Modulating paclitaxel bioavailability for targeting prostate cancer

Four novel water-soluble peptide-paclitaxel conjugates were designed and synthesized as prostate-specific antigen (PSA)-activated prodrugs for prostate cancer therapy. These prodrugs were composed of a peptide, HSSKLQ or SSKYQ, each of which is selectively cleavable by PSA; a self-immolative linker, either para-aminobenzyl alcohol (PABS) or ethylene diamine (EDA); and the parent drug, paclitaxel. Introduction of a PABA or EDA linker between the peptide and paclitaxel in prodrugs 2-5 resulted in products with an increased rate of hydrolysis by PSA. The stability of prodrugs 2 and 3, with the PABA linker, was poor in the serum-containing medium because of the weak carbonate bond between the PABA and paclitaxel; however, this disadvantage was overcome by introducing a carbamate bond using an EDA linker in prodrugs 4 and 5. Thus, the incorporation of an EDA linker increased both the stability and PSA-mediated activation of these prodrugs. The cytotoxicity of each prodrug, as compared to paclitaxel, was determined against a variety of cell lines, including the PSA-secreting CWR22Rv1 prostate cancer cell line. The EDA-derived prodrug of paclitaxel 5 was stable and capable of being efficiently converted to an active drug that killed cells specifically in the presence of PSA, suggesting that this prodrug and similarly designed PSA-cleavable prodrugs may have potential as prostate cancer-specific therapeutic agents.     

Synthesis and preclinical characterization of a paclitaxel prodrug with improved antitumor activity and water solubility

The development of novel chemotherapy strategies based on prodrugs remains a major challenge for effective treatment of malignancies. We tested the hypothesis that this can be achieved by a prodrug of paclitaxel where one biologically active center, represented by the C7 hydroxyl group, was blocked by a dihydroxypropyl side chain which can be hydrolytically cleaved by a pH-dependent, slow-release mechanism. The prodrug was synthesized by condensation of solketal chloroformate with the C7 hydroxyl group of paclitaxel followed by a ring-opening reaction to the dihydroxyl derivative. The cytotoxicity of the prodrug was similar to paclitaxel, when tested in vitro against a variety of human tumor cell lines. In vitro cell cycle analysis indicated that concentrations within the micromolar range of both drug and prodrug are required to induce sufficient G2M arrest. The hydrophilic paclitaxel prodrug proved to be more than 50-fold more water soluble than the parental drug and effectively converted to paclitaxel by pH dependent hydrolysis. Importantly, the prodrug could be used at a 3-fold higher maximum tolerated dose (MTD) and revealed a markedly improved antitumor activity in mice compared to paclitaxel. Taken together, our results demonstrate, that a hydrolytically activated paclitaxel prodrug exhibits greater water solubility and superior antitumor activity than the parental drug.     

Reductively activated disulfide prodrugs of paclitaxel

A series of unsymmetrical polar disulfide prodrugs 2–5 of paclitaxel were designed and synthesized as reductively activated prodrugs. These compounds behaved as prodrugs in vitro on L2987 lung carcinoma cells. In vivo evaluation in mice demonstrated that the mutual prodrug 5 with captopril exhibited significant regressions and cures.     

Novel Developments in the Use of Antimetabolites

Antimetabolites are the most widely used and most efficacious group of anticancer drugs. Antimetabolites are also the oldest rationally designed anticancer drugs, targeted against RNA and DNA, and can, therefore, be considered as the first generation of targeted drugs. Unfortunately, resistance often develops, leading to the design of new antimetabolites, which either have a novel mechanism of action, bypass resistance or in combination enhance the effect of other drugs, such as another antimetabolite, other DNA, or protein kinase targeted anticancer drugs. Several novel antimetabolites are in clinical development. The cytidine-analog fluorocyclopentenylcytosine (RX-3117) is active in gemcitabine-resistant tumors and is activated by uridine-cytidine-kinase, can be incorporated into RNA and DNA and can downregulate DNA-methyltransferase-1. TAS-114 is a new generation dUTPase inhibitor. dUTPase normally prevents incorporation of dUTP and of the 5FU-nucleotide FdUTP into DNA. However, inhibition of dUTPase will enhance their incorporation, thereby increasing thymine-less cell-death. The formulation TAS-102 (trifluorothymidine and thymidine-phosphorylase-inhibitor) acts by incorporation into DNA and has shown efficacy in tumors progressing on 5FU therapy. Gemcitabine and cytarabine prodrugs were tested in model systems and have entered clinical evaluation. The elaidic-acid prodrugs of gemcitabine (CP-4126, CO101) and cytarabine (elacytarabine) failed in randomized Phase III studies. Two other gemcitabine prodrugs LY2334737 (gemcitabine with a valproic acid at the 5′-position) and NUC1031 (a 5′-arylphosphoamidate prodrug, with a side-chain at the 5′-phosphate) are in early clinical development.

In summary, several novel antimetabolites show promise in clinical development, either because of a novel mechanism of action, or clever combination or by innovative prodrug design.     

Enzymatic activation of second-generation dendritic prodrugs: Conjugation of self-immolative dendrimers with poly(ethylene glycol) via click chemistry

Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer's core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.     

Identification of novel enzyme-prodrug combinations for use in cytochrome P450-based gene therapy for cancer

Gene-directed enzyme prodrug therapy can be used to increase the therapeutic activity of anti-cancer prodrugs that undergo liver cytochrome P450 (CYP)-catalyzed prodrug to active drug conversion. The present report describes a cell-culture-based assay to identify CYP gene-CYP prodrug combinations that generate bystander cytotoxic metabolites and that may potentially be useful for CYP-based gene therapy for cancer. A panel of rat liver microsomes, comprising distinct subsets of drug-inducible hepatic CYPs, was evaluated for prodrug activation in a four-day 9L gliosarcoma cell growth inhibition assay. A strong NADPH- and liver microsome-dependent increase in 9L cytotoxicity was observed for the CYP prodrugs cyclophosphamide, ifosfamide, and methoxymorpholinyl doxorubicin (MMDX) but not with three other CYP prodrugs, procarbazine, dacarbazine, and tamoxifen. MMDX activation was potentiated approximately 250-fold by liver microsomes from dexamethasone-induced rats (IC(50) (MMDX) approximately 0.1nM), suggesting that dexamethasone-inducible CYP3A enzymes contribute to activation of this novel anthracycline anti-tumor agent. This CYP3A dependence was verified in studies using liver microsomes from uninduced male and female rats and by using the CYP3A-selective inhibitors troleandomycin and ketoconazole. These findings highlight the advantages of using cell culture assays to identify novel CYP prodrug-CYP gene combinations that are characterized by production of cell-permeable, cytotoxic metabolites and that may potentially be incorporated into CYP-based gene therapies for cancer treatment.     

New Taxol (paclitaxel) prodrugs designed for ADEPT and PMT strategies in cancer chemotherapy

Two new glucuronide paclitaxel prodrugs have been synthesized. Linked to the 2'-OH of the drug by a carbonate function, they include a self-immolative spacer bearing an arylnitro or arylamino group between the drug and the glucuronic acid residue. Both prodrugs were well detoxified and easily cleaved in the presence of beta-D-glucuronidase with fast removal of the spacer, releasing paclitaxel. The arylamino spacer-containing prodrug, more stable than the corresponding nitro analogue, was selected for further studies.     

Acid-activatable prodrug nanogels for efficient intracellular doxorubicin release

Endosomal pH-activatable doxorubicin (DOX) prodrug nanogels were designed, prepared, and investigated for triggered intracellular drug release in cancer cells. DOX prodrugs with drug grafting contents of 3.9, 5.7, and 11.7 wt % (denoted as prodrugs 1, 2, and 3, respectively) were conveniently obtained by sequential treatment of poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-co-ethyl glycinate methacrylamide) (PEG-b-P(HEMA-co-EGMA)) copolymers with hydrazine and doxorubicin hydrochloride. Notably, prodrugs 1, 2, and 3 formed monodispersed nanogels with average sizes of 114.4, 75.3, and 66.3 nm, respectively, in phosphate buffer (PB, 10 mM, pH 7.4). The in vitro release results showed that DOX was released rapidly and nearly quantitatively from DOX prodrug nanogels at endosomal pH and 37 °C in 48 h, whereas only a minor amount (ca. 20% or less) of drug was released at pH 7.4 under otherwise the same conditions. Confocal laser scanning microscope (CLSM) observations revealed that DOX prodrug nanogels delivered and released DOX into the cytosols as well as cell nuclei of RAW 264.7 cells following 24 h incubation. MTT assays demonstrated that prodrug 3 had pronounced cytotoxic effects to tumor cells following 72 h incubation with IC(50) data determined to be 2.0 and 3.4 μg DOX equiv/mL for RAW 264.7 and MCF-7 tumor cells, respectively. The corresponding polymer carrier, PEG-b-P(HEMA-co-GMA-hydrazide), was shown to be nontoxic up to a tested concentration of 1.32 mg/mL. These endosomal pH-activatable DOX prodrug nanogels uniquely combining features of water-soluble macromolecular prodrugs and nanogels offer a promising platform for targeted cancer therapy.     

Development of first photoresponsive prodrug of paclitaxel

A prodrug of paclitaxel which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (O-acyl isoform of paclitaxel) has been synthesized. The prodrug was selectively converted to isotaxel by visible light irradiation (430 nm) with the cleavage of coumarin. Finally, paclitaxel was released by subsequent spontaneous O-N intramolecular acyl migration.     

Bioactivation of carbamate-based 20(S)-camptothecin prodrugs

Two new prodrugs of CPT were synthesized, based on carbamate linkages between the 20-hydroxy group of CPT and a linker designed to be enzymatically removed by either Penicillin-G-Amidase or catalytic antibody 38C2. Cell growth inhibition assays showed an up-to-2250-fold difference in toxicity between the prodrugs and the active drug. A significant increase in toxicity was observed upon incubation of the enzyme or the catalytic antibody with the corresponding prodrug. The described derivatives of CPT further our knowledge in the design of prodrugs for use in selective approaches for targeted chemotherapy.     

Structural modifications modulate stability of glutathione-activated arylated diazeniumdiolate prodrugs

JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical development as an anti-cancer drug candidate. This prodrug was designed to be activated by glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH activation and the drugs' biological activity. We show that nitro-to-cyano substitution increases the half-life of the prodrug in the presence of GSH without compromising the compound's in vivo antitumor activity.     

NMR spectroscopic study of cyclodextrin inclusion complexes with A-007 prodrugs

One- and two-dimensional NMR spectroscopy was used to demonstrate the formation of inclusion cyclodextrin complexes with several A-007 prodrugs. These complexes are comprised from the encapsulation of the two phenol moieties of the A-007 prodrugs within the cyclodextrin cavity. Considering the size of the two phenol moieties of the A-007 prodrugs compared to the sizes of alpha-, beta-, and gamma-cyclodextrin cavities, we observed complementary binding of the A-007 prodrug with only beta-cyclodextrin, which was also demonstrated spectroscopically. The beta-cyclodextrin inclusion complexes increased the prodrug solubility and modified the prodrug half-life in water. Therefore, beta-cyclodextrin inclusion complexes can be used as an essential form of A-007 prodrug delivery.     

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: synthesis,stability, enzymatic reduction,and antiproliferative activity in cell culture

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.     

Prodrug activation enzymes in cancer gene therapy

Among the broad array of genes that have been evaluated for tumor therapy, those encoding prodrug activation enzymes are especially appealing as they directly complement ongoing clinical chemotherapeutic regimes. These enzymes can activate prodrugs that have low inherent toxicity using both bacterial and yeast enzymes, or enhance prodrug activation by mammalian enzymes. The general advantage of the former is the large therapeutic index that can be achieved, and of the latter, the non-immunogenicity (supporting longer periods of prodrug activation) and the fact that the prodrugs will continue to have some efficacy after transgene expression is extinguished. This review article describes 13 different prodrug activation schemes developed over the last 15 years, two of which - activation of ganciclovir by viral thymidine kinase and activation of 5-fluorocytosine to 5-fluorouracil - are currently being evaluated in clinical trials. Essentially all of these prodrug activation enzymes mediate toxicity through disruption of DNA replication, which occurs at differentially high rates in tumor cells compared with most normal cells. In cancer gene therapy, vectors target delivery of therapeutic genes to tumor cells, in contrast to the use of antibodies in antibody-directed prodrug therapy. Vector targeting is usually effected by direct injection into the tumor mass or surrounding tissues, but the efficiency of gene delivery is usually low. Thus it is important that the activated drug is able to act on non-transduced tumor cells. This bystander effect may require cell-to-cell contact or be mediated by facilitated diffusion or extracellular activation to target neighboring tumor cells. Effects at distant sites are believed to be mediated by the immune system, which can be mobilized to recognize tumor antigens by prodrug-activated gene therapy. Prodrug activation schemes can be combined with each other and with other treatments, such as radiation, in a synergistic manner. Use of prodrug wafers for intratumoral drug activation and selective permeabilization of the tumor vasculature to prodrugs and vectors should further increase the value of this new therapeutic modality.     

Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug

Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was approximately 1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.     

Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer

A promising agent for use in prostate cancer therapy is the Hedgehog (Hh) signaling pathway inhibitor, cyclopamine. This compound, however, has the potential for causing serious side effects in non-tumor tissues. To minimize these bystander toxicities, we have designed and synthesized two novel peptide-cyclopamine conjugates as prostate-specific antigen (PSA)-activated prodrugs for use against prostate cancer. These prodrugs were composed of cyclopamine coupled to one of two peptides (either HSSKLQ or SSKYQ) that can be selectively cleaved by PSA, converting the mature prodrug into an active Hedgehog inhibitor within the malignant cells. Of the two prodrugs, Mu-SSKYQ-Cyclopamine was rapidly hydrolyzed, with a half-life of 3.2h, upon incubation with the PSA enzyme. Thus, modulating cyclopamine at the secondary amine with PSA-cleavable peptides is a promising strategy for developing prodrugs to target prostate cancer.     

Structure of human dCK suggests strategies to improve anticancer and antiviral therapy

Human deoxycytidine kinase (dCK) phosphorylates the natural deoxyribonucleosides deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA) and is an essential enzyme for the phosphorylation of numerous nucleoside analog prodrugs routinely used in cancer and antiviral chemotherapy. For many of these compounds, the phosphorylation step catalyzed by dCK is the rate-limiting step in their overall activation pathway. To determine the factors that limit the phosphorylation efficiency of the prodrug, we solved the crystal structure of dCK to a resolution of 1.6 A in complex with its physiological substrate deoxycytidine and with the prodrugs AraC and gemcitabine. The structures reveal the determinants of dCK substrate specificity. Especially relevant to new prodrug development is the interaction between Arg128 and the hydrogen-bond acceptor at the sugar 2'-arabinosyl position of AraC and gemcitabine. On the basis of the structures, we designed a catalytically superior dCK variant that could be used in suicide gene-therapy applications.     

Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.     

Enzyme-catalyzed prodrug approaches for the histamine H3-receptor agonist (R)-alpha-methylhistamine

Five novel prodrug types of the potent and selective histamine H3-receptor agonist (R)-alpha-methylhistamine (1) were prepared and pharmacologically tested in vitro as well as in vivo. In particular, an amide of fatty acid, mono- and dicarbamates, an (acyloxy)alkylcarbamate, and a diphthalidyl derivative were synthesized, all of which require initial prodrug activation through an enzyme-catalyzed reaction in contrast to formerly developed azomethine prodrugs which are cleaved by chemical hydrolysis only. Further drug liberation may ensue spontaneously in a cascade to give 1. Since they have diverse stabilities the prodrugs were investigated for drug liberation in vitro under neutral, acidic, and basic conditions at different temperatures as well as with liver homogenates. In vivo investigation of prodrugs after oral administration to mice proved that the fatty amide 2, the Nalpha-methylcarbamate 4a, and the Nalpha-(1-(acetyloxy)ethylcarbamate) 5 showed moderate to high plasma levels of 1. Compound 5 displayed even more than 2.5 times the AUC for 1 than that of the reference azomethine prodrug BP2.94 in the periphery and also displayed a detectable drug level in the central nervous system. It was shown that prodrug approaches based on an initial enzyme-catalyzed liberation step are successfully applicable to different pro-moieties for improved bioavailability and prolonged half-live. These approaches may also be used for other aminergic compounds of this class to optimize pharmacokinetic behavior.     

Design and synthesis of the tumor-activated prodrug of dihydropyrimidine dehydrogenase (DPD) inhibitor,RO0094889 for combination therapy with capecitabine

A series of tumor-activated prodrugs of the inhibitors of dihydropyrimidine dehydrogenase (DPD), an enzyme catabolizing 5-fluorouracil (5-FU: 4g), has been designed and synthesized. RO0094889 (11c) is a prodrug of 5-vinyluracil (4c), a known DPD inhibitor, and was designed to generate 4c selectively in tumor tissues by sequential conversion of 11c by three enzymes: esterase, cytidine deaminase and thymidine phosphorylase, the latter two of which are known to be highly expressed in various tumor tissues. When capecitabine (1), a tumor-activated prodrug of 5-FU, was co-administered orally with 11c, 5-FU in tumor tissues was significantly increased with only a slight increase of 5-FU in plasma as compared with oral capecitabine alone.