Synthesis, chemical and enzymatic hydrolysis, and bioavailability evaluation in rabbits of metronidazole amino acid ester prodrugs with enhanced water solubility.

A series of amino acid esters (3a-e) have been synthesized and evaluated as potential prodrugs of metronidazole with the aim of improving aqueous solubility and therapeutic efficacy. The aqueous solubility and the lipophilicity (expressed as the log P value) of metronidazole and its esters were investigated. In general the prodrugs revealed enhanced water solubility compared with metronidazole. N,N-diethylglycinate hydrochloride (3a) and 4-ethylpiperazinoacetate (3e) derivatives displayed higher aqueous solubility, which exceeded that of the parent drug by factors of approximately 140 and 100, respectively. All the esters revealed lower log P values than metronidazole except for the 4-phenylpiperazinoacetate derivative (3f), which was 6.5-times more lipophilic than metronidazole. The hydrolysis kinetics of the esters were studied in aqueous phosphate buffer (pH 7.4) and 80% human plasma at 37 degrees C. In all cases the hydrolysis followed pseudo-first-order kinetics and resulted in a quantitative reversion to metronidazole as evidenced by HPLC analysis. The prodrugs exhibited adequate chemical stability (half-life, t1/2, 4-16 h) in aqueous phosphate solution of pH 7.4. In 80% human plasma they were hydrolysed within a few minutesto metronidazole. The esters 3d (methylpiperazinoacetate derivative) and 3f were exempted since their t1/2 values were approximately 2.5 and 8.5 h, respectively. A comparative pH-rate profile study of N,N-diethylglycinate hydrochloride (3a) and 4-ethyl-piperazinoacetate (3e) derivatives in aqueous buffer solution over the pH range 2.2-10 was investigated. The results indicated that 3a showed marked stability at pH 2-6 followed by accelerated hydrolysis at pH 7.4. The basic ester 3e was found to be less stable at lower pH values but exhibited comparative stability at physiological pH. Moreover, in-vivo experiments in rabbits revealed a higher metronidazole plasma level with sustained release characteristics within the prodrug-treated animals (10- and 2.5-fold) as compared with the parent drug-treated group. In conclusion, the designed amino acid esters 3a and 3c-e might be considered as good candidates for water-soluble prodrug forms of metronidazole.     

Discovery of 1-[2-[(1S)-(3-dimethylaminopropionyl)amino-2-methylpropyl]-4-methylphenyl]-4-[(2R)-methyl-3-(4-chlorophenyl)-propionyl]piperazine as an orally active antagonist of the melanocortin-4 receptor for the potential treatment of cachexia

A potent and selective antagonist of the melanocortin-4 receptor, 1-[2-[(1S)-(3-dimethylaminopropionyl)amino-2-methylpropyl]-6-methylphenyl]-4-[(2R)-methyl-3-(4-chlorophenyl)propionyl]piperazine (10d), was identified from a series piperazinebenzylamine attached with a N,N-dimethyl-beta-alanine side chain. This compound possessed high water solubility and exhibited good metabolic profiles. In animals, 10d showed moderate to good oral bioavailability and promoted food intake in tumor-bearing mice after oral administration.     

Fluorouracil prodrugs for the treatment of proliferative vitreoretinopathy: formulation in silicone oil and in vitro release of fluorouracil

Three new N(1)-alkylcarbonyl-5-fluorouracil derivatives that are prodrugs of 5-fluorouracil (FU), one of them being a co-drug FU-retinoic acid (RA), were studied as potentially effective drugs against postsurgical proliferative vitreoretinopathy (PVR). The stability of N(1)-octenoylFU (3), N(1)-lauroylFU (2), and N(1)-retinoylFU (4) in aqueous medium, their solubility in silicone oil (SiO), the kinetics of FU release in an in vitro system were determined. Compound 3 is very rapidly soluble in SiO. Its saturation concentration, reached after 6h, is 233 +/- 13 microg g(-1) SiO. Compound 2 is not very soluble in SiO but its kinetic of solubilization is fast. Its saturation concentration, reached after 2 days, is 27 +/- 2 microg g(-1) SiO. Compound 4 is poorly soluble in SiO. A concentration plateau, with a mean value of 4 microg g(-1) SiO, is reached after 4 days. The addition in SiO of 5% of a perfluorinated perhydrogenated alkene greatly improves the solubilization of compound 4. Two different types of FU release are observed. For compound 3, the release is fast and is achieved after 1 day. For compounds 2 and 4, the release is slower and is ended at 10 and 27 days, respectively. The solubility of the prodrugs in SiO is not correlated with their lipophilicity, whereas the release rate of FU decreased with increased lipophilicity of the prodrug. The most promising prodrug is compound 4 that slowly releases two active drugs (FU and RA) with a t (1/2 release) of 5.8 days. It might be interesting for the treatment of PVR. However, an in vivo study on an animal model of PVR is necessary to prove the efficacy of this formulation and to study its toxicity.     

Selective water-soluble gelatinase inhibitor prodrugs

SB-3CT (1), a selective and potent thiirane-based gelatinase inhibitor, is effective in animal models of cancer metastasis and stroke; however, it is limited by poor aqueous solubility and extensive metabolism. We addressed these issues by blocking the primary site of metabolism and capitalizing on a prodrug strategy to achieve >5000-fold increased solubility. The amide prodrugs were quantitatively hydrolyzed in human blood to a potent gelatinase inhibitor, ND-322 (3). The arginyl amide prodrug (ND-478, 5d) was metabolically stable in mouse, rat, and human liver microsomes. Both 5d and 3 were nonmutagenic in the Ames II mutagenicity assay. The prodrug 5d showed moderate clearance of 0.0582 L/min/kg, remained mostly in the extracellular fluid compartment (Vd = 0.0978 L/kg), and had a terminal half-life of >4 h. The prodrug 5d had superior pharmacokinetic properties than those of 3, making the thiirane class of selective gelatinase inhibitors suitable for intravenous administration in the treatment of acute gelatinase-dependent diseases.     

Inhibitors of cyclic AMP phosphodiesterase. 4. Synthesis and evaluation of potential prodrugs of lixazinone (N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro-2- oxoimidazo[2,1-b]quinazolin-7-yl)-oxy]butyramide, RS-82856)

The cyclic AMP phosphodiesterase (cAMP PDE) inhibitor and cardiotonic agent lixazinone (N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro-2- oxoimidazo[2,1-b]quinazolin-7-yl)oxy]butyramide, RS-82856, 1) and its acid and base addition salts were found to be insufficiently soluble in formulations suitable for intravenous administration. These results prompted an investigation into potential prodrugs with enhanced aqueous solubility designed to deliver 1 by three distinct mechanisms: (1) decarboxylation of alpha-carboxamides; (2) hydrolytic loss of a solubilizing N-1-(acyloxy)methyl or (N,N-dialkylamino)methyl moiety; or (3) intramolecular closure of a guanidino ester or amide. The target compounds were evaluated as delivery systems for 1 by three criteria: (1) chemical conversion rate to 1 under physiological conditions; (2) inhibition of type IV cAMP PDE at a fixed time point; and (3) in vivo inotropic activity in anesthetized dogs by both intravenous and oral administration. Release of 1 from 4a (series 1) was found to be too slow to be of value as a prodrug of 1, since decarboxylation could be induced only by strong acid, conditions under which hydrolytic ring opening was found to severely compete. Conversely, 1 was released too readily on exposure of (N,N-dialkylamino)methyl derivatives such as 8d (series 2) to physiological conditions, although no large increase in aqueous solubility was realized. Finally, both the physicochemical and in vitro studies indicated that ring closure of the guanidinium esters and amides 17a-k (series 3) to 1 was quantitative and pH- and time-dependent, suggesting the possibility of delivery of the open, water-soluble prodrug form, followed by closure to 1 in plasma. Detailed examination of these agents in vivo, however, demonstrated that only those compounds that rapidly cyclized to 1, as measured by plasma levels of 1, exhibited inotropic activity, indicating that the open prodrug form was not efficiently absorbed upon oral administration.     

A novel prodrug approach for tertiary amines. 2. Physicochemical and in vitro enzymatic evaluation of selected N-phosphonooxymethyl prodrugs.

Quaternary amine prodrugs resulting from N-phosphonooxymethyl derivatization of the tertiary amine functionality of drugs represents a novel approach for improving their water solubility. Separate reports have demonstrated the synthetic feasibility and rapid and quantitative prodrug to parent drug conversion in rats and dogs. This work is a preliminary evaluation of the physicochemical and in vitro enzymatic reversion properties of selected prodrugs. The loxapine prodrug had over a 15 000-fold increase in aqueous solubility relative to loxapine free base at pH 7.4. The loxapine prodrug was also shown to be quite stable at neutral pH values. The time for degradation product (parent drug) precipitation from an aqueous prodrug formulation would be expected to dictate the shelf life. Using this assumption, together with solubility and elevated temperature chemical stability studies, the shelf life of a parenteral formulation of the loxapine prodrug was projected to be close to 2 years at pH 7.4 and 25 degrees C. In addition, the prodrugs of cinnarizine and loxapine have been shown to be substrates for alkaline phosphatase, an enzyme found throughout the human body, and revert to the parent compound in its presence. The results from these evaluations demonstrate that the derivatives examined have many of the ideal properties required for potential clinical application.     

Synthesis of congeners and prodrugs. 3. Water-soluble prodrugs of taxol with potent antitumor activity

Taxol has shown good in vivo antitumor activity in a number of test systems. The formulation of taxol for antitumor testing has been difficult. Esterification at either C-2' or C-7 resulted in loss of in vitro tubulin assembly activity but not cytotoxicity. These observations suggested that esters at C-2' and/or C-7, which would tend to promote water solubility, might serve as useful prodrugs of taxol. The reaction of taxol with either succinic anhydride or glutaric anhydride in pyridine solution at room temperature gave the crystalline mono 2'-adducts 1b and 1f, respectively. Salts of these acids (1b, 1f, 1i) were formed by the addition of 1 equiv of the corresponding base, followed by evaporation and/or freeze-drying of the solvent(s). The salts had improved antitumor activity as compared to the free acids. The triethanolamine and N-methylglucamine salts showed greatly improved aqueous solubility and were more active than the sodium salts. The glutarate series was preferred because of the higher activity and the higher yields obtained. 2'-Glutaryltaxol (1f) was coupled with 3-(dimethylamino)-1-propylamine, using CDI, to form in excellent yield the amino amide 1o. The hydrochloride salt (1p) showed good solubility and was extremely potent and active. At 10 mg/kg, in the B16 screen, 1p gave a T/C of 352 with 5 out of 10 cures. In the MX-1 breast xenograft assay, this prodrug gave values of -100 at doses of 40 and 20 mg/kg, with all live animals being tumor free.     

A new class of potent vascular endothelial growth factor receptor tyrosine kinase inhibitors: structure-activity relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-c]carbazole-5-ones and the identification of CEP-5214 and its dimethylglycine ester prodrug clinical candidate CEP-7055

A series of potent vascular endothelial growth factor R2 (VEGF-R2) tyrosine kinase inhibitors from a new indenopyrrolocarbazole template is reported. The structure-activity relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-c]carbazole-5-ones revealed an optimal R9 substitution with ethoxymethyl 19 (VEGF-R2 IC(50) = 4 nM) and isopropoxymethyl 21 (VEGF-R2 IC(50) = 8 nM) being the most potent inhibitors in the series. The VEGF-R2 activity was reduced appreciably by increasing the size of the R9 alkoxy group or by alpha-methyl branching adjacent to the ring. The combined R9 alkoxymethyl and N12 hydroxypropyl substitutions were required for potent VEGF-R2 activity, and the corresponding thioether analogues were weaker than their ether counterparts. Compound 21 (R9 isopropoxymethyl, CEP-5214) was identified as a potent, low-nanomolar pan inhibitor of human VEGF-R tyrosine kinases, displaying IC(50) values of 16, 8, and 4 nM for VEGF-R1/FLT-1, VEGF-R2/KDR, and VEGF-R3/FLT-4, respectively, with cellular activity equivalent to the isolated enzyme activity. Compound 21 exhibited good selectivity against numerous tyrosine and serine/threonine kinases including PKC, Tie2, TrkA, CDK1, p38, JNK, and IRK. To increase water solubility and oral bioavailability, the N,N-dimethylglycine ester 40 was prepared. In pharmacokinetic studies in mice and rats, increased plasma levels of 21 were observed after oral administration of 40. Compound 21 demonstrated significant in vivo antitumor activity in numerous tumor models and was advanced into phase I clinical trials as the water-soluble N,N-dimethylglycine ester prodrug 40 (CEP-7055).     

Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP

Cyclosaligenyl-2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate (cycloSal-d4TMP) is a potent and selective inhibitor of human immunodeficiency virus replication in cell culture and differs from other nucleotide prodrug approaches in that it is designed to selectively deliver the nucleotide 5'-monophosphate by a controlled, chemically induced hydrolysis. Its antiviral efficacy in cell culture is at least as good as, if not superior to, that of d4T. CycloSal-d4TMP was found to lead to the efficient intracellular release of d4TMP in a variety of cell lines, including both wild-type CEM and thymidine kinase-deficient CEM/TK(-) cells. Under similar experimental conditions, exposure of CEM/TK(-) cells to d4T failed to result in significant d4TTP levels. The intracellular conversion of cycloSal-d4TMP proved to be both time and dose dependent. The half-life of d4TTP generated intracellularly from d4T- or cycloSal-d4TMP-treated CEM cells was approximately 3.5 h, and the intracellular ratios of d4TTP/d4TMP in cells exposed to cycloSal-d4TMP gradually increased from 1 to 3.4 upon prolonged incubation. Radiolabeled cycloSal-d4TMP could be separated as its two R(p) and S(p) diastereomers on high-performance liquid chromatography. The R(p) diastereomer of cycloSal-d4TMP was 3- to 7-fold more efficient in releasing d4TMP and generating d4TTP than the S(p) cycloSal-d4TMP diastereomer. This correlated well with the 5-fold more pronounced antiviral activity of the R(p) diastereomer versus the S(p) diastereomer. d4TMP is a poor substrate for the cytosolic 5'(3')-deoxyribonucleotidase (V(max)/K(m) for d4TMP: 0.08 of V(max)/K(m) for dTMP) and is only slowly hydrolyzed to d4T. This contributes to the efficient conversion of the prodrug of d4TTP.     

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.     

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.     

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.     

Synthesis and biological evaluation of prodrugs of zidovudine

A series of prodrugs of zidovudine (AZT) has been synthesized in an effort to enhance the uptake of the prodrugs by the HIV-1 infected cells and to increase the plasma half-life of AZT. The 5'-OH function of AZT was esterified with various acids in the presence of DCC and 4-(dimethylamino)pyridine (DMAP). The prodrug moieties included (a) morpholine and N-phenylpiperazine-1-acetic acid, (b) 1,4-dihydro-1-methyl-3-nicotinic acid, (c) retinoic acid, and (d) certain amino acids. The anti-HIV-1 activity of the esters was determined in peripheral blood lymphocytes. The IC50 for AZT in this system was 0.12 microM whereas for prodrugs it ranged from 0.05 to 0.2 microM. The prodrugs were generally less cytotoxic than AZT except the retinoic acid ester. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t1/2 ranging from 10 to 240 min. Drug uptake studies in H9 cells with radiolabeled analogues demonstrated that the retinoic acid ester achieved approximately 4-fold higher intracellular concentration than [3H]AZT. However, 1,4-dihydro-1-methyl-3-[(pyridylcarbonyl)oxy] ester (5) was the most active agent of this series and had a higher therapeutic index than AZT.     

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.     

Synthesis and in vitro evaluation of novel morpholinyl- and methylpiperazinylacyloxyalkyl prodrugs of 2-(6-methoxy-2-naphthyl)propionic acid (Naproxen) for topical drug delivery

Various novel morpholinyl- (3a,b) and methylpiperazinylacyloxyalkyl (3c-f) esters of 2-(6-methoxy-2-naphthyl)propionic acid were synthesized and evaluated in vitro for topical drug delivery as potential prodrugs of naproxen (1). Compounds 3a-f were prepared by coupling the corresponding naproxen hydroxyalkyl ester with the morpholinyl- or (4-methyl-1-piperazinyl)acyl acid in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) and quantitatively hydrolyzed (t(1/2) = 1-26 min) to naproxen in human serum. Compounds 3c-f showed higher aqueous solubility and similar lipophilicity, determined by their octanol-buffer partition coefficients (log P(app)), at pH 5.0 when compared to naproxen. At pH 7.4 they were significantly more lipophilic than naproxen. The best prodrug 3c led to a 4- and 1.5-fold enhancement of skin permeation when compared to naproxen at pH 7.4 and 5.0, respectively. The present study indicates using a methylpiperazinyl group yields prodrugs that are partially un-ionized under neutral and slightly acidic conditions, and thus, a desirable combination is achieved in terms of aqueous solubility and lipophilicity. Moreover, the resulting combination of biphasic solubility and fast enzymatic hydrolysis of the methylpiperazinylacyloxyalkyl derivatives gave improved topical delivery of naproxen.     

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.     

The effect of SBE4-β-CD on i.m. prednisolone pharmacokinetics and tissue damage in rabbits: Comparison to a co-solvent solution and a water-soluble prodrug

The i.m. pharmacokinetics of prednisolone (5 mg/kg) in eight rabbits after its administration in a co-solvent (40:10:50; PEG 400/ethanol/water) mixture, in a slightly hypertonic 0.09 M SBE4-β-CD (a sulfobutyl ether derivative variably substituted on the 2-, 3- and the 6-positions of β-cyclodextrin) solution and from a water-soluble prodrug, the 21-phosphate ester, disodium salt were studied. Muscle damage as measured by changes in plasma creatine kinase (CK) levels caused by the administration of the three solutions was also assessed. The prednisolone plasma AUC values over 24 h from the SBE4-β-CD formulation and the phosphate ester were 87.0 ± 12.6 and 78.0 ± 14.1% of that from co-solvent, respectively. The apparent bioavailability of prednisolone over 24 h from the SBE4-β-CD formulation and its prodrug was not significantly different from that of the co-solvent. The changes in CK levels from the SBE4-β-CD were identical to those from normal saline, however, the co-solvent mixture caused significantly elevated CK levels. The presence or absence of prednisolone had no effect on the relative CK levels for the cyclodextrin solution and the normal saline. There was a small effect noted for the co-solvent, with and without prednisolone. These results confirm that i.m. administered drugs, such as prednisolone, appear to be rapidly, quantitatively and safely released from SBE4-β-CD inclusion complexes. SBE4-β-CD may provide an alternative to the use of co-colvents and possibly even prodrugs for the i.m. delivery of sparingly water-soluble drugs such as prednisolone.     

Antibody‐Directed Enzyme Prodrug Therapy: A Promising Approach for a Selective Treatment of Cancer Based on Prodrugs and Monoclonal Antibodies

The antibody‐directed enzyme prodrug therapy allows a selective liberation of cytotoxic agents from non‐toxic prodrugs in cancerous tissue by targeted antibody–enzyme conjugates. We have developed a series of novel glycosidic prodrugs based on the natural antibiotic CC‐1065 and the duocarmycins, which are up to 4800 times less toxic than the drugs liberated from these prodrugs in the presence of the activating enzyme (e.g., β‐d ‐galactosidase). Furthermore, the drugs show very high cytotoxicities with IC₅₀ values of as low as 4.5 pm . In this report, we summarize our recent results on the development and biological evaluation of these novel third‐generation prodrugs with higher water solubility, higher difference in cytotoxicity between the prodrugs and the corresponding drugs and improved cytotoxicity of the drugs as compared with previous compounds.     

Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT

Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.     

Water-soluble gambogic acid PEGylated prodrugs: synthesis, characterization, physicochemical properties and in vitro hydrolysis

A series of poly(ethylene glycol) (PEG) prodrugs of gambogic acid (GA) with different molecular weight which used L-leucine as spacer were synthesized and characterized by FT-IR, 1H NMR and TOF MS. Drug loading capability, analyzed by UV spectrum, was 17.48, 9.26, 3.99, and 1.79%, aqueous solubility of the prodrugs was determined to be 1750, 1250, 800, and 645 mg/ml, respectively. The drug release from prodrugs was investigated under simulated in vivo conditions whose half-time (t1/2) in plasma ranged from 1.26 to 6.12 h. The effect of temperature on drug release was studied at four different temperatures and activation energy was determined as well. The stability of the prodrugs was improved in parallel with increasing molecular weight of PEG while prodrug yields and drug loading capability were reduced.