Prodrugs of anthracyclines for use in antibody-directed enzyme prodrug therapy

A series of new prodrugs of daunorubicin and doxorubicin which are candidates for antibody-directed enzyme prodrug therapy (ADEPT) is reported. These compounds (25a,b,c and 32a,b,c) have been designed to generate cytotoxic drugs after activation with beta-glucuronidase. As expected, recovery of the active drug was observed after enzymatic cleavage by Escherichia coli beta-glucuronidase as well as by a fusion protein which has been obtained from human beta-glucuronidase and humanized CEA-specific binding region. The six prodrugs are highly stable and are more than 100-fold less cytotoxic than doxorubicin against murine L1210 cell lines. The ortho-substituted phenyl carbamates 25a,b,c are better substrates for beta-glucuronidase than the corresponding para-substituted analogues. After taking into account additional factors such as stability in plasma and kinetics of enzymatic cleavage, we selected the o-nitro prodrug 25c for clinical trials.     

New prodrug activation gene therapy for cancer using cytochrome P450 4B1 and 2-aminoanthracene/4-ipomeanol

Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD50 of 2.5 microM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood-brain barrier.     

Mustard prodrugs for activation by Escherichia coli nitroreductase in gene-directed enzyme prodrug therapy

Twenty nitrogen mustard analogues derived from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954, 1) were evaluated as candidate prodrugs for gene-directed enzyme prodrug therapy (GDEPT) in Chinese hamster V79 cell lines engineered to express Escherichia coli nitroreductase (NR). Structural variations within the series included the use of N-dihydroxypropyl and (N-dimethylamino)ethyl carboxamide side chains, the use of chloro, bromo, mesyl, and iodo leaving groups on the mustards, and regioisomeric changes. The compounds were assayed for cytotoxicity (IC50) with the NR-expressing and controls of non-NR-expressing cell lines. The proportion of NR-expressing cells required in a mixture for nonexpressing cells to experience 50% of their cytotoxicity (termed the TE50) was used to assess the compounds' ability to induce a bystander effect. This study suggests that 5-[N,N-bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (8), 5-[N,N-bis(2-iodoethyl)amino]-2,4-dinitrobenzamide (9), 2-[N,N-bis(2-bromoethyl)-amino]-3,5-dinitrobenzamide (13), and 2-[N,N-bis(2-iodoethyl)amino]-3,5-dinitrobenzamide (14) showed considerable improvements over 1, exhibiting greater potency, higher IC50 ratios, and lower TE50s, and are thus superior prodrugs to 1 for GDEPT.     

Radiation-activated prodrugs as hypoxia-selective cytotoxins: model studies with nitroarylmethyl quaternary salts

Bioreductive drugs are designed to be activated by enzymatic reduction in hypoxic regions of tumours, but activation of these drugs is not always fully suppressed by oxygen in normal tissues. A further limitation is that bioreductive drug activation depends on suitable reductases being expressed in the hypoxic zone. This essay proposes an alternative approach in which prodrugs are reduced, and thereby activated, in hypoxic regions by ionizing radiation rather than by enzymes. This strategy is theoretically attractive, but design requirements for such radiation-activated cytotoxins are challenging. In particular, the reducing capacity of radiation at clinically relevant doses is small, which necessitates the development of prodrugs capable of releasing very potent cytotoxins efficiently in hypoxic tissue. It is shown that nitroarylmethyl quaternary (NMQ) salts possess many of the features required of a radiation-activated prodrug. In some heterocyclic NMQ compounds the cytotoxicity of the latent cytotoxic amine effector is suppressed by > 100-fold in the prodrug form, and the effector is released rapidly by fragmentation following reduction by a single electron. Appreciable cytotoxic activation of NMQ prodrugs can be achieved by irradiation at clinically relevant doses in anoxic plasma. Some of the further drug design challenges required to develop a clinical agent based on this approach are outlined.     

Toward targeted "oxidation therapy" of cancer: peroxidase-catalysed cytotoxicity of indole-3-acetic acids

PURPOSE: The study aimed to identify suitable prodrugs that could be used to test the hypothesis that peroxidase activity in cells, either endogenous or enhanced by immunological targeting, can activate prodrugs to cytotoxins. We hypothesized that prototype prodrugs based on derivatives of indole-3-acetic acid (IAA), when activated by peroxidase enzymes (e.g., from horseradish, HRP) should produce peroxyl radicals, with deleterious biological consequences. METHODS AND MATERIALS: V79 hamster cells were incubated with IAA or derivatives +/- HRP and cytotoxicity assessed by a clonogenic assay. To assess the toxicity of stable oxidation products, prodrugs were also oxidized by HRP without cells, and the products then added to cells. RESULTS: The combination of prodrug and enzyme resulted in cytotoxicity, but neither indole nor enzyme in isolation was toxic under the conditions used. Although lipid peroxidation was stimulated in liposomes by the prodrug/enzyme treatment, it could not be measured in mammalian cells. Adding oxidized prodrugs to cells resulted in cytotoxicity. CONCLUSIONS: Although the hypothesis that prodrugs of this type could enhance oxidative stress via lipid peroxidation was not established, the results nonetheless demonstrated oxidatively-activated cytotoxicity via indole acetic acid prodrugs, and suggested these as a new type of substrate for antibody-directed enzyme-prodrug therapy (ADEPT). The hypothesized free-radical fragmentation intermediates were demonstrated, but lipid peroxidation associated with peroxyl radical formation was unlikely to be the major route to cytotoxicity.     

Theophylline for irreversible chronic airflow limitation: a randomized study comparing n of 1 trials to standard practice

Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies on this unique approach to ADEPT.     

N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin: synthesis, chemical and enzymatic stability studies

Four N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin are prepared and characterized. Their enzymatic and chemical stability are assessed using high-performance liquid chromatography. The prodrug derivatives are shown to degrade stoichiometrically to Leu-enkephalin in phosphate buffer [t1/2 (0.05 M phosphate buffer without KCl): acetone prodrug (II) 930 min; cyclopentanone prodrug (III): 216 min; cyclohexanone prodrug (IV): 432 min; 4-methylcyclohexanone prodrug (V): 792 min]. Furthermore, the prodrugs are shown to afford global stabilization of the Leu-enkephalin molecule towards the enzymes, aminopeptidase N and angiotensin converting enzyme, primarily responsible for degradation of Leu-enkephalin at the blood-brain barrier and in plasma. Therefore, the 4-imidazolidinones, being metabolic stable and bioreversible, may be suitable prodrug candidates for delivery of Leu-enkephalin to important target areas such as the brain, if given intravenously.     

Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-alpha-phenylalanine). Thus, the kcat/Km value for the wild type hCPA1 with MTX-alpha-phenylalanine is 0.44 microM-1 s-1, and kcat/Km values for hCPA1-T268G with MTX-alpha-3-cyclobutylphenylalanine and MTX-alpha-3-cyclopentyltyrosine are 1.8 and 0.16 microM-1 s-1, respectively. The cytotoxic efficiency of hCPA1-268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with the in vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.     

Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies

BACKGROUND: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. METHODS: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. RESULTS AND CONCLUSIONS: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.     

The antitumour activity of the prodrug N-L-leucyl-doxorubicin and its parent compound doxorubicin in human tumour xenografts

The antitumour activity of the investigational agent N-L-leucyl-doxorubicin (Leu-DOX) was compared with that of doxorubicin (DOX) in human tumour xenografts growing subcutaneously in athymic nude mice. Leu-DOX was developed as a prodrug of DOX, and may be converted into the clinically active parent compound by hydrolytic enzymes present in or on tumour cells. It has been suggested that a better therapeutic index with a reduced cardiac toxicity and higher efficacy might be obtained. Both compounds were administered intravenously weekly for 2 weeks, each at maximum tolerated doses of 8 mg/kg and 28 mg/kg for DOX and Leu-DOX, respectively. The panel of xenografts represented three different tumour types. Leu-DOX showed antitumour activity, defined as tumour growth inhibition > 50% and specific growth delay > 1.0, in 10 of the 16 tumours, including two of five breast, five of seven small cell and three of four non-small cell lung carcinomas. In comparison, DOX was active in one breast, four small cell lung and two lung adenocarcinoma xenografts. In all the DOX sensitive lung tumours, Leu-DOX showed higher efficacy than the parent compound. Based on the results of the present study, and since phase I clinical trials with Leu-DOX have already been performed, phase II clinical evaluation of Leu-DOX in patients with breast and lung cancer is recommended.     

Cancer chemotherapy using suicide genes

Suicide gene therapy is a unique form of drug delivery system that allows for negative selection of malignant cells using a prodrug approach. Malignant cells are transduced with a gene encoding an enzyme that can metabolize an otherwise nontoxic prodrug into a toxic metabolite. The prototype of this system is the herpes simplex virus thymidine kinase gene (HSV-tk). Suicide genes may be introduced into tumor cells either by viral vectors or nonviral methods. Current work is underway to fine tune both the delivery systems and optimize the efficacy of the production of the toxic metabolites. Suicide gene therapy is an exciting strategy currently in clinical trial in the treatment of a number of tumors.     

Ocular penetration and bioconversion of prostaglandin F2alpha prodrugs in rabbit cornea and conjunctiva

The objective of this study was to identify prostaglandin F2alpha (PGF2alpha) prodrugs that have an optimal ocular absorption profile and therefore could be potentially useful for the treatment of glaucoma. Rabbit cornea, conjunctiva, and iris/ciliary body were mounted in a flow-through chamber to evaluate the permeability and bioconversion of PGF2alpha and its prodrugs. The prodrugs tested were PGF2alpha 1-isopropyl, 1,11-lactone, 15-acetyl, 15-pivaloyl, 15-valeryl, and 11,15-dipivaloyl esters. After 4 h in the donor or acceptor compartments, the products and formation of PGF2alpha were analyzed by HPLC. Effects on intraocular pressure and ocular surface hyperemia were also determined. All prodrugs penetrated the rabbit cornea faster than PGF2alpha by 4- to 83-fold. All prodrugs penetrated conjunctiva faster than PGF2alpha, except the 15-acetyl ester prodrug, which was equally permeable. No direct correlation between drug lipophilicity and permeability across the cornea or conjunctiva was apparent. The most metabolically stable prodrug was the 1,11-lactone, followed by the 11,15-dipivaloyl, 15-pivaloyl, 15-acetyl, 1-isopropyl, and the 15-valeryl esters, the latter of which was extensively converted to PGF2alpha. A separation index for various prodrugs was calculated from the ratio of the bioavailable PGF2alpha for ocular hypotension to the bioavailable PGF2alpha for hyperemia. The highest separation index was observed for the 1,11-lactone prodrug (2.33), followed by the 11,15-dipivaloyl ester prodrug (1.80). Thus the 1,11-lactone and 11,15-dipivaloyl ester prodrugs appeared to be superior to the others in providing bioavailable PGF2alpha for ocular hypotension, while minimizing hyperemia. The favorable separation index for these compounds appeared to be due to their metabolic stability at the corneal surface and conjunctiva combined with sufficient bioavailability for ocular hypotension.     

Intracellular signaling events in superoxide generation and adhesion of channel catfish, Ictalurus punctatus rafinesque, neutrophils to the extracellular matrix protein fibrinogen

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.     

Secondary leukaemia characterised by monosomy 7 occurring post-autologous stem cell transplantation for AML

Secondary leukaemia has rarely been reported as a complication of autologous stem cell transplantation for AML. We report two cases of AML who presented with well-characterised cytogenetic abnormalities at presentation: t(8;21) and t(15;17) respectively, and who, after achieving complete morphological and cytogenetic remissions post-autograft, developed MDS/AML associated with monosomy 7. This secondary change is most frequently seen following alkylating agent therapy for solid tumours. The secondary leukaemia seen in our patients may thus be due to exposure of the residual stem cells to the alkylating agents used in the transplant conditioning.     

Changing responsiveness of developing midbrain dopaminergic neurons for extracellular growth factors

Mitomycin C (MMC) is the prototype bioreductive DNA alkylating agent. To exploit its unique properties and maximize patient responses, different therapeutic approaches have been investigated. Recently, the focus has concentrated on monitoring the levels of the proteins metabolizing the drug and relating these to activity in a regimen referred to as enzyme-directed bioreductive drug development. To be successful, it is important to understand the enzymology of metabolic activation not only in cell lines but also in solid tumour models. A general mechanism of action for MMC has now emerged that is activated regardless of the source of reducing equivalents, comprising three competing pathways that give rise to unique reactive intermediates and different DNA adducts. Partitioning into the pathways is dictated by chemical considerations such as pH and drug concentration. DT-diaphorase stands out in this mechanism, since it is much less effective at metabolizing MMC at neutral pH. At least five different enzymes can catalyse MMC bioreduction in vitro, and as many activities may be present in solid tumours, including a series of novel mitochondrial reductases such as a cytochrome P450 reductase. Competition between reductases for MMC appears to be based solely on protein levels rather than enzyme kinetics. Consequentially, DT-diaphorase can occupy a central role in MMC metabolic activation since it is often highly overexpressed in cancer cells. Although a good correlation has been observed in cell lines between DT-diaphorase expression and aerobic cytotoxicity, this does not hold consistently in vivo for any single bioreductive enzyme, suggesting revision of the enzyme-directed hypothesis as originally formulated.     

Stereoselectivity in cutaneous hydrolysis and transdermal transport of propranolol prodrug

This review is written to evaluate the stereoselectivity in cutaneous hydrolysis and transdermal transport of propranolol prodrug. This discussion will be useful in the development of knowledge about stereoselective cutaneous hydrolysis and its influence on stereoselective transdermal transport of many other chiral prodrugs and drugs. Propranolol prodrugs undergo stereoselective hydrolysis in hairless mouse skin homogenate and in excised skin samples during permeation; the stereoselectivity is markedly biased towards hydrolysis of the (R) isomer. Unlike the liver, the esterase activity of the skin is high in its cytosolic fraction. Most of the lipophilic propranolol prodrugs cause stereoselective permeation across hairless mouse skin. A mechanism of stereoselective permeation of propranolol prodrug across the skin has been proposed, which indicates that the stereoselectivity in permeation is resulted from the stereoselective hydrolysis of lipophilic prodrug during permeation.     

5'-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter

PURPOSE: General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5' -amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. METHODS: Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleoside antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycly ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5' -amino acid ester prodrugs was characterized in three different experimental systems; in situ rat perfusion model, CHO/hPEPT1 cells and Caco-2 cells. RESULTS: Testing 5' -amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPT1, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H+/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5' - amino acid ester prodrugs enhanced the transcellular transport of the parent drug. CONCLUSIONS: This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.     

Risk factors of severe hypoglycaemia in adult patients with Type I diabetes--a prospective population based study

Novel thiazolidine prodrugs were prepared by the condensation of L-cysteine with aldose disaccharides. Using a disaccharide in prodrug construction allows for a terminal cyclic sugar moiety to be present on the prodrug, which may allow the delivery of the agent to specific receptors, such as the asialoglycoprotein receptor (ASGPR) of hepatocytes, that require specific structural motifs for recognition. Three L-cysteine prodrugs were synthesized with a pendant cyclic galactose moiety; two related glucose-bearing prodrugs were synthesized for comparison. The prodrugs were designed to release L-cysteine, which is then available to support glutathione (GSH) biosynthesis and provide cytoprotection against a variety of toxic insults. Protection studies in Swiss-Webster mice used acetaminophen (575 mg/kg), a well-documented hepatotoxin which depletes GSH at overdose. Three prodrugs performed exceptionally well against acetaminophen-induced hepatotoxicity, as measured by increased survival and improved histological profiles of liver tissue after 48 h. In further experimentation, two of the disaccharide-based prodrugs, prepared from alpha- and beta-lactose, were compared with the monosaccharide-based compound prepared from ribose. Co-administration of the selected prodrugs with a 400 mg/kg dose of acetaminophen to Swiss-Webster mice prevented the short-term depletion in hepatic GSH and also reduced hepatotoxicity as determined by histological damage and serum levels of alanine aminotransferase. A single dose of the prodrugs alone had no effect on hepatic drug metabolizing enzymes [glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase (QOR), UDP-glucuronosyltransferase (UGT), and cytochrome P450], but, concordant with the reduction of hepatotoxicity, the latentiated forms prevented the significant elevation in QOR activity and mRNA and GST mRNA elicited by acetaminophen itself. GST activity, UGT activity and mRNA, and cytochrome P450 concentration were all unaffected by acetaminophen or the prodrugs. These studies identified novel L-cysteine prodrugs with potentially useful hepatoprotective activity. However, no structure-activity relationships were obvious. In addition, the occurrence of targeted delivery to hepatocytes remains ambiguous.     

Molecular basis of the catalytic differences among DT-diaphorase of human, rat, and mouse

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acceptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.     

N-Substituted 2-(2,6-dinitrophenylamino)propanamides: novel prodrugs that release a primary amine via nitroreduction and intramolecular cyclization

A series of N-dinitrophenylamino acid amides [(4-CONHZ-2, 6-diNO2Ph)N(R)C(X,Y)CONHPhOMe] were prepared as potential bioreductive prodrugs and reduced radiolytically to study their rates of subsequent intramolecular cyclization. Compounds bearing a free NH group (R = H) underwent rapid cyclization in neutral aqueous buffers (t1/2 < 1 min) following 4-electron reduction, with the generation of a N-hydroxydihydroquinoxalinone and concomitant release of 4-methoxyaniline. Amine release from analogous N-methyl analogues (R = Me) was relatively slow. These results are consistent with intramolecular cyclization of a monohydroxylamine intermediate. The high rates of cyclization/extrusion by these very electron-deficient hydroxylamines suggest that the process is greatly accelerated by the presence of an H-bonding "conformational lock" between the anilino NH group and the adjacent o-nitro group (Kirk and Cohen, 1972). Changes in the phenylcarboxamide side chain or in C-methylation in the linking chain had little effect on the rate of cyclization. The model compounds had 1-electron reduction potentials in the range appropriate for cellular reduction (-373 mV for a measured example) and appeared suitable for development as prodrugs that release amine-based effectors following enzymic or radiolytic reduction. Prodrug examples containing 4-aminoaniline mustard and 5-amino-1-(chloromethyl)benz[e]indoline alkylating units were evaluated but were not activated efficiently by cellular nitroreductases. However, cell killing by the radiation-induced reduction of the latter prodrug was demonstrated.