut by the action of azo reductases produced by microflora. This releases the active agent, aminosalicylic acid, which has an anti-inflammatory effect on the colon, and sulfapyridine. The advantage of this prodrug approach is that the combination of cleavage of the azo linkage and generation of aminosalicylic acid prior to absorption prevents the systemic absorption of the agent and helps concentrate the active agent at the site of action. Carbonyl Compounds A number of different functionalities have been evaluated as prodrug derivatives of carbonyls (e.g., aldehydes and ketones), although this approach has not found wide clinical use. These have generally involved derivatives in which the hybridized carbonyl carbon is converted to a sp3 hybridized carbon attached to two heteroatoms, such as oxygen, nitrogen, or sulfur. Under hydrolysis conditions, these functionalities are reconvened to the carbonyl compounds. An example of this approach is methenamine which releases formaldehyde in the urine, which acts as an antibacterial agent by reacting with nucleophiles present in bacteria. The agent is administered in enteric-coated capsules to protect it from premature hydrolysis in the acidic environment of the stomach. After dissolution of the enteric-coated capsules in the intestine, the agent is absorbed and moves into the bloodstream, eventually ending up in the urine, where the acidic pH catalyzes the chemical hydrolysis to give 11 formaldehyde. Use of this prodrug approach prevents the systemic release of formaldehyde and reduces toxicity. BIOPRECURSOR PRODRUGS Bioprecursor prodrugs do not contain a carrier or promoiety but rather contain a latent functionality that is metabolically or chemically transformed to the active drug molecule. The types of activation often involve oxidative activation, reductive activation, phosphorylation, and in some cases chemical activation. Of these, oxidation is commonly seen, since a number of endogenous enzymes can carry out these transformations. Phosphorylation has been widely exploited in the development of antiviral agents, and many currently available agents depend on this type of activation. The abundance of oxidizing enzymes in the body has made this type of bioactivation a popular route. Isozymes of cytochrome P450 can oxidize a wide variety of functionalities, generally to produce more polar compounds that can be excreted directly or undergo phase 2 conjugation reactions and subsequently undergo elimination. This occurs in a fairly predictable manner and, therefore, has been successfully exploited in prodrug approaches. A good example of a prodrug that requires oxidative activation is the NSAID nabumetone. NSAIDs produce stomach irritation, which in patients with preexisting conditions or patients taking large amounts of NSAIDs for extended periods may be severe. This irritation is associated in part with the presence of an acidic functionality in these agents. The carboxylic acid functionality commonly found in these agents is un-ionized in the highly acidic environment of the stomach. As a result, these agents are more lipophilic in nature and may pass into the cells of the gastric mucosa. The intracellular pH of these cells is more basic than that of the stomach lumen, and the NSAID becomes ionized. This results in backflow of H+ from the lumen into these cells, with concomitant cellular damage. This type of damage could be prevented if the 12 carboxylic acid function could be eliminated from these agents; this functional group is required for activity. Nabumetone contains no acidic functionality and passes through the stomach without producing the irritation normally associated with this class of agents. Subsequent absorption occurs in the intestine, and metabolism in the liver produces the active compound. This approach did not completely eliminate the gastric irritation associated with nabumetone, since it is due only in part to a direct effect on the stomach. Inhibition of the target enzyme, cyclooxygenase, while having an anti-inflammatory effect, also results in the increased release of gastric acid, which irritates the stomach. So, while nabumetone induces less gastric irritation than other NSAIDs, this undesirable effect was not completely eliminated by a prodrug approach. Such an effect was also seen above with the NSAID sulindac, whose Gl irritation was reduced but not completely eliminated. Reductive activation is occasionally seen as a method of prodrug activation but, because there are fewer reducing enzymes, is generally less common than oxidative activation. One of the best-known examples of reductive activation is for the antineoplastic agent mitomycin C, which is used in the treatment of bladder and lung cancer. Mitomycin C contains a quinone functionality that undergoes reduction to give a hydroquinone. This is important because of the differential effect of the quinone and hydroquinone on the electron pair of the nitrogen. Whereas the quinone has an electron- withdrawing effect on this electron pair, the hydroquinone has an electron-releasing effect, which allows these electrons to participate in the expulsion of methoxide and the subsequent loss of the carbamate to generate a reactive species that can alkylate DNA. 13  The cascade of events that leads to an alkylating active drug species is initiated by the reduction of the quinone functionality in mitomycin C. The selectivity of mitomycin for hypoxic cells is minimal. The selectivity is determined in part by the reduction potential of the quinine, which can be influenced by the substituents attached to the ring. In an effort to modify the reduction potential of mitomycin C. various analogues have been prepared and tested for antineoplastic activity. It was hoped that the reduction potential could be altered so that the analogues would only be activated in hypoxic conditions, such as those found in slow-growing solid tumors that are poorly vascularized. In these tissues with a low oxygen content it was thought that reductive metabolism might be more prevalent than in nor mat tissues, so the agents would be selectively activated and, therefore, selectively toxic. Although mitomycin was the first agent used clinically to be recognized as requiring reductive activation, it is only modestly selective for hypoxic cells. A much more selective agent tirapazamine is currently undergoing phase III clinical trials. Tirapazamine is reported to be 100 to 2(X) times more selective for hypoxic cells than for normal cells. The mechanism of activation involves a one-electron reduction that is catalyzed by a number of enzymes, including cytochrome P-450 and cytochrome P- 450 reductase to give a radical species. This species, which is shown as a carbon- centered radical, can initiate breaks in the DNA chain under hypoxic conditions. Under aerobic conditions, hydroxide radical is formed, which can initiate chain breaks. 14  Phosphorylation is a common metabolic function of the body, which is used to produce high-energy phosphodiester bonds such as those present in ATP and GTP. The body then typically uses these molecules to phosphorylate other molecules and, in the process of doing so, activates these molecules. The type of activation achieved depends on the molecule phosphorylated, but in many cases. phosphorylation introduces a leaving group, which can be displaced by an incoming nucleophile. This is seen, in the synthesis of DNA and RNA, in which nucleotides are added to the 3' end of a growing chain of DNA or RNA. Phosphorylation is commonly required for the bioactivation of antiviral agents. These agents are commonly nucleosides, which must be converted to the nucleotides to have activity. Most often, antiviral agents disrupt the synthesis or function of DNA or RNA, which is generally accomplished by conversion to the triphosphate. Since normal cells are also involved in the synthesis of DNA and RNA, compounds have been sought that would be converted to the triphosphates, the active form, in greater amounts in infected cells than in normal cells. Therefore, nucleosides that have higher affinity for the viral kinase enzymes than the mammalian kinases are desirable and have greater selective toxicity. This can be seen in the prodrug idoxuridine, which was the first agent to show clinical effectiveness against viruses. The nucleoside enters the cell, where it is phosphorylated. In virally infected cells, this phosphorylation is accomplished preferentially by viral thymidine kinase, because the idoxuridine is a better substrate for the viral enzyme than for the corresponding mammalian enzyme. Therefore, the drug is activated to a greater extent in the virally infected cells and achieves some selective toxicity, although this selectivity is rather low, and there is significant toxicity to normal cells. Once the drug is phosphorylated to the triphosphate stage, it can inhibit 15 DNA synthesis in a number of ways, including inhibition of viral DNA polymerase and incorporation into DNA, which results in incorrect base pairing that disrupts the ability of DNA to function as a template for DNA and RNA synthesis. In addition to the selective toxicity mentioned, the prodrug approach offers the additional advantage of increased cell penetration. The prodrug can easily enter the cell via active transport mechanisms, whereas the active nucleotides are unable to use this process and arc too polar to cross the membrane via passive diffusion. A good example of chemical activation is seen with the protein pump inhibitors such as omeprazole. In this case, chemical activation is provided by the highly acidic environment in and around the parietal cell of the stomach. This allows protonation of nitrogen on the benzimidazole ring followed by attachment of the pyridine nitrogen. Ring opening then gives the sulfenic acid that subsequently cyclizes with the loss of water. Attachment by a sulfhydryl group present on the proton pump of the parietal cell then occurs and inactivates this enzyme, preventing further release of H+ into the GI tract, which is useful in treating gastric ulceration. 16 CHEMICAL DELIVERY SYSTEM The knowledge gained front drug metabolism and prodrug studies may be used to target a drug to its site of action. Site specific chemical delivery systems take advantage of higher levels of activity in a metabolic or chemical pathway at the target site. A prodrug form of the active drug is designed to serve as a substrate in that specific pathway, thus yielding a high concentration of active drug at the target site. Site- specific chemical delivery requires that the prodrug reaches the target site and that the enzymatic or chemical process exists at the target site for conversion of the prodrug to the active drug. Many factors are involved in the relative success of site-specific drug delivery, including extent of target organ perfusion rate of conversion of prodrug to active drug in both target and nontarget sites, and input/output rates of prodrug and drug from the target sites. Site-specific chemical delivery systems represent but one approach to the selective delivery of drug molecules to their site of action for increased therapeutic effectiveness and limited side effects. Other than chemical drug delivery, many carrier systems have been evaluated for drug delivery, including proteins, polysaccharides, 17 liposomes, emulsions, cellular carriers (erythrocytes and leukocytes), magnetic control targeting, and implanted mechanical pump. As the fate of drugs in the human body has become more clearly understood, research activity to improve the delivery of active drug to the target site has increased. The basic goal of these efforts is to protect the drug from the nonspecific biological environment and to protect the nonspecific bioenvironment from the drug to achieve some site-specific drug delivery. Site-specific drug delivery has been evaluated extensively for drugs with narrow therapeutic windows, such as many of the anticancer drugs. The site-specific delivery of the active drug via its prodrug counterpart requires that the prodrug be readily transported to the site of action and rapidly absorbed at the site. On arrival at the target site, the prodrug should be selectively converted to drug relative to its rate of conversion at nontarget sites. Since high metabolic activity occurs in highly perfused tissues such as liver and kidney, delivery to these organs has a natural advantage. Unfortunately, prodrug delivery of active drug to other organs or tissues is disadvantaged for the same reasons. Furthermore, it is highly desirable to have the active drug, once formed, migrate from the target site at a slow rate. On the basis of all these requirements, clearly site- specific delivery of drug to the target by a prodrug chemical delivery system is a far more complex undertaking than designing a prodrug to improve one aspect of its overall properties. Yet there are several excellent examples of site-specific chemical delivery systems in use in modem drug therapy. The target sites include cancer cells, GI tract, kidney and urinary tract, bacterial cells, viral material, ocular tissue, and the blood—brain barrier. The prodrug methenamine, can be considered a site-specific chemical delivery system for the urinary tract antiseptic agent formaldehyde. The low pH of the urine promotes the hydrolysis of methenamine to formaldehyde, the active antibacterial agent. The rate of hydrolysis increases with increased acidity (decreased pH), and this can be promoted by administration of urinary pH-lowering agents or by diet. The pH of the plasma is buffered to about 7.4, and the rate of hydrolysis is low, preventing systemic toxicity from formaldehyde. This compound is administered in enteric-coated 18 tablets that prevent dissolution and, therefore, premature hydrolysis in the highly acidic environment of the stomach. A number of prodrugs for cancer chemotherapy have been designed for selective delivery to active drug to tumor tissue, based on higher levels of activating enzyme in the tumor cell than in normal tissues. Many enzymatic systems show higher activity in tumor cells than in normal tissue because of the higher growth rates associated with tumor tissue. Peptidases and proteolytic enzymes are among those systems showing higher activity in and near tumor cells. Thus, one means of attempting to produce higher rates of drug incorporation into tumors than in surrounding normal tissue involves deriving a drug molecule with an amino acid or peptide fragment. Capecitabine is an example of a prodrug chemical delivery system that requires a series of enzymatic steps for conversion to the active antitumor drug species. 5-Fluorouracil.Tumors located in tissues with high levels of the required enzymes should respond best to treatment with capecitabine. Esterase activity occurs primarily in the liver, allowing the intact ester capecitabine to be the absorbed species following oral administration. The ester hydrolysis product itself shows some specific toxicity toward GI tract tissue, which prevents this molecule from serving as an effective prodrug delivery form of 5-fluorouracil. The other two enzymes involved in the formation of 5-fluorouracil occur in high concentrations in target tissues such as cervix, breast, kidney, and colon. There is considerable current interest in the general concept of tumor-activated prodrugs, and a number of strategies have been proposed for drug targeting in tumor cells. One of the more interesting approaches is linking an exogenous (nonhuman) enzyme to a tumor-specific antibody. Based on immunological response, the antibody 19 would carry the enzyme to the tumor surface, where it would be available for prodrug activation. Prodrugs activated by this exogenous enzyme would be converted to the active species only at the tumor site. Since the activating exogenous enzyme is not normally found in human tissue, maximum accuracy in drug targeting should be achieved in this antibody directed enzyme prodrug therapy. The antiviral drugs, such as idoxuridine, are an interesting example of site- specific chemical delivery. These drugs serve as substrates for phosphorylating enzymes found in viruses, and the phosphorylated species is the active antiviral agent. The active phosphorylated species is incorporated into viral DNA, disrupting viral replication and, thus, producing the antiviral effect. These drugs do not undergo phosphorylation by mammalian cells, so the prodrug is specific for those sites where it serves as a substrate for phosphorylation enzymes. One of the requirements for site- specific chemical delivery discussed above was the proper input/output ratios for prodrug and active drug species at the target. The relative physicochemical properties of prodrug and its phosphorylated derivative suggest an appropriate input/output ratio for site specificity. The prodrug can readily penetrate the virus, and the increased polarity of the phosphorylated derivative would serve to retain that active species inside the virus. The combination of increased polarity and viral retention of the active phosphorylated species likely reduces any human toxicity that might he associated with this active species. The amino acid drug L-dopa can be considered a site-specific chemical delivery system that delivers the drug dopamine to the brain. The brain has an active transport system that operates to incorporate L amino acids into the central nervous system (CNS), and L-dopa is transported into the brain in this manner. Once across the blood— brain barrier. L-dopa undergoes decarboxylation, to yield the active metabolite, dopamine. Direct systemic administration of dopamine does not produce significant levels of the drug in the brain because of its high polarity and poor membrane permeability as well as its facile metabolic degradation by oxidative deamination. Dopamine formed on the inside of the blood—brain barrier is held there, however, because of the poor membrane permeability of this drug. Although some specificity for 20 brain tissue is achieved by this delivery method, peripheral side effects of L-dopa are the direct result of decarboxylation to dopamine in other organ systems. In this case, the enzyme activating system is not localized at the target site, and its presence in other tissues and organs leads to undesirable side effects. Another example of the chemical delivery of a drug to the brain and CNS is the prodrug form of 2-PAM (pro-2-PAM), an important antidote for the phosphate and carbamate acetylcholinesterase inhibitors used in insecticides and nerve gases. The polar properties of 2-PAM, a permanent cationic species, prevent this drug from being absorbed following oral administration and restrict the drug from access to the brain, even after IV administration. Pro-2-PAM is a dihydropyridine derivative that undergoes metabolic and chemical oxidation to yield the active drug 2-PAM. The nonionic pro-2-PAM can easily cross the blood—brain barrier, and oxidation to 2-PAM within the brain essentially traps the active cationic drug species inside the brain. Oxidation of the dihydropyridine ring of pro-2-PAM occurs throughout the mammalian system, not just in the brain, and the levels of the resulting 2-PAM are approximately the same in peripheral tissue as in the brain. IV administration of pro- 2-PAM, however, yields brain levels of 2-PAM that are approximately 10 times higher than those achieved by IV administration of the parent drug. The delivery of drugs across the blood—brain barrier has been a significant issue in the design of many therapeutic compounds. Only very lipophilic drugs can cross into the brain without the aid of some active uptake process, such as the one that operates to incorporate essential amino acids into the CNS. The facile oxidation of the dihydropyridine ring system has been extensively investigated as a general process for 21 chemical delivery of a number of drugs to the CNS. The approach has been described as a chemical delivery system, not just a prodrug designed to penetrate the blood— brain barriers. This process is a multistep procedure involving delivery of the drug— dihydropyridine derivative to the brain via facile diffusion across the blood—brain barrier, followed by oxidation to the quaternary pyridine cation, which is trapped in the brain. The drug is then released from the pyridine cation by a second metabolic/chemical event. A number of functional groups can be added to the dihydropyridine to facilitate the derivatization of various functional groups found in CNS drugs. Since many CNS drugs are amines, amides of dihydropyridine carboxylic acids are often prepared and used to deliver the drugs across the blood—brain barrier into the brain. Additionally, these amide derivatives often serve to protect the amines from metabolic degradation before they reach the target site. Primary amines such as dopamine and norepinephrine and ninny others are readily metabolized and degraded by oxidative deamination before reaching the CNS. The dihydropyridine derivative of a dopamine ester has access to the CNS via passive absorption of the tertiary amine, which on oxidation restricts the resulting pyridinium amide to the brain. Amide hydrolysis then delivers the active form of the drug at or near its site of action. The amide hydrolysis step may be slower than the dihydropyridine oxidation step, and thus a reservoir of pyridinium amide precursor may be available for conversion to the active drug species. The use of prodrug concepts has been very successful in the delivery of active drug species to the human eye following local application. Lipophilic esters of epinephrine, such as the dipivaloyl ester, show better corneal penetration following direct application to the eye than the more polar parent drug epinephrine. The esterases necessary for the hydrolysis of the prodrug are readily available in the eye and skin. The more polar drug species, epinephrine, is then localized within the lipophilic membrane barriers of the eye, and the drug remains available at the target site to produce its antiglaucoma effects. The local application of the prodrug species to the skin or eye allows metabolic processes to activate the drug without concern for competitive reactions at other tissues or sites of loss. 22  The delivery of drugs to the colon and lower GI tract has taken advantage of the unique enzymatic processes found in colon bacteria. The glucosidase activity of these bacteria allows hydrolysis of glucoside derivatives of drugs in the colon and provides higher concentrations of active drug. A number of steroid drugs demonstrate increased effectiveness in the lower GI tract following administration as their glucoside derivatives. The polar glucoside derivatives of the steroids are not well absorbed into the bloodstream from the GI tract and remain available to serve as substrates for the bacteria that are found primarily in the human colon. The prodrug approach for the delivery of anticancer drugs to the site of action has been used in a number of cases in an effort to increase effectiveness and lower side effects. Several enzyme systems that show higher activity in and near the cancer cells have been evaluated for their ability to activate the prodrug species. In most cases, the enzyme activity level is simply higher near the faster growing cancer cells, but the presence of the enzymes in normal tissue prevents the of complete site specificity for these agents. Polymeric prodrugs 23  Soluble synthetic polymers have been widely employed as drug carrier systems. Polymer chemistry allows the development of tailor-made conjugates in which target moieties as well as drugs are introduced into the carrier molecule. In the case of enhanced permeability retention in e.g. tumor vasculature, the introduction of drugs into the polymer may be sufficient. As non-specific adherence to cells is an undesirable property, excessive charge or hydrophobicity should be avoided in the design of polymeric carriers. For cancer therapy, the well-established N(-2-hydroxypropyl)methacrylamide (HMPA) polymers have been extensively studied. PK1, a 28-kDa HPMA copolymer containing doxorubicin is now in clinical testing. Other drugs that have been incorporated in these polymers are platinates and xanthine oxidase, respectively. Furthermore, conjugates (so called SMANCS) of the anticancer drug neocarzinostatin (NCS) and styrene-comaleic acid/anhydride (SMA) have been developed for therapy of liver cancer. New polymers developed in the last few years include the cationic low molecular mass chitosan polymers for DNA delivery and highly branched, low dispersity dendrimers consisting of various chemical origins. Fab’ antibody fragments can copolymerize with HPMA and drug-containing monomers to yield a targetable HPMA copolymer–Mce6 conjugate. In vitro studies showed that, as a result, the photosensitizer Mce6 was more efficiently internalized by OVCAR-3 carcinoma cells than the non-targeted copolymer and hence had greater cytotoxicity. 24  Polymers or synthetic copolymers are believed to accumulate in solid tumors due to enhanced vascular permeability of tumor blood vessels combined with a lack of lymphatic drainage in the tumor tissue. Polymer-based targeting strategies can be divided into two main categories, i.e. polymer–protein conjugates (so far the most widely studied) and polymer–drug conjugates, particularly those containing conventional anti-tumor agents. Polymer–drug conjugation can be used to alter the biodistribution, elimination rate and rate of metabolism of covalently bound drugs. In the case of protein adducts, polymer conjugation can prolong the protein plasma elimination half-life, reduce proteolytic degradation and may have the added benefit of reducing immunogenicity. Polyethylene glycol (PEG) is the most widely used polymer for protein conjugation. Soluble polymer conjugates have also been proposed as macromolecular pro- drugs for controlled release and targeting of various low molecular weight, (non- protein) chemicals. In this case, polymer conjugation not only serves to alter drug biodistribution by restricting cellular capture to the lysosomotropic route, but the polymer–drug linkage can also be designed to allow site-specific enzymatic or hydrolytic cleavage. Thus, both the rate and the site of drug delivery can in principle be controlled. Enhanced permeability of the microvasculature at certain sites, particularly within solid tumors, can be exploited to facilitate site-specific accumulation of polymer–drug conjugates. Other (co)polymers of interest besides PEG 25 are SMANCS (styrene-co-maleic anhydride neocarzinostatin; zinostatin stimalar) and HPMA (N-(2-hydroxypropyl) methylacrylamide). Polymer-directed enzyme prodrug therapy (PDEPT) consists of two-step antitumor approach combining a polymeric prodrug and polymer-enzyme to generate cytotoxic drug at the tumor site. PDEPT proposes initial administration of the polymeric prodrug to promote tumor targeting before administration of the activating polymer-enzyme conjugate. Targeting moieties such as sugars (galactose, mannose), proteins and antibodies have been incorporated into the conjugates to promote receptor-mediated recognition. Thus, cell- or organ- specific localization of therapy may be achieved. It should be noted however, that various cell types in the liver and spleen are important target cells for sugar-derivatized proteins and that hepatic clearance will always compete with extrahepatic distribution. Polymer conjugates are most useful in the context of immuno-conjugates. Other protein constructs such as fusion proteins can assist their future development. Soluble polymer conjugates have been introduced into clinical practice in the last decade. Several PEG conjugates have been evaluated clinically for 26 cancer therapy including a PEG conjugate of asparaginase in the treatment of acute lymphoblastic leukaemia in patients hypersensitive to the native enzyme, and a PEG conjugate of IL-2. A phase I clinical and pharmacokinetic study of PK1 comprising doxorubicin covalently bound to N-(2-hydroxypropyl)-methacrylamide copolymer by a peptidyl linker, was carried out in 36 patients with refractory or resistant cancers. PK1 demonstrated anti-tumor activity, and that polymer–drug conjugation decreased doxorubicin dose-limiting toxicities. Phase II studies are in progress. Polymer cross-linking leads to a decrease in the water solubility of many readily soluble polysaccharides, low water solubility being a requirement for colon-specific drug delivery. Dextrans, the mucopolysaccharide chondroitin sulfate, guar gum, pectin and inulin have all been investigated in cross-linked forms. Again, with a higher degree of cross-linking, the swelling properties of these polymers tend to be lower and this leads to a slower degradation rate and thus slower release of the drug. Poor water- soluble drugs are usually released by an erosion-type mechanism. DRUG TARGETING One of the major goals in drug design is to find ways of targeting drugs to the exact locations in the body where they are most needed. The principle of targeting drugs can be traced back to Paul Ehrlich who developed antimicrobial drugs that were selectively toxic for microbial cells over human cells. Drugs can also be made more selective to distinguish between different targets within the body. A major goal in cancer chemotherapy is to target drugs efficiently against tumor cells rather than normal cells. One method of achieving this is to design drugs which make use of specific molecular transport systems. The idea is to attach the active drug to an important 'building block' molecule that is needed in large amounts by the rapidly dividing tumor cells. This could be an amino acid or a nucleic acid base Of course, normal cells require these building blocks as well, but tumor cells often grow more quickly than normal cells and require the building blocks more urgently. Therefore, the uptake is greater in tumor cells. 27  A more recent idea has been to attach the active drug (or a poison such as ricin) to monoclonal antibodies, which can recognize antigens unique to the tumor cell. Once the antibody binds to the antigen, the drug or poison is released to kill the cell. The difficulties in this approach include the identification of suitable antigens and the production of antibodies in significant quantity. Another tactic which has been used to target anticancer drugs is to administer an enzyme-antibody conjugate where a suitable enzyme is chosen to activate an anticancer prodrug and the antibody is chosen to direct the enzyme to the tumor. If a drug is to be targeted against an infection of the gastrointestinal tract, it must be prevented from being absorbed into the blood supply. This can easily be done by using a fully ionized drug which is incapable of crossing cell membranes. For example, highly ionized sulfonamides are used against gastrointestinal infections because they are incapable of crossing the gut wall. It is often possible to target drugs such that they act peripherally and not in the central nervous system. By increasing the polarity of drugs, they are less likely to cross the blood-brain barrier and this means they are less likely to have central nervous system side effects. Achieving selectivity for the central nervous system over the peripheral regions of the body is not so straightforward. Antibody-directed enzyme prodrug therapy (ADEPT) Antibody-directed enzyme prodrug therapy has been used in an attempt to develop drugs that specifically target cancer cells. The method of approach is based on the observation that many prodrugs are enzyme activated. It uses an antibody–enzyme conjugate to deliver the enzyme to the target. Once a sufficient concentration of the enzyme has reached the tumor, the prodrug is administered. When the prodrug reaches the tumor, it is converted by the enzyme carried by the antibody to the active drug. For example, the anticancer agent etoposide is a semisynthetic derivative of podophyllotoxin, a compound isolated from the north American plant Podophyllum peltatum. In the ADEPT approach, its phosphorylated derivative is used as the prodrug because it is inactive but can be converted to etoposide by alkaline phosphatase (AP). This enzyme is delivered using an antibody–alkaline phosphatase conjugates whose antibody section recognizes specific antigens on the tumor’s surface. Subsequent administration of the etoposide phosphate is followed by liberation of etoposide at the 28 surface of the tumor. The etoposide acts by diffusing into the tumor cell and destroying it. The ideal ADEPT approach depends on: 1. Finding an enzyme that does not usually occur in the body but is capable of liberating the drug from the drug–carrier prodrug complex. 2. The enzyme being relatively stable under physiological conditions. 3. Producing a drug–carrier complex that is not metabolized to any extent by the enzyme systems in the body. Unfortunately these conditions are difficult to fulfill and to date clinical results for the first generation of antibody-directed prodrugs have been disappointing. One disadvantage of ADEPT is that there may be an immune response to the antibody– enzyme conjugate as the enzyme is a foreign body. However, the risk of this happening may be reduced by the use of humanised antibodies and human enzymes such as alkaline phosphatase and b-glucuronidase. Further disadvantages are the lack of information concerning tumor antibodies and obtaining an enzyme that is sufficiently active to liberate sufficient of the active drug at the target site. 29 Antibody-directed abzyme prodrug therapy (ADAPT) Abzymes are antibodies which have a catalytic property. It is possible that prodrugs could be designed that act as antigens for these antibodies and are activated by the catalytic properties of the antibody. This can be done by immunizing mice with a transition-state analogue of the reaction that is desired, followed by isolation of the monoclonal antibodies by hybridization techniques. Since the antibody targets the prodrug rather than antigens on the cancer cell, this fails to target drugs to cancer cells. However, it should be possible to construct hybrid antibodies where one arm recognizes antigens on cancer cells while the other arm recognizes the prodrug and activates it. This approach is still in its early stages, but it has several potential advantages over ADEPT. For example, it should be possible to design catalytic mechanisms that do not occur naturally; allowing highly selective activation of prodrugs at tumors. It also removes the risk of an immune response due to foreign enzymes. At present the catalytic activity of abzymes is too low to be useful and much more research has to be carried out. Gene-directed enzyme prodrug therapy (GDEPT) Gene-directed enzyme prodrug therapy (GDEPT) involves the delivery of a gene to the cancer cell. Once delivered, the gene codes for an enzyme capable of transforming a prodrug into an active drug. As the enzyme will be produced inside the cell, the prodrug is required to enter the cell. The main challenge in GDEPT is delivering the gene selectively to tumor cells. In one method, the gene is packaged inside a virus such as a retrovirus or adenovirus. In the case of adenoviruses, the desired genes could be spliced into the viral DNA such that the virus inserts into host cell DNA on infection. The virus is also genetically modified such that it is no longer virulent and can do no harm to normal cells. Non- viral vectors have also been tried, such as cationic lipids and peptides. So far, it has not been possible to achieve the required selectivity for cancer cells over normal cells, and so the delivery vector has to be administered directly to the tumor. The enzymes which are ultimately produced by the introduced genes should not be present in normal cells, so that prodrug activation only occurs in tumor cells. One 30 advantage of GDEPT over ADEPT is the fact that foreign enzymes could be generated inside cancer cells and hidden from the immune response. The thymidine kinase enzyme produced by herpes simplex virus has been studied intensively in GDEPT. This enzyme activates the antiviral drugs aciclovir and ganciclovir. Since these drugs are poor substrates for mammalian thymidine kinase, activation will only be significant in the tumor cells containing the viral form of the enzyme. Several clinical trials have been carried out using this approach. One problem associated with GDEPT is that it is unlikely that all tumor cells will receive the necessary gene to activate the prodrug. It is therefore important that the anticancer drug is somehow transferred between cells in the tumor—a so called 'bystander' effect. This may occur by a variety of means such as release of the activated drug from the infected cell, direct transfer through intercellular gap junctions or by the release of drug-carrying vesicles following cell death. GDEPT has been used to introduce the genes for the bacterial enzymes nitroreductase and carboxypeptidase G2 into cancer cells. Prodrugs were then administered which were converted to alkylating agents by the resulting enzymes. One of the problems with carboxypeptidase G2 is the difficulty some of the prodrugs have in crossing cell membranes. In order to overcome this problem the gene was modified such that the resulting enzyme was incorporated into the cell membrane with the active site revealed on the outer surface of the cell. Gene therapy aimed at activating the prodrug irinotecan tries and improves the process by which the urethane is hydrolyzed to the active drug. This could involve the introduction of a gene encoding a more active carboxypeptidase enzyme into tumor cells. For example, rabbit liver carboxypeptidase is 100-1000 times more efficient than the human form of the enzyme. 31