Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.     

Antitumor effects of an antibody-carboxypeptidase g2 conjugate in combination with a benzoic acid mustard prodrug

The F(ab’)₂ fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme produrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino]-benzoyl-l-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/ blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.     

PDEPT: polymer-directed enzyme prodrug therapy. 2. HPMA copolymer-beta-lactamase and HPMA copolymer-C-Dox as a model combination

Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumor approach that uses a combination of a polymeric prodrug and polymer-enzyme conjugate to generate a cytotoxic drug rapidly and selectively at the tumor site. Previously we have shown that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound cathepsin B can release doxorubicin intratumorally from an HPMA copolymer conjugate PK1. Here we describe for the first time the synthesis and biological characterization of a PDEPT model combination that uses an HPMA-copolymer-methacryloyl-glycine-glycine-cephalosporin-doxorubicin (HPMA-co-MA-GG-C-Dox) as the macromolecular prodrug and an HPMA copolymer conjugate containing the nonmammalian enzyme beta-lactamase (HPMA-co-MA-GG-beta-L) as the activating component. HPMA-co-MA-GG-C-Dox had a molecular weight of approximately 31 600 Da and a C-Dox content of 5.85 wt %. Whereas free beta-L has a molecular weight of 45 kDa, the HPMA-co-MA-GG-beta-L conjugate had a molecular weight in the range of 75-150 kDa, and following purification no free enzyme was detectable. Against the cephalosporin C or HPMA-co-MA-GG-C-Dox substrates, the HPMA-co-MA-GG-beta-L conjugate retained 70% and 80% of its activity, respectively. In vivo (125)I-labeled HPMA-co-MA-GG-beta-L showed prolonged plasma concentration and greater tumor targeting than (125)I-labeled beta-L due to the enhanced permeability and retention (EPR) effect. Moreover, administration of HPMA-co-MA-GG-C-Dox iv to mice bearing sc B16F10 melanoma followed after 5 h by HPMA-co-MA-GG-beta-L led to release of free Dox. The PDEPT combination caused a significant decrease in tumor growth (T/C = 132%) whereas neither free Dox nor HPMA-co-MA-GG-C-Dox alone displayed activity. The PDEPT combination displayed no toxicity at the doses used, so further evaluation of this approach to establish the maximum tolerated dose (MTD) is recommended.     

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.     

Metabolism of GS-7340, a novel phenyl monophosphoramidate intracellular prodrug of PMPA, in blood

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HIV. In the cells, PMPA is efficiently phosphorylated by intracellular kinases to produce PMPApp, the pharmacologically active metabolite. Despite its demonstrated antiviral potency, PMPA has limited cell permeability presumably resulting from the presence of two negative charges on the phosphonyl group. To enhance intracellular concentrations of PMPA, we developed a prodrug, selectively metabolized inside cells. GS-7340 (9-[(R)-2-[[[[(S)-1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-methoxy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as the intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in peripheral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was examined following exposure of whole blood to PMPA or GS-7340 at concentrations similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in plasma, produced high levels of PMPA and its phosphorylated metabolites in PBMC but not in RBC. No intact prodrug was present in PBMC. The only other species present in PBMC was monoalaninyl PMPA. The levels of PMPA and the phosphorylated metabolites were over 20 times greater than those after incubation with PMPA. The dog and human blood data were similar. The intracellular levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10-fold concentration range indicating a lack of saturability of uptake and phosphorylation. Since PMPApp is the species responsible for antiviral activity of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.     

Effect of glucose-cysteine adduct as a cysteine prodrug in rats

Summary Effect of intraperitoneal administration (5 mmol/kg of body weight) of glucose- cysteine adduct (glc-cys) as a cysteine prodrug in rat tissues was studied. Cysteine levels in liver and kidney increased to 1.08 and 1.98mol per g or ml, respectively, at 2h after the administration. GSH levels did not change substantially. However, when glc-cys was injected to rats treated with diethyl maleate, a GSH-depleting agent, the decreased GSH levels were restored rapidly. The recoveries in liver and kidney were 72% at 1h and 66% at 2h, respectively, after glc-cys administration. Metabolism of glc-cys was assessed by urinary excretion of glc-cys, sulfate and taurine. Average excretion of glc-cys was 2.86mmol/kg/24h after glc-cys administration. Increased excretions of sulfate and taurine were 0.77 and 0.14mmol/kg/24h, respectively. Data show that, although glc-cys excretion was relatively rapid, glc-cys was effectively utilized for GSH synthesis in GSH-depleted tissues.     

Synthesis of a novel cyclic prodrug of RGD peptidomimetic to improve its cell membrane permeation

The objective of this work was to synthesize cyclic prodrug 2 derived from the parent RGD peptidomimetic 1 and to evaluate its chemical and enzymatic stabilities and antithrombic activity. Cyclic prodrug 2 was formed to improve the cell membrane permeation of RGD peptidomimetic 1 by transiently masking the unfavorable physicochemical properties of compound 1. Cyclic prodrug 2 was synthesized by linking the amino and carboxylic acid groups of parent 1 via the (acyloxy)alkoxy promoiety. The prodrug-to-drug conversion of cyclic prodrug 2 was evaluated in isolated esterase and human plasma in the absence and presence of the esterase inhibitor paraoxon. The rate of hydrolysis of cyclic prodrug 2 was significantly faster in plasma (t(1/2)=33.5+/-0.6 min) than in PBS (t(1/2)=314+/-11 min). Cyclic prodrug 2 was converted by esterase to the parent compound 1 and this conversion was inhibited by an esterase inhibitor, paraoxon. The IC50 (4 micro M) of cyclic prodrug 2 was higher than the IC50 (1.9 micro M) of parent drug 1. The antithrombic activity of cyclic prodrug 2 depends on the incubation time in platelet-rich plasma; the activity increases with incubation time, suggesting that the prodrug-to-drug conversion is time-dependent and mediated by esterase. Cyclic prodrug 2 was more stable under acidic and neutral conditions than under basic conditions, suggesting that handling and formulation of this prodrug should be undertaken under acidic conditions.     

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.     

Synthesis and evaluation of an injectable everolimus prodrug

A novel water-soluble everolimus prodrug, glutathione-everolimus, was designed and synthesized by introducing an endogenous tripeptide with an acetyl as the linker. The improvement in water solubility allowed the conjugate to be developed into an injectable drug. The results of biological evaluation in vitro and in vivo suggested that the prodrug was more effective and long acting than everolimus. Meanwhile, the pharmacokinetics study in vivo confirmed that the delivery of everolimus through the injection of the prodrug can overcome the low bioavailability of oral everolimus.     

Synthesis and characterization of a catalytic antibody-HPMA copolymer-Conjugate as a tool for tumor selective prodrug activation

Selective chemotherapy remains a key issue for successful treatment in cancer therapy. The use of targeting approaches like the enhanced permeability and retention (EPR) effect of macromolecules, is consequently needed. Here, we report the preparation of a novel catalytic antibody-polymer conjugate for selective prodrug activation. HPMA copolymer was conjugated to catalytic antibody 38C2 through an amide bond formation between epsilon-amino group of lysine residue from the antibody molecule and a p-nitrophenyl ester of the polymer. The conjugate was purified over a size exclusion column using FPLC. In the isolated fraction, one or two molecules of polymer were conjugated to one molecule of antibody based on gel analysis. The resulting conjugate retained most of its catalytic activity (75-81%) in comparison to the free antibody. The activity was monitored with a fluorogenic substrate and a prodrug activation assay using HPLC. Furthermore, the conjugate was evaluated in vitro for its ability to activate an etoposide prodrug using two different cancer cell lines. Cells growth inhibition using the prodrug and the conjugate was almost identical to inhibition by the free antibody and the prodrug. For the first time, a catalytic antibody was conjugated to a passive targeting moiety while retaining its catalytic ability to activate a prodrug. The conjugate described in this work can be used for selective activation of prodrug in the PDEPT (polymer directed enzyme prodrug therapy) approach by replacing the enzyme component with catalytic antibody 38C2.     

Orally active factor Xa inhibitors: investigation of a novel series of 3-amidinophenylsulfonamide derivatives using an amidoxime prodrug strategy

A series of novel and potent 3-amidinophenylsulfonamide derivatives of factor Xa inhibitors were designed and synthesized using an amidoxime prodrug strategy. We focused on systemic clearance of parent compounds in rats, and performed in vivo pharmacokinetic screening. Incorporation of a carboxymethoxy group markedly improved systemic clearance (compound 43), and the related amidoxime 44 showed sufficient prodrug conversion. Compound 45, the double prodrug of 43, exhibited practicable bioavailability after oral administration in rats. Among the various compounds under investigation, KFA-1982 was selected for clinical development.     

Exogenous cytosine deaminase gene expression in Bifidobacterium breve I-53-8w for tumor-targeting enzyme/prodrug therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.     

Efficient clearance of poly(ethylene glycol)-modified immunoenzyme with anti-PEG monoclonal antibody for prodrug cancer therapy

The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol). The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity. Conjugate levels in tumors decreased to 36% of peak levels at this time. Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72. 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors. Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72. 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy.     

Development of a novel albumin-binding prodrug that is cleaved by urokinase-type-plasminogen activator (uPA)

A water-soluble albumin-binding prodrug of doxorubicin [EMC-Gly-Gly-Gly-Arg-Arg-DOXO (EMC, 6-maleimidocaproic acid)] was developed that is cleaved specifically by the tumor-associated protease urokinase-type plasminogen activator (uPA).     

Design, synthesis, and bioavailability evaluation of coumarin-based prodrug of meptazinol

Based on the known coumarin-based prodrug system, a new meptazinol (Z)-3-[2-(propionyloxy) phenyl]-2-propenoic ester (3) was designed and synthesized as prodrug to minimize the first-pass effect of meptazinol (1) and improve the oral bioavailability. The prodrug (3) showed a 4-fold increase in oral bioavailability over the parent drug meptazinol in rats.     

The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level.

The liver is an important target tissue of cadmium. The compound O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2 diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO) prodrug that is metabolized by hepatic P450 enzymes to release NO in hepatocytes. In vivo, V-PYRRO/NO can protect against the toxicity of various hepatotoxicants, including cadmium. Since NO is an effective vasodilator, whether this protective effect against cadmium toxicity is at the level of the hepatic vascular system or actually within the liver cells has not been defined. Thus, we studied the effects of V-PYRRO/NO pretreatment on cadmium-induced toxicity and apoptosis in cultured rat liver epithelial (TRL 1215) cells. Cells were pretreated with V-PYRRO/NO at 500 or 1000 microM for up to 24 h, then exposed to cadmium (as CdCl2) for additional 24 h and cytotoxicity was measured. Cadmium was significantly less cytotoxic in V-PYRRO/NO (1000 microM) pretreated cells (LC50=6.1+/-0.6 microM) compared to control cells (LC50=3.5+/-0.4 microM). TRL 1215 cells acted upon the prodrug to release NO, producing nitrite levels in the extracellular media after 24 h of exposure to 500 or 1000 microM V-PYRRO/NO measured at 87.0+/-4.2 and 324+/-14.8 microM, respectively, compared to basal levels of 7.70+/-0.46 microM. V-PYRRO/NO alone produced small increases in metallothionein (MT), a metal-binding protein associated with cadmium tolerance. However, V-PYRRO/NO pretreatment greatly enhanced cadmium induction of MT. V-PYRRO/NO pretreatment also markedly reduced apoptotic cell death induced by cadmium (5 microM), apparently by blocking cadmium-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Thus, the prodrug, V-PYRRO/NO, protects against the adverse effects of cadmium directly within rat liver cells apparently through generation of NO and, at least in part, by facilitation of cadmium-induced MT synthesis.     

Stereoselective disposition of flurbiprofen from a mutual prodrug with a histamine H2-antagonist to reduce gastrointestinal lesions in the rat

The in vitro and in vivo stereoselective hydrolysis characteristics of the mutual prodrug FP-PPA, which is a conjugate of flurbiprofen (FP) with the histamine H₂-antagonist PPA, to reduce gastrointestinal lesions induced by FP were investigated and compared with those of FP methyl ester (rac-FP-Me) and FP ethyleneglycol ester (rac-FP-EG). The rac-FP derivatives were hydrolyzed preferentially to the (+)-S-isomer in plasma and to the (−)-R-isomer in liver and small intestinal mucosa. Interestingly, in the gastric mucosa, the stereoselectivity of hydrolysis of (−)-R-FP-PPA was opposite from that of rac-FP-Me and rac-FP-EG, which suggested that the stereoselective hydrolysis of FP-PPA was helpful in reducing gastric damage induced by (+)-S-FP. However, hydrolysis of all rac-FP derivatives was found to be catalyzed by carboxylesterases in the gastric mucosa. The stereoselective disposition of FP enantiomers early after intravenous administration of rac-FP-PPA could be explained by the stereoselective formation of (−)-R-FP from rac-FP-PPA in the liver. (−)-R-FP-PPA was completely hydrolyzed to form (−)-R-FP in vivo, while 78% of (+)-S-FP-PPA was hydrolyzed to (+)-S-FP, with a corresponding decrease in the area under the curve. Twenty-five percent of (+)-S-FP-PPA might be eliminated as the intact prodrug or its metabolites other than FP. The most important bioconversion of FP-PPA occurred in plasma, and additional hydrolysis of the R-enantiomer in liver resulted in the stereoselectivity observed following both i.v. and p.o. administration. © 1996 Wiley-Liss, Inc.     

Antibody-directed enzyme prodrug therapy (ADEPT)

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with¹²⁵I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab′)₂ fragment showed localization in these xenografts. The AB57-F(ab′)₂ fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with¹²⁵iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon). A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab′)₂-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress.     

Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug.

CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol x min(-1) x mg(-1) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.     

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 2. Synthesis and characterization of a novel imide-type prodrug for improving oral absorption

As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorption of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubility (19μg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy (T/C=-6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising candidate and are performing further studies. Herein, we report the design, synthesis, and characterization of novel imide-type prodrugs.