Ribonucleoside 3′-Phosphates as Pro-Moieties for an Orally Administered Drug

Oral administration of chemotherapeutic agents is the mainstay for the treatment of disease. Sustained release formulations have been crucial for the safe and effective dosing of orally administered drugs.[1] Such formulations allow for the prolonged maintenance of therapeutic drug concentrations, reducing the required dosages per day and thereby enhancing patient compliance. Sustained release formulations also provide tighter control over the pharmacokinetics of a drug, thereby minimizing side effects.[1] Aqueous solubility is likewise a critical attribute for an orally available drug.[2] Robust absorption across the intestinal epithelium relies upon a high concentration of the drug to drive diffusion into enterocytes and, eventually, into the circulatory system. On average, 35–40% of lead compounds have aqueous solubilities of <5 mg/mL, which is defined by the U.S. Pharmacopeia as being slightly soluble or worse.[3] Accordingly, the bioavailability and consequent efficacy of many compounds relies on enhancing their aqueous solubility.[2c] The formation of a phosphomonoester can improve the oral bioavailability of poorly water-soluble chemotherapeutic agents.[2b,2c,4] Endogenous phosphatases near the surface of enterocytes can catalyze the hydrolysis of the phosphoryl group, releasing the lipophilic drug and allowing for its efficient absorption into the body. Several prodrugs approved by the U.S. Food and Drug Administration rely on this strategy, including estramustine, fosamprenavir, and prednisolone phosphate.[4] Recently, we reported on the potential utility of a phosphodiester as the pro-moiety for a drug administered intravenously.[5] Specifically, we found that the coupling of 4-hydroxytamoxifen to uridine 3′-phosphate enabled its timed-release in serum by human pancreatic ribonuclease (RNase 1[6]; EC 3.1.27.5). This modification also increased the aqueous solubility of 4-hydroxytamoxifen. RNase 1 is an ideal endogenous enzyme to elicit pro-moiety release. A major excreted enzyme, RNase 1 has a concentration of 6.4 mg/mL in human pancreatic juice and 0.2 mg/mL in saliva, according to a radioimmunoassay.[7] Moreover, like its renowned homologue bovine pancreatic ribonuclease (RNase A[8]), RNase 1 catalyzes the cleavage of RNA by a transphosphorylation reaction[9] with little specificity for its leaving group.[10] Herein, we report on the utility of several ribonucleoside 3′-phosphates as pro-moieties for a model orally available drug, metronidazole. Metronidazole is a commonly used antibiotic for a variety of protozoa and anaerobic bacterial infections, including Bacteroides fragilis, Helicobacter pylori, Clostridium difficile, Trichomonas vaginalis, and Entamoeba histolytica.[11] In 1997, Flagyl ER, an extended release formulation of metronidazole, was approved by the FDA as a superior treatment for bacterial vaginosis. Still, metronidazole has several common side effects, such as nausea, diarrhea, and metallic taste. Moreover, metronidazole therapy can occasionally cause more severe side effects, such as pancreatitis, neutropenia, neuropathies, or CNS toxicities.[12] These adverse effects could be attenuated with better control over the pharmacokinetics of metronidazole.[13] Hence, in this proof-of-concept study, we elected to attach metronidazole to ribonucleoside 3′-phosphates to assess the attributes of this promoiety for orally available drugs (Figure 1). Figure 1 Scheme showing catalysis of the cleavage of a ribonucleoside 3′-(metronidazole phosphate) (NpMet) by RNase 1 to yield a nucleoside 2′,3′-cyclic phosphate (N>p) and Met. Each ribonucleoside 3′-(metronidazole phosphate) (NpMet) was synthesized in four steps from commercially available metronidazole (Met) and a ribonucleoside phosphoramidite (Scheme 1). Briefly, Met was coupled to the phosphoramidite by using N-methylbenzimidazolium triflate (MBIT) as a catalyst.[14] The coupled product was oxidized with iodine and deprotected stepwise. The final products were purified by chromatography on silica gel. This route was used to synthesize three different NpMets: cytidine 3′-(metronidazole phosphate) (CpMet, 18% non-optimized yield), uridine 3′-(metronidazole phosphate) (UpMet, 80%), and adenosine 3′-(metronidazole phosphate) (ApMet, 64%). Scheme 1 Route for the synthesis of NpMets. We expected the ribonucleoside 3′-phosphate moiety of an NpMet to endow the prodrug with greater hydrophilicity than the parent drug, which could improve its oral bioavailability. To investigate this issue, we calculated the partition (log P) and distribution (log D) coefficients of Met, CpMet, UpMet, and ApMet.[15] The calculated log P and log D values for the NpMets were indeed significantly lower than those of the parent drug (Table 1), indicative of increased hydrophilicity and decreased tendency to aggregate. Table 1 Calculated Partition and Distribution Coefficients of Met and NpMets[15] To be the basis for an effective timed-release prodrug strategy, the pro-moiety needs to be released by the activating enzyme over time. Hence, we used 1H NMR spectroscopy to assess the rate at which RNase 1 catalyzed the release of Met from the prodrugs (Figure S1). We assumed that pancreatic juice is diluted in the intestine, which led us to use RNase 1 at concentrations of 0.1 mg/mL and 0.01 mg/mL in these assays. Because inorganic phosphate inhibits RNase A with a Ki value of 2.3 mM,[16] we initially investigated the effect of phosphate in simulated intestinal fluid (SIF) on the rates of UpMet unmasking (Figure 2A). Compared to a buffer with no inorganic phosphate (19.5 mM Tris–HCl, pH 7.4, 2.5% v/v D2O), the rate of Met release in SIF was only marginally slower. RNase 1 cleaves after pyrimidine residues more readily than after purine residues.[6,7] Accordingly, we predicted that RNase 1 would unmask CpMet and UpMet faster than ApMet. For both concentrations of RNase 1, we did indeed observe that the cytidine and uridine prodrugs were unmasked faster than the adenosine prodrug (Figure 2B). Moreover, unlike the uridine 3′-phosphate–4-hydroxytamoxifen conjugate that cleaved spontaneously in aqueous solutions lacking ribonucleases, the NpMet conjugates were stable in SIF, which has pH 7.5, and in simulated gastric fluid (SGF), which has pH 1.1. The absence of appreciable degradation (<5%) in either medium (Figure S2) is attributable to the alkoxyl group of metronidazole being a much worse leaving group than the aryloxyl group of 4-hydroxytamoxifen. Figure 2 Progress curves for the release of Met from NpMets under various conditions as determined by 1H NMR spectroscopy. (A) Comparison of UpMet cleavage rates in Tris–HCl buffer, pH 7.4, and simulated intestinal fluid (SIF). (B) Comparison of CpMet, ... Finally, we sought to assess the antimicrobial activity of our NpMet prodrugs on B. fragilis. This penicillin-resistant Gram-negative bacillus is common in anaerobic infections, like those that originate from the gastrointestinal tract. We determined the minimum inhibitory concentration (MIC) of UpMet and ApMet, as well as Met (Figure S3). We found that both UpMet and ApMet had considerably higher MIC values than did Met (Table 2), demonstrating that the prodrugs were relatively inactive. Incubating UpMet with 0.1 mg/mL RNase 1 overnight resulted in an MIC similar to that of Met. Finally, adding UpMet to a culture medium containing RNase 1 gave an intermediate MIC, demonstrating the in situ release of the drug. Table 2 MIC Values of NpMets for Bacteroides fragilis