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At least 19 recordsLinked to original sources

PubMed1995

Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT).

Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics.

Antibodies, Monoclonal↗

PubMed2003

Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy.

Cytochrome P450-based gene therapy can substantially increase the sensitivity of tumor cells to P450-activated cancer chemotherapeutic prodrugs such as cyclophosphamide (CPA) without increasing host toxicity. While the role of 4-OH-CPA, the primary active metabolite of CPA, in eliciting tumor cell death is well established, the effect of 4-OH-CPA exposure on the capacity of P450-expressing tumor cells for continued metabolism and activation of CPA has not been investigated. The present study addresses this question and characterizes the impact of CPA dose and treatment schedule on the ability of P450-expressing tumor cells to sustain prodrug activation over time. 9L gliosarcoma cells expressing human P450 2B6 and treated with CPA in a continuous manner exhibited a time- and CPA dose-dependent decrease in P450-catalyzed CPA 4-hydroxylase activity. This decrease reflects a selective, 4-OH-CPA-induced loss of cellular P450 protein content. By contrast, when the P450-expressing tumor cells were treated with CPA as a single 8 hours exposure, cellular CPA 4-hydroxylase activity and P450 protein expression were substantially prolonged when compared to continuous prodrug treatment. This schedule-dependent effect of CPA was influenced by the level of P450 protein expressed in the tumor cells. At high P450 protein and activity levels, which could be achieved by culturing the tumor cells at high cell density, net production and release of 4-OH-CPA into the culture media was increased substantially. This increase fully offset the decline in CPA 4-hydroxylase activity as the tumor cells underwent CPA-induced apoptotic death. These findings demonstrate the impact of CPA dose and treatment schedule on the efficacy of P450 gene-directed enzyme prodrug therapy, with bolus CPA treatment being compatible with sustained expression of P450 protein and maintenance of P450-dependent prodrug activation by the target tumor tissue.

Animals↗

PubMed1998

A comparison of the bioconversion rates and the Caco-2 cell permeation characteristics of coumarin-based cyclic prodrugs and methylester-based linear prodrugs of RGD peptidomimetics.

PURPOSE: To compare the bioconversion rates in various biological media and the Caco-2 cell permeation characteristics of coumarin based cyclic prodrugs (3a, 3b) and methylester-based linear prodrugs (1b, 2b) of two RGD peptidomimetics (1a, 2a). METHODS: Bioconversion rates of the prodrugs to the RGD peptidomimetics were determined in Hank balances salt solution (HBSS), pH 7,4, at 37 degrees C and in various biological media (human blood plasma, rat liver homogenate, Caco-2 cell homogenate) known to have esterase activity. Transport rates of the prodrugs and the RGD peptidomimetics were determined using Caco-2 cell monolayers, an in vitro cell culture model of the intestinal mucosa. RESULTS. In HBSS, pH 7,4, the coumarin-based cyclic prodrugs 3a and 3b degraded slowly and quantitatively to the RGD peptidomimetics 1a and 2a, respectively (3a, t1/2 = 630+2-14 min; 3b, t1/2 = 301 +/-12 min). The methylester-based linear prodrugs 1b and 2b were more stable to chemical hydrolysis (1b and 2b, t1/2 > 2000 min). Both the coumarin-based cyclic prodrugs and the methylester-based linear prodrugs degraded more rapidly in biological media containing esterase activity (e.g., 90% human blood plasma: 1b, t1/2 < 5 min; 2b, t1/2 < 5 min; 3a, t1/2 < 91+/-1 min; 3b, 1/2 < 57+/-2 min). When the apical (AP)-to-basolateral (BL) permeation characteristics were determined using Caco-2 cell monolayers, it was found that the methylester prodrugs 1b and 2b underwent esterase bioconversion (>80%) to the RGD peptidomimetics 1a and 2a, respectively, In contrast, the cyclic prodrugs 3a and 3b permeated the cell monolayers intact. Considering the appearance of both the prodrug and the RGD peptidomimetic on the BL side, the methylester prodrugs 1b and 2b were approximately 12-fold more able to permeate than were the RGD peptidomimetics 1a and 2a. When similar analysis of the transport data for the coumarin prodrugs 3a and 3b was performed, they were shown to be approximately 6-fold and 5-fold more able to permeate than were the RGD peptidomimetics 1a and 12a, respectively. CONCLUSION: The coumarin-based cyclic prodrugs 3a and 3b were chemically less stable, but metabolically more stable, then the methylester based linear prodrugs. The esterase stability of the cyclic prodrugs 3a and 3b means that they are transported intact across the Caco-2 cell monolayer in contrast to the methylester prodrugs 1b and 2b, which undergo facile bioconversion during their transport to the RGD peptidomimetics. However, both prodrug systems successfully delivered more (5-12-fold) of the RGD peptidomimetic and/or the precursor (prodrug) than did the RGD peptidomimetics themselves.

Animals↗

PubMed1999

A novel prodrug approach for tertiary amines. 3. In vivo evaluation of two N-phosphonooxymethyl prodrugs in rats and dogs.

N-phosphonooxymethyl derivatives of tertiary amine containing drugs have been identified as a novel prodrug approach for improving aqueous solubility. The in vivo reversion of two prodrugs to the corresponding parent compounds following iv and im administration to rats and dogs was investigated. Equimolar doses of parent drugs (loxapine or cinnarizine) and the corresponding prodrugs were each administered via a rapid iv infusion to rats and dogs. Equimolar doses of loxapine and its prodrug were each administered im to rats only. Blood samples were collected over 12 h, and plasma was assayed for both parent drug and intact prodrug by HPLC. Comparison of the plasma AUC for the parent drugs following administration of the parent drugs and prodrugs allowed estimation of the apparent bioavailability of parent drug from prodrug dosing. Plasma levels of the prodrugs fell below the limit of detection 5 min after iv infusion with an approximate half-life of 1 min. The mean AUCs following iv and im dosing of parent drugs were not statistically different from the parent drug AUCs obtained after prodrug dosing. The results are consistent with rapid and quantitative prodrug to parent drug reversion following administration of the phosphonooxymethyl prodrugs to the rats and dogs. This information, together with previous studies on the synthesis and physicochemical evaluation of the prodrugs, suggests that this novel prodrug strategy is a very promising approach for overcoming solubility limitations seen with many tertiary amine containing drugs at physiological pH values.

Amines↗

PubMed1997

In vitro and in vivo evaluations of the metabolism, pharmacokinetics, and bioavailability of ester prodrugs of L-767,679, a potent fibrinogen receptor antagonist: an approach for the selection of a prodrug candidate.

The present study demonstrates the utility of an in vitro-in vivo correlative approach in the selection of an optimum prodrug candidate of L-767,679 (N-([7-(piperazin-1-yl)-3,4-dihydro-1(1H)-isoquinolinone-2-yl]acetyl)-3(S)-(ethynyl)-beta-alanine), a potent fibrinogen receptor antagonist. As an initial screening step, a comparative in vitro hepatic metabolism study was conducted for L-767,679 and a series of aliphatic and aromatic ester prodrugs in dogs, monkeys, and humans. In all species, the active acid L-767,679, but not the ester prodrugs, was resistant to metabolism. Only the methyl, ethyl, and isopropyl esters were converted exclusively to the active acid in liver microsomal preparations from dogs and humans, and thus were selected for further studies. In the preparations from monkeys, all of the esters investigated were metabolized efficiently to both the active acid and several other products. The absolute formation rates of L-767,679 from the esters followed the rank order: methyl approximately ethyl > isopropyl in all species, and in humans > dogs for the three esters. The three ester prodrugs did not undergo appreciable hydrolysis in blood or upon incubation with intestinal S9 from any of the studied species. In vivo evaluation of the previous three aliphatic esters in dogs and monkeys supported the in vitro findings. L-767,679 was metabolically stable in both dogs and monkeys. After intravenous administration of the prodrugs to either species, the extent of acid formation was higher in dogs than in monkeys. In addition, the extent of L-767,679 formed from these prodrugs followed the rank order: methyl approximately ethyl > isopropyl. Similar results were obtained after oral dosing of the prodrugs, such that the bioavailability of L-767,679 was higher in dogs than in monkeys, and the bioavailability was higher after the ethyl ester than after the isopropyl prodrug in both species. In either species, both ethyl and isopropyl ester prodrugs were better absorbed than L-767,679. Overall, the results suggested that the bioavailability of the active acid after administration of an ester prodrug was dictated primarily by two factors, viz.:1) the relative rates of ester hydrolysis versus competing metabolic reactions and 2) the absolute rates of ester hydrolysis. In the case of L-767,679 prodrugs, absorption was not a limiting factor. Consequently, the bioavailability of L-767,679 after oral administration of the ester prodrugs would likely be greater in humans than in dogs, and in humans would be higher with the ethyl ester than with the isopropyl ester. On this basis, the ethyl ester was considered as a promising candidate for clinical evaluation as a fibrinogen receptor antagonist prodrug.

Administration, Oral↗

PubMed1987

Effects of vehicles and prodrug properties and their interactions on the delivery of 6-mercaptopurine through skin: bisacyloxymethyl-6-mercaptopurine prodrugs.

A series of S6,9-bisacyloxymethyl-6-mercaptopurine (6,9-bis-6-MP) prodrug derivatives was synthesized and characterized. The solubilities of the derivatives in solvents (vehicles), which exhibited a wide range of polarities from water to oleic acid, were measured. The abilities of the prodrugs to deliver 6-mercaptopurine (6-MP) from the vehicles have also been determined, and experimental fluxes and permeability coefficients (Kp) have been calculated for a large number of prodrug: vehicle combinations. Generally the best prodrugs of the series in terms of delivering 6-MP, regardless of the vehicle, were the first two members--the bisacetyl- and the bispropionyloxymethyl-6-mercaptopurine prodrugs. This result has been attributed mainly to the increased water solubility of these two prodrugs compared with that of 6-MP and the other prodrugs, since all of the prodrugs are much more lipid soluble than 6-MP. For three vehicles--isopropyl myristate, propylene glycol, and water--there was a good correlation between log experimental Kp for the delivery of 6-MP by the prodrugs from those vehicles and the theoretical solubility parameters of the prodrugs. The stabilities of the bisacetyl-(2), bisproprionyl-(3), and bisbutyryloxymethyl-6-mercaptopurine (4) derivatives were determined in buffer and in buffer containing enzymes leached from the dermis. Prodrug 2 was more stable than 3 or 4 in the buffer containing the enzymes, while 4 was more stable than 2 or 3 in the plain buffer.

Animals↗

PubMed1988

Effects of vehicles and prodrug properties and their interactions on the delivery of 6-mercaptopurine through skin: S6-acyloxymethyl-6-mercaptopurine prodrugs.

A homologous series of S6-acyloxymethyl-6-mercaptopurine (6-mono-6-MP) and two 9-acyloxymethyl-6-mercaptopurine (9-mono-6-MP) prodrugs have been synthesized and characterized. The ability of the 6-mono-6-MP prodrugs to deliver 6-mercaptopurine (6-MP) through hairless mouse skin from isopropyl myristate (IPM) and propylene glycol (PG) has been evaluated. There was a good correlation between the log experimental permeability coefficients from the diffusion data and calculated solubility parameters of the prodrugs. Although there was no statistical difference between the rates of delivery of 6-MP by the acetyl through valeryl 6-mono-6-MP prodrugs from IPM, the butyryl and valeryl prodrugs were significantly better at delivering 6-MP from PG. For a given solubility parameter value, the 6-mono-6-MP prodrugs were less soluble in water and IPM, and more soluble in PG than the previously studied S6,9-bisacyloxymethyl-6-MP (6,9-bis-6-MP) prodrugs. On the other hand, for a given solubility parameter, the 6,9-bis-6-MP prodrugs were generally more effective at delivering 6-MP from IPM and PG. The single 9-mono-6-MP prodrug that was evaluated was much less effective at delivering 6-MP than either the 6-mono- or 6,9-bis-6-MP prodrugs. Thus, it is much less important to mask the imidazole than the thionamide functional group in 6-MP to enhance the topical delivery of 6-MP using a prodrug approach.

Animals↗

PubMed2003

Development of water-soluble prodrugs of the HIV-1 protease inhibitor KNI-727: importance of the conversion time for higher gastrointestinal absorption of prodrugs based on spontaneous chemical cleavage.

We designed and synthesized a series of water-soluble prodrugs of the HIV-1 protease inhibitor KNI-727 (1), which is a sparingly water-soluble drug with a water solubility of 5.5 microg/mL. These prodrugs, which contain a water-soluble auxiliary with two tandem-linked units, i.e., a self-cleavable spacer and a solubilizing moiety with an ionized amino function, exhibited a marked increase in water solubility (>10(4)-fold) compared with the parent drug 1. The mechanism of conversion to the parent drug 1 is not enzymatic but through a chemical cleavage at the spacer via an intramolecular cyclization-elimination reaction through an imide formation under physiological conditions. To diversify the conversion time for the parent drug regeneration, chemical modification of the auxiliary was carried out focusing on the introduction of cyclic tertiary amines, which can modify the basicity and/or conformational flexibility of the terminal amino function at the solubilizing moiety, and the change in bond length, which can attenuate the five-membered ring intermediate formation in the cleavage. These newly synthesized water-soluble prodrugs exhibited a practical water solubility with values greater than 50 mg/mL and enabled the constant regeneration of the parent drug 1 with diversified conversion times ranging from 4 min to 34 h as t(1/2) values under physiological conditions. All the water-soluble prodrugs tested regenerated the parent drug 1 in vivo as well as in vitro. A clear increase in the gastrointestinal absorption was observed in prodrugs 8, 12, and 13 with bioavailability (BA) values of 23%, 26%, and 29%, respectively. These BA values were 1.5-1.9-fold higher than that in the administration of the parent drug 1 alone. Other prodrugs showed only a similar or decreased BA compared to the parent drug 1. From these results, we found that not only a high water solubility but also an appropriate conversion time of the prodrug with a relatively narrow limit of around 35 min via intraduodenal administration was necessary for significant improvement of the gastrointestinal absorption in water-soluble prodrugs based on the spontaneous chemical cleavage. This is the first successful water-soluble prodrug that suggests an increased BA value greater than the parent drug in HIV-1 protease inhibitors and is the first study to show the importance of optimal conversion time in water-soluble prodrugs. Consequently, a water-soluble strategy that can control the conversion time would be extensively applicable to improve the gastrointestinal absorption of sparingly water-soluble drugs. The present information is an intriguing discovery and is one of the key factors that will contribute to the future design of practical water-soluble prodrugs.

Animals↗

PubMed2001

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy.

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 10(2)-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.

Animals↗

PubMed1997

Antibody-directed enzyme prodrug therapy: pharmacokinetics and plasma levels of prodrug and drug in a phase I clinical trial.

Antibody-directed enzyme prodrug therapy (ADEPT) was administered to ten patients in a phase I clinical trial. The aim was to measure plasma levels of the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA) and the bifunctional alkylating drug (CJS11) released from it by the action of tumour-localised carboxypeptidase G2 (CPG2) enzyme. New techniques were developed to extract the prodrug and drug from plasma by solid-phase absorption and elution and to measure CPG2 activity in plasma and tissue. All extracts were analysed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). CPG2 activity was found in metastatic tumour biopsies but not in normal tissue, indicating that localisation had been successful. The clearing agent SB43-gal, given at 46.5 mg/m2, achieved the aim of clearing non-tumour-localised enzyme in the circulation, indicating that conversion of prodrug to drug could take place only at the site of localised conjugate. Plasma prodrug did not always remain above its required threshold of 3 microM for the "therapeutic window" of 120 min after dosing, but the presence of residual prodrug after the first administration of each day indicated that this could be achieved during the remaining four doses over the following 8 h. Despite considerable inter-patient prodrug plasma concentration variability, the elimination half-life of the prodrug was remarkably reproducible at 18 +/- 8 min. Rapid appearance of the drug in plasma indicated that successful conversion from the prodrug had taken place, but also undesirable leakback from the site of localisation into the bloodstream. However, drug plasma levels fell rapidly by at least 50% at between 10 and 60 min with a half-life of 36 +/- 14 min. Analysis of the plasma extracts by LC/MS indicated that this technique might be used to confirm qualitatively the presence of prodrug, drug and their metabolites.

Antibodies, Monoclonal↗

PubMed2005

Nitroarylmethylcarbamate prodrugs of doxorubicin for use with nitroreductase gene-directed enzyme prodrug therapy.

A series of nitrobenzyl- and nitroimidazolylmethyl carbamate prodrugs of doxorubicin were prepared and evaluated for their potential use in nitroreductase (NTR) mediated gene-directed enzyme prodrug therapy (GDEPT). The carbamate prodrugs and doxorubicin were tested in a cell line panel comprising parental and NTR transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79/V79-NTR(puro)) and murine (EMT6/EMT6-NTR(puro)) cell line pairs, and were compared with the established NTR substrates CB 1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard SN 29427. The low solubility of the prodrugs (from 3 to 39 microM) precluded the determination of IC(50) values against the parent cell lines in some instances. All of the prodrugs were unstable in culture medium with 5% added fetal calf serum over a 24h period, although release of doxorubicin was not observed. The prodrugs were 20- to >336-fold less toxic than doxorubicin in the human cells lines SKOV3 and WiDr, with overall less deactivation seen in the V79 cell line (11- to >286-fold) and EMT6 cell line (1.8- to >178-fold). Prodrugs with the nitrobenzyl unit directly conjugated to doxorubicin showed modest selectivity for NTR across the cell line panel (1- to 5.9-fold) but this was increased to between >10- and >370-fold with the interpolation of an 4-aminobenzyl spacer unit between the bioreductive unit and doxorubicin. A 2-nitroimidazolylmethyl carbamate provided deactivation of doxorubicin (8- to 124-fold) but showed only modest selectivity for NTR (2- to 14-fold) across the panel. The interpolation of a 4-aminobenzyl spacer gave slightly lower deactivation (3- to 64-fold) and similar selectivity for NTR (>1.2- to >12-fold) for 2- and 5-nitroimidazolylmethyl prodrugs. The activity of two nitrobenzyl prodrugs containing an aminobenzyl spacer, providing excellent selectivity for NTR+ve cells in culture, was evaluated against EMT6 tumours comprising ca. 10% NTR+ve cells, but neither showed statistically significant levels of killing even of NTR+ve cells. This lack of activity in tumours, despite potent and selective activity in culture, indicates that pharmacokinetic optimization is needed to achieve in vivo efficacy against solid tumours with this new class of NTR prodrugs.

Animals↗

PubMed1993

Renal selective N-acetyl-L-gamma-glutamyl prodrugs: studies on the selectivity of some model prodrugs.

1. In this study, a number of structurally different N-acetyl-L-gamma-glutamyl prodrugs were investigated with respect to selective uptake by the kidney in male Wistar rats. 2. All prodrugs were tested in vitro in rat kidney slices and kidney homogenate to study their uptake and conversion. It was found that the prodrugs of para-nitroaniline (agPNA), aminophenyl acetic acid (agAFA), sulphamethoxazole (agSM), sulphadimethoxine (agSDM), propranolol (agPP) and metoprolol (agMP) were accumulated by a probenecid-sensitive carrier. The prodrug of 4'-aminoantipyrine (agAAP) was not accumulated by a probenecid- or buthionine sulphoximine-sensitive carrier. Unlike all other prodrugs, agAAP and agMP were not, or only a very limited extent converted to the parent compound in vitro. 3. agPNA, agAFA and agPP were also investigated in vivo. The tissue distribution of the prodrugs and the parent drugs was established, as was their urinary excretion and pharmacokinetic behaviour. agPNA and agAFA showed selective uptake by the kidney, in contrast to agPP which accumulated in the liver. The distribution of the parent compounds following prodrug administration was as follows: agPNA was found in kidney and plasma: agAFA in kidney only; agPP in liver only. 4. The factors which determine the selectivity of N-acetyl-L-gamma-glutamyl prodrugs are discussed. The main factors are: the transport into the kidney, the conversion rate, the residence time of the prodrug in the kidney and the presence or absence of competition for uptake and conversation by other tissues, e.g. the liver. It is concluded that this prodrug approach offers the possibility of delivering drugs selectively to the kidney, but also that it is not universally applicable.

Ampyrone↗

PubMed2004

Design, synthesis, and characterization of a series of cytochrome P(450) 3A-activated prodrugs (HepDirect prodrugs) useful for targeting phosph(on)ate-based drugs to the liver.

A new class of phosphate and phosphonate prodrugs, called HepDirect prodrugs, is described that combines properties of rapid liver cleavage with high plasma and tissue stability to achieve increased drug levels in the liver. The prodrugs are substituted cyclic 1,3-propanyl esters designed to undergo an oxidative cleavage reaction catalyzed by a cytochrome P(450) (CYP) expressed predominantly in the liver. Reported herein is the discovery of a prodrug series containing an aryl substituent at C4 and its use for the delivery of nucleoside-based drugs to the liver. Prodrugs of 5'-monophosphates of vidarabine, lamivudine (3TC), and cytarabine as well as the phosphonic acid adefovir were shown to cleave following exposure to liver homogenates and exhibit good stability in blood and other tissues. Prodrug cleavage required the presence of the aryl group in the cis-configuration, but was relatively independent of the nucleoside and absolute stereochemistry at C4. Mechanistic studies suggested that prodrug cleavage proceeded via an initial CYP3A-catalyzed oxidation to an intermediate ring-opened monoacid, which subsequently was converted to the phosph(on)ate and an aryl vinyl ketone by a beta-elimination reaction. Studies in primary rat hepatocytes and normal rats comparing 3TC and the corresponding HepDirect prodrug demonstrated the ability of these prodrugs to effectively bypass the rate-limiting nucleoside kinase step and produce higher levels of the biologically active nucleoside triphosphate.

Adenine↗

PubMed1995

Cyclization-activated prodrugs: N-(substituted 2-hydroxyphenyl and 2-hydroxypropyl)carbamates based on ring-opened derivatives of active benzoxazolones and oxazolidinones as mutual prodrugs of acetaminophen.

N-(Substituted 2-hydroxyphenyl)- and N-(substituted 2-hydroxypropyl)carbamates based on masked active benzoxazolones (model A) and oxazolidinones (model B), respectively, were synthesized and evaluated as potential drug delivery systems. A series of alkyl and aryl N-(5-chloro-2-hydroxyphenyl)carbamates 1 related to model A was prepared. These are open drugs of the skeletal muscle relaxant chlorzoxazone. The corresponding 4-acetamidophenyl ester named chlorzacetamol is a mutual prodrug of chlorzoxazone and acetaminophen. Chlorzacetamol and two other mutual prodrugs of active benzoxazolones and acetaminophen were obtained in a two-step process via condensation of 4-acetamidophenyl 1,2,2,2-tetrachloroethyl carbonate with the appropriate anilines. Based on model B, two mutual prodrugs of acetaminophen and active oxazolidinones (metaxalone and mephenoxalone) were similarly obtained using the appropriate amines. All the carbamate prodrugs prepared were found to release the parent drugs in aqueous (pH 6-11) and plasma (pH 7.4) media. The detailed mechanistic study of prodrugs 1 carried out in aqueous medium at 37 degrees C shows a change in the Brönsted-type relationship log t1/2 vs pKa of the leaving groups ROH: log t1/2 = 0.46pKa-3.55 for aryl and trihalogenoethyl esters and log t1/2 = 1.46pKa-16.03 for alkyl esters. This change is consistent with a cyclization mechanism involving a change in the rate-limiting step from formation of a cyclic tetrahedral intermediate (step k1) to departure of the leaving group ROH (step k2) when the leaving group ability decreases. This mechanism occurs for all the prodrugs related to model A. Regeneration of the parent drugs from mutual prodrugs related to model B takes place by means of a rate-limiting elimination-addition reaction (E1cB mechanism). This affords acetaminophen and the corresponding 2-hydroxypropyl isocyanate intermediates which cyclize at any pH to the corresponding oxazolidinone drugs. As opposed to model A, the rates of hydrolysis of mutual prodrugs of model B clearly exhibit a catalytic role of the plasma. It is concluded from the plasma studies that the carbamate substrates can be enzymatically transformed into potent electrophiles, i.e., isocyanates. In the case of the present study, the prodrugs are 2-hydroxycarbamates for which the propinquity of the hydroxyl residue and the isocyanate group enforces a cyclization reaction. This mechanistic particularity precludes their potential toxicity in terms of potent electrophiles capable of modifying critical macromolecules.

Acetaminophen↗

PubMed2003

Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form.

The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.

Aniline Mustard↗

PubMed1997

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.

Animals↗

PubMed2006

Development of an etoposide prodrug for dual prodrug-enzyme antitumor therapy.

Enzyme-prodrug approaches to cancer therapy, theoretically, have the potential to mediate tumor-selective cytotoxicity. However, even if tumor-specific prodrug activation is achieved, enzyme-prodrug systems investigated thus far comprised a single enzyme and a specific prodrug. Although targeted, such systems constitute single-agent therapy, which may be ineffective and/or may promote development of drug resistance. Therefore, a goal of our laboratories was to design and characterize a novel dipiperidinyl derivative of etoposide [1,4'-dipiperidine-1'-carboxylate-etoposide (dp-VP16)] that would act as a prodrug. We envisioned that dp-VP16 would be converted to the active chemotherapeutic agent VP-16 by the same rabbit carboxylesterase (rCE) that we have previously shown to efficiently activate the prodrug irinotecan (CPT-11). This dp-VP16 prodrug might then be used in combination with CPT-11, with both drugs activated by a single enzyme. We evaluated the ability of pure rCE and two human carboxylesterases, hCE1 and hiCE (hCE2), to activate dp-VP16 in vitro, and in neuroblastoma cell lines designed to express/overexpress each enzyme. In SK-N-AS neuroblastoma cell transfectants, expression of rCE or hiCE decreased the IC50 of dp-VP16 as a single agent by 8.3- and 3.4-fold, respectively, in growth inhibition assays. Purified hCE1 did not metabolize dp-VP16 in vitro and did not affect its IC50 in intact cells. The combination indices of sequential exposure to CPT-11 followed by dp-VP16 ranged from approximately 0.4 to 0.6, suggesting that this combination produced greater-than-additive cytotoxicity in neuroblastoma cells expressing rCE. These data provide proof-of-principle that enzyme-prodrug therapy approaches comprised of prodrugs with complementary mechanisms of cytotoxicity that are activated by a single enzyme can be developed.

Animals↗

PubMed1991

Comparison of half-lives and cytotoxicity of N-chloroethyl-4-amino and N-mesyloxyethyl-benzoyl compounds, products of prodrugs in antibody-directed enzyme prodrug therapy (ADEPT).

The synthesis of two novel drugs, 4-[bis[2-(mesyloxy)ethyl]amino]benzoic acid (7) and 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]amino]benzoic acid (8) is described here. They are the active drugs of two prodrugs (9 and 10) designed for use as anti-cancer agents. The prodrugs (9, 10 and 11) were made as a series of compounds which are bifunctional alkylating agents in which the activating effect of the ionized carboxyl function is masked through an amide bond to a glutamic acid residue. These relatively inactive prodrugs were designed to be activated to their corresponding alkylating agent active drugs (7, 8 and 12 respectively) at a tumour site by prior administration of a monoclonal antibody conjugated to a bacterial enzyme. This system is called antibody-directed enzyme prodrug therapy (ADEPT). The chemical half-lives of the prodrugs and their active drugs were measured in order to determine their relative reactivities. The half-lives ranged from 21 to 324 min for the active drugs and from 42 to 1158 min for the prodrugs. The viability of two different tumour cell lines was monitored with each active drug and prodrug. The IC50 values varied from 65 to 625 microM for the active drugs: no IC50 values could be obtained for the prodrugs, using a rapid incubation procedure. Each in vitro technique demonstrated the ability of the glutamic acid moiety to deactivate the drugs, forming effective prodrugs.

Antibodies, Monoclonal↗