Improving Drug Delivery While Tailoring Prodrug Activation to Modulate Cmax and Cmin by Optimization of (Carbonyl)oxyalkyl Linker-Based Prodrugs of Atazanavir.

Structure-property relationships associated with a series of (carbonyl)oxyalkyl amino acid ester prodrugs of the marketed HIV-1 protease inhibitor atazanavir (1), designed to enhance the systemic drug delivery, were examined. Compared to previously reported prodrugs, optimized candidates delivered significantly enhanced plasma exposure and trough concentration (Cmin at 24 h) of 1 in rats while revealing differentiated PK paradigms based on the kinetics of prodrug activation and drug release. Prodrugs incorporating primary amine-containing amino acid promoieties offered the benefit of rapid bioactivation that translated into low circulating levels of the prodrug while delivering a high Cmax value of 1. Interestingly, the kinetic profile of prodrug cleavage could be tailored for slower activation by structural modification of the amino terminus to either a tertiary amine or a dipeptide motif, which conferred a circulating depot of the prodrug that orchestrated a sustained release of 1 along with substantially reduced Cmax and a further enhanced Cmin.

(Carbonyl)oxyalkyl linker-based amino acid prodrugs of the HIV-1 protease inhibitor atazanavir that enhance oral bioavailability and plasma trough concentration.

We describe the design, synthesis and pharmacokinetic (PK) evaluation of a series of amino acid-based prodrugs of the HIV-1 protease inhibitor atazanavir (1) derivatized on the pharmacophoric secondary alcohol using a (carbonyl)oxyalkyl linker. Prodrugs of 1 incorporating simple (carbonyl)oxyalkyl-based linkers and a primary amine in the promoiety were found to exhibit low chemical stability. However, chemical stability was improved by modifying the primary amine moiety to a tertiary amine, resulting in a 2-fold enhancement of exposure in rats following oral dosing compared to dosing of the parent drug 1. Further refinement of the linker resulted in the discovery of 22 as a prodrug that delivered the parent 1 to rat plasma with a 5-fold higher AUC and 67-fold higher C24 when compared to oral administration of the parent drug. The PK profile of 22 indicated that plasma levels of this prodrug were higher than that of the parent, providing a more sustained release of 1 in vivo.

Design, Synthesis, and Pharmacokinetic Evaluation of Phosphate and Amino Acid Ester Prodrugs for Improving the Oral Bioavailability of the HIV-1 Protease Inhibitor Atazanavir.

Phosphate and amino acid prodrugs of the HIV-1 protease inhibitor (PI) atazanavir (1) were prepared and evaluated to address solubility and absorption limitations. While the phosphate prodrug failed to release 1 in rats, the introduction of a methylene spacer facilitated prodrug activation, but parent exposure was lower than that following direct administration of 1. Val amino acid and Val-Val dipeptides imparted low plasma exposure of the parent, although the exposure of the prodrugs was high, reflecting good absorption. Screening of additional amino acids resulted in the identification of an l-Phe ester that offered an improved exposure of 1 and reduced levels of the circulating prodrug. Further molecular editing focusing on the linker design culminated in the discovery of the self-immolative l-Phe-Sar dipeptide derivative 74 that gave four-fold improved AUC and eight-fold higher Ctrough values of 1 compared with oral administration of the drug itself, demonstrating a successful prodrug approach to the oral delivery of 1.

Coupling of an Acyl Migration Prodrug Strategy with Bio-activation To Improve Oral Delivery of the HIV-1 Protease Inhibitor Atazanavir.

HIV-1 protease inhibitors (PIs), which include atazanavir (ATV, 1), remain important medicines to treat HIV-1 infection. However, they are characterized by poor oral bioavailability and a need for boosting with a pharmacokinetic enhancer, which results in additional drug-drug interactions that are sometimes difficult to manage. We investigated a chemo-activated, acyl migration-based prodrug design approach to improve the pharmacokinetic profile of 1 but failed to obtain improved oral bioavailability over dosing the parent drug in rats. This strategy was refined by conjugating the amine with a promoiety designed to undergo bio-activation, as a means of modulating the subsequent chemo-activation. This culminated in a lead prodrug that (1) yielded substantially better oral drug delivery of 1 when compared to the parent itself, the simple acyl migration-based prodrug, and the corresponding simple l-Val prodrug, (2) acted as a depot which resulted in a sustained release of the parent drug in vivo, and (3) offered the benefit of mitigating the pH-dependent absorption associated with 1, thereby potentially reducing the risk of decreased bioavailability with concurrent use of stomach-acid-reducing drugs.

Absorption and cleavage of enalapril, a carboxyl ester prodrug, in the rat intestine: in vitro, in situ intestinal perfusion and portal vein cannulation models.

In recent years prodrug strategy has been used extensively to improve the pharmacokinetic properties of compounds exhibiting poor bioavailability. Mechanistic understanding of the absorption and the role of intestine and liver in the activation of oral prodrugs is crucial. Enalapril, a carboxyl ester prodrug, is reported to be metabolized by human carboxylesterase-1 (CES1) but not by carboxylesterase-2 (CES2) to its active metabolite enalaprilat. Further, it has been reported that the small intestines of both rat and human contain mainly CES2. The objective of this work was to understand whether enalapril remains unchanged as it is absorbed through the intestine into the portal circulation. This was evaluated using different intestinal preparations, an in situ intestinal perfusion experiment and a portal vein cannulated rat model. No turnover of enalapril was seen with commercial rat intestinal S9 and microsomes, but reasonable turnover was observed with freshly prepared rat intestinal and mucosal homogenate and S9. In the intestinal perfusion study, both enalapril and enalaprilat were observed in the mesenteric plasma with the data suggesting 32% hydrolysis of enalapril in the intestine. In the portal vein cannulated rat, about 51% of enalapril absorbed into intestine was converted to enalaprilat. Overall, it was demonstrated that even though enalapril has been shown to be a specific substrate for CES1, it is converted to enalaprilat to a significant extent in the intestine. Such experimental techniques can be applied by other scientific groups who are working on prodrugs to determine the region and extent of activation. Copyright © 2015 John Wiley & Sons, Ltd.

Characterization of recombinantly expressed rat and monkey intestinal alkaline phosphatases: in vitro studies and in vivo correlations.

Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent K(m) values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.

Effect of multidose cilostazol on pharmacokinetic and lipid profile of atorvastatin in male Wistar rats.

OBJECTIVES: Atorvastatin (ATV) and cilostazol (CLZ) are often co-prescribed to treat conditions such as peripheral arterial disease. In the present study, the drug-drug interaction potential of multi-dose CLZ on both pharmacokinetics and the lipid-lowering ability of single-dose ATV is demonstrated. METHOD: The pharmacokinetic parameters of ATV were determined in Wistar rats after per-oral pre-treatment with CLZ for 7 days in order to assess the interaction potential between ATV and CLZ. In-vitro metabolic inhibition and everted gut sac studies were conducted to elucidate the mechanism of this interaction. Biochemistry analyser was used to estimate lipid profiles in Wistar rats. A validated LC-MS/MS method was employed to simultaneously quantify both ATV and CLZ in rat plasma matrix. KEY FINDINGS: A statistically significant increase in systemic exposure to ATV after a single dose was observed in CLZ pre-treated rats. In-vitro metabolism studies using rat liver microsome (RLM) demonstrated statistically significant inhibition of ATV metabolism when co-incubated with CLZ. No change in apparent permeability of ATV was observed in the presence of CLZ. The blood lipid profile study after ATV administration indicated a statistically significant decrease in total cholesterol, triglycerides and LDL-cholesterol. CONCLUSIONS: Multi-dose administration of CLZ influences the pharmacokinetics and lipid-lowering properties of ATV. Collectively, an apparent interaction between selected drugs was evident.

Drug-drug interaction study to assess the effects of atorvastatin co-administration on pharmacokinetics and anti-thrombotic properties of cilostazol in male Wistar rats.

Cilostazol (CLZ) and atorvastatin (ATV) are often co-prescribed to treat conditions such as peripheral arterial disease. In the present study, the drug-drug interaction potential of multi-dose ATV co-administration with CLZ on both pharmacokinetics and the anti-thrombotic property of CLZ is demonstrated. The pharmacokinetic parameters of CLZ (6 mg/kg, twice daily) were determined in male Wistar rats after 7 days co-administration with ATV (5 mg/kg, once daily) in order to assess the interaction potential between CLZ and ATV on chronic treatment. In vitro metabolic inhibition and everted gut sac studies were conducted to elucidate the mechanism of this interaction. Pharmacodynamic drug-drug interaction was evaluated on anti-thrombotic models including time to occlusion, platelet aggregation and rat tail bleeding time. A validated LC-MS/MS method was employed simultaneously to quantify both ATV and CLZ in rat plasma matrix. A statistically significant increase in systemic exposure (Css(max) by ~1.75 fold; AUC by ~3.0 fold) to CLZ was observed in ATV pre-treated rats. In vitro metabolism studies using liver microsomes (RLM and HLM) demonstrated statistically significant inhibition of CLZ metabolism when co-incubated with ATV. No change in apparent permeability of CLZ was observed in the presence of ATV. Atorvastatin showed a significant delay in artery occlusion time without altering CLZ's bleeding time and platelet aggregation profile. Collectively the results of these studies provide metabolic insight into the nature of drug-drug interaction between the selected drugs. Co-administration with ATV influences the pharmacokinetics and anti-thrombotic property of CLZ. A thorough clinical investigation is required before extrapolation of data to humans.

Simultaneous Determination of Celecoxib, Erlotinib, and its Metabolite Desmethyl-Erlotinib (OSI-420) in Rat Plasma by Liquid chromatography/Tandem Mass Spectrometry with Positive/Negative Ion-Switching Electrospray Ionisation.

A new method for the simultaneous determination of celecoxib, erlotinib, and its active metabolite desmethyl-erlotinib (OSI-420) in rat plasma, by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionization mode, was developed and validated. Protein precipitation with methanol was selected as the method for preparing the samples. The analytes were separated on a reverse-phase C(18) column (50mm×4.6mm i.d., 3µ) using methanol: 2 mM ammonium acetate buffer, and pH 4.0 as the mobile phase at a flow rate 0.8 mL/min. Sitagliptin and Efervirenz were used as the internal standards for quantification. The determination was carried out on a Theremo Finnigan Quantam ultra triple-quadrupole mass spectrometer, operated in selected reaction monitoring (SRM) mode using the following transitions monitored simultaneously: positive m/z 394.5→278.1 for erlotinib, m/z 380.3→278.1 for desmethyl erlotinib (OSI-420), and negative m/z -380.1→ -316.3 for celecoxib. The limits of quantification (LOQs) were 1.5 ng/mL for Celecoxib, erlotinib, and OSI-420. Within- and between-day accuracy and precision of the validated method were within the acceptable limits of < 15% at all concentrations. The quantitation method was successfully applied for the simultaneous estimation of celecoxib, erlotinib, and desmethyl erlotinib in a pharmacokinetic study in Wistar rats.