Secion 82 Mechanisms of Drug Activation The local anesthetic benzocaine (8.13.R=H;one trade name is Americaine)has been 507 converted into water-soluble amide prodrug forms with various mino13.R +NH CHR'CO):amidase-catalyzed hydrolysis in human serum occurs rapidly CO,E benzocaine(R=H) 8.13 b.Prodrugs for Improved Absorption and Distribution The skin is designed to maintain the body fluids and prevent absorption of xenobioties into the general circulation.Consequently,drugs applied to the skin are poorly absorbed Even steroids have low dermal permeability,particularly if they contain hydroxyl groups that can interact with the skin or binding sites in the keratin.Corticosteroids for the topical treatment of inflammatory,allergic,and pruritic skin conditions can be made more suitable for topical absorption by esterification or acetonidation.For example,both fluocinolone acetonide(8.14. R =H;one trade name is Synalar)and fluocinonide(8.14.R COCH3:Lidex)are prodrugs used for inflammatory and pruritic manifestations.Once absorbed through the skin,an esterase releases the drug. OR CH CH HO CH CH F fluocinolone acetonide(R=H) fluocinonide(R=COCHa) 8.14 Dipivaloylepinephrine (dipivefrin,8.15,R =Me3CCO:Propine).a prodrug for the antiglaucoma drug epinephrine(8.15.R =H:one trade name is Epifrin),is able to pen- etrate the comnea better than epinephrine.The cornea and aqueous humor have significant esterase activity25] RO OH RO NHCHs dipivefrin(R=MegCCO) epinephrine(R=H) 8.15 The targeting of drugs for a specific site in the body by conversion to a prodrug is plausible Prodrugs for Site Specificity when the physicochemical properties of the parent drug and prodrug are optimal for the target c
508 Chapter 8 Prodrugs and Drug Delivery Systems iteKeepin mind,however,that when the lipophilicity of a drug is increased.it will improve passive transport of the drug nonspecifically to all tissues. Oxyphenisatin (8.16.RH:Lavema)is a bowel sterilant that is active only when admin. istered rectally.However.when the hydroxyl groups are acetylated (8.16.RAc.one trade name is Noloc).the prodrug.oxyphenisatin acetate.can be administered orally,and it is hydrolyzed at the site of action in the intestines to oxyphenisatin. RO oxyphenisatin(R=H) 8.16 One important membrane that must be traversed for drug delivery into the brain is the bloodbrain barriera unique lipid-like protective barrier that prevents hydrophilic com- pounds from entering the brain unless they are actively transported.The blood-brain barrier also contains active enzyme systems to protect the central nervous system even further.Conse- quently,molecular size and lipophilicity are often necessary,not sufficient.criteria for gaining entry into the brain.1271 .As was discussed in Chapter 5.Section 5.5.C.3.a,p.287.increasing the brain concentra- tion of the inhibitory neurotransmitter y-aminobutyric acid(GABA)results in anticonvulsant activity.However.GABA is too polar to cross the blood-brain barrier,so it is not an effective anticonvulsant drug.As mentioned above,progabide(8.6)is an effective lipophilic analog of GABA that crosses the blood-brain barrier,releases GABA inside the brain,and shows anti- convulsant activity 28 Another related example of anticonvulsant drug design is a glyceryl lipid(8.17.R linolenoyl)containing one GABA molecule and one vigabatrin molecule, a mechanism-based inactivator of GABA aminotransferase and anticonvulsant drug (see Chapter 5.Section 5.5.C.3.a.p.287).129)This compound inactivates GABA aminotransferase in vitro only if brain esterases are added to cleave the vigabatrin from the glyceryl lipid.It also is 300 times more potent than vigabatrin in vivo presumably because of its increased ability to enter the brain. OCOR NH NH: 8.17 In the above examples.the lipophilicity of the drugs wasincreased sothat they could diffuse through various membranes.Another approach for site-specific drug delivery is to designa prodrugthat requresactivationby an enyme found predominantly at the desired site ofaction Foreumr ceohigher of phosphatases and amidases than doceConseuprodrgofeytoen ou be directedtotumorce
Seion2 Mechanism rug Activatlon hr ofhertothe prodrctivaion pro Detyb diphosphate (RP)designed for p very of 509 (RH:one tradeame is Stibestrol)to prosatie careinoma g ou thistppaot been veryThe ppopnateprodnugsaretopolarioreachtheemymesne,theeniteenynatcelectmiy sinsufficientand the tumor cell perfusion rateis poor RO diethylstilbestrol diphosphate(R=PO3) diethylstilbestrol(R=H) 8.18 Several strategies,under the rubric of enzyme-prodrug therapies,have been developed to achieve selective activation of prodrugs atadesired site.typically intumorcelsAllofthese approaches involve two steps:In the first step aprodrug-activating enzyme is incorporated into the target tumor cells,and in the second step a nontoxic prodrug,which is a substrate of the exogenous enzyme that was incorporated into the tumors,is administered systemically.The prodrug is selectively converted into the active anticancer drug in a high local concentration inside the tumor cell.Certain criteria are important for this general approach tobe effective: (1)The prodrug-activating enzyme should be either of nonhuman origin or a human protein that is absent or expressed only at low concentrations in normal tissues:(2)the prodrug- activating enzyme must achieve adequate expression in the targeted tumor cells and have high catalytic activity;(3)the prodrug should be a good substrate for the enzyme incorporated in the tumors but not be activated by endogenous enzymes outside of the tumors;(4)the prodrug must be able to cross the tumor cell membrane for intracellular activation:(5)the cytotoxicity difference between the prodrug and its corresponding active drug should be high:(6)the activated drug should be highly diffusible or be actively taken up by adjacent nonexpressing cancer cells for what is known as a bystander killing effect,the ability of the drug to kill neighboring nonexpressing cells;and (7)the half-life of the active drug should be long enough to induce a bystander killing effect but short enough to avoid the drug leaking out of the tumor cells and causing damage elsewhere. One strategy of this type is called antibody-directed enzyme prodrug therapy(abbreviated ADEPT).In the first stepn antibody raised against a particular tumorcen isd with (attached to)the enzyme that is needed to activate an antitmor prodrug.Afer the antibody-enzyme conjugae is administered and has accumulatedonhemreeltheexcess conjugate not bound to the tumor cell is given enough time to clear from the blood and normal tissues.The prodrug is then administered.The enzyme conjuged with the antibody at the tumor cell surface catalyzes the conversion of the prodrug to the drug when it reaches the morcel The advantage of this method,relative todirectdof theprodg isthe increased selectiviy for release of high conof the drug at the rgeedcel Thissoevident ifenoughtime isaowed forracebdyyme
10 Chapter B Prodrugs and Drug Delivery Systems carboxypeptidase G2 L-Glu +C0 CO.H CO,H electron-donating: 8.19 nctivates nitrogen mustard clectron-withdrawing: deactivates nitrogen mustard Scheme 8.4Nitrogen mustard activation by carboxypeptidase G2 for use with ADEPT conjugate that is not bound to the tumor cells.An increase in the clearance rate of unbound antibody-enzyme conjugate would permit the administration of a higher concentration of the prodrug for a longer period of time.Galactosylation of the antibody leads to more rapid and efficient clearance of the unbound antibody-enzyme conjugate by galactose receptors in the liver.33] The drawbacks to ADEPT are the potential for immunogenicity and rejection of the antibody-enzyme conjugate,the potential for leakback of the active drug formed at the tumor,and the requirement of i.v.administration as well as the complexity of the treatment. Nonetheless,this approach is in the clinical trial stage. An example of ADEPT is the delivery of a nitrogen mustard as a glutamic acid conju- gate (8.19)after administration of a humanized monoclonal antibodyl34]conjugated to the bacterial enzyme carboxypeptidase G2(Scheme 8.4)1351 Humanization of antibodies raised from external sources minimizes immunogenicity.Note that the enzyme selected for prodrug activation is a bacterial enzyme so that there may be selectivity for prodrug activation by this enzyme in preference to that by human carboxypeptidase at sites other than at the tumor cells. The drawback to this approach is the potential for increased incidence of immunogenicity and rejection as a result of using a bacterial enzyme.This problem could be alleviated with the use of a humanized catalytic antibody 361 in place of the bacterial enzyme for activation of the prodrug.The reaction shown in Scheme 8.4 was also effected using a humanized catalytic antibody in a process termed antibody-directed abzyme37 prodrug therapy (ADAPT). An even more effective approach for attaining selectivity for prodrug activation at the tumor cell would be to use a humanized catalytic antibody that not only does not exist in humans,but catalyzes a reaction not known to occur in humans.That way,the only site where the prodrug could be activated would be where the catalytic antibody resides,presumably directed with a monoclonal antibody to the desired tumor cells.Antibody 38C2139 is a broad specificity catalytic antibody that catalyzes sequential retro-aldol and retro-Michael reactions. acombination of reactions not catalyzed by any known human enzyme.The abzyme was found to be long lived nvivtoactivate prodrugs selectively.and to potentiate the killing of coon and prostate cancer cell lines4 An example of the activation reactions catalyzed by this abzyme is shown in Scheme 8.5 for a doxorubicin prodrug(8.20). A related strategy for improving the selectivity of cancer chemotherapy is called gene directed me prodrug therapy (GDEPT aso ealled suicide gene therapy)In this approachagene encodin the prodrug-activating enzyme is integrated into the genome of the argetnder theoof tumor-selective promotors orby viral transfection
Section 82 Mechanisms of Drug Activation 511 OH etro-ald CH O 8.20 OH HO NI doxorubicin Scheme 8.5Catalytic antibody 38C2 activation of a doxorubicin prodrug by tandem retro-aldol/ retro-Michael reactions for use with ADAPT OMe nitroreductase OMe HO c NH: amino-seco-CBI-TMI 821 Scheme 8.6 Nitroreductase activation of a prodrug for use with GDEPT These cells.then.express the enzyme that activates the prodrug added in the second stepas A common enzyme used for activation of a prodrug by GDEPT is an aerobic flavin- described above for ADEPT. dependent nitroreductase from Eco B,that catalyzes the reduction ofaromatic nrogroups toherreondnghydroxymngroupForexmpe the(mh carbamate prodrug of the minor groove alkylating agent amino-seco-CBI-TMI(821)isstable until nitroreductase reduces the aromatic nitro group to the corresponding hydroxymino group.This initates the elimintion of the cabamate togive CO and the free alkylating agent (Scheme 8.6).1431