Impact of Simulated Intestinal Fluids on Dissolution, Solution Chemistry, and Membrane Transport of Amorphous Multidrug Formulations.

The solution behavior and membrane transport of multidrug formulations were herein investigated in a biorelevant medium simulating fasted conditions. Amorphous multidrug formulations were prepared by the solvent evaporation method. Combinations of atazanavir (ATV) and ritonavir (RTV) and felodipine (FDN) and indapamide (IPM) were prepared and stabilized by a polymer for studying their dissolution (under non-sink conditions) and membrane transport in fasted state simulated intestinal fluid (FaSSIF). The micellar solubilization by FaSSIF enhanced the amorphous solubility of the drugs to different extents. Similar to buffer, the maximum achievable concentration of drugs in combination was reduced in FaSSIF, but the extent of reduction was affected by the degree of FaSSIF solubilization. Dissolution studies of ATV and IPM revealed that the amorphous solubility of these two drugs was not affected by FaSSIF solubilization. In contrast, RTV was significantly affected by FaSSIF solubilization with a 30% reduction in the maximum achievable concentration upon combination to ATV, compared to 50% reduction in buffer. This positive deviation by FaSSIF solubilization was not reflected in the mass transport-time profiles. Interestingly, FDN concentrations remain constant until the amount of IPM added was over 1000 µg/mL. No decrease in the membrane transport of FDN was observed for a 1:1 M ratio of FDN-IPM combination. This study demonstrates the importance of studying amorphous multidrug formulations under physiologically relevant conditions to obtain insights into the performance of these formulations after oral administration.

Quantum simulations of SARS-CoV-2 main protease Mpro enable high-quality scoring of diverse ligands.

The COVID-19 pandemic has led to unprecedented efforts to identify drugs that can reduce its associated morbidity/mortality rate. Computational chemistry approaches hold the potential for triaging potential candidates far more quickly than their experimental counterparts. These methods have been widely used to search for small molecules that can inhibit critical proteins involved in the SARS-CoV-2 replication cycle. An important target is the SARS-CoV-2 main protease Mpro, an enzyme that cleaves the viral polyproteins into individual proteins required for viral replication and transcription. Unfortunately, standard computational screening methods face difficulties in ranking diverse ligands to a receptor due to disparate ligand scaffolds and varying charge states. Here, we describe full density functional quantum mechanical (DFT) simulations of Mpro in complex with various ligands to obtain absolute ligand binding energies. Our calculations are enabled by a new cloud-native parallel DFT implementation running on computational resources from Amazon Web Services (AWS). The results we obtain are promising: the approach is quite capable of scoring a very diverse set of existing drug compounds for their affinities to M pro and suggest the DFT approach is potentially more broadly applicable to repurpose screening against this target. In addition, each DFT simulation required only ~ 1 h (wall clock time) per ligand. The fast turnaround time raises the practical possibility of a broad application of large-scale quantum mechanics in the drug discovery pipeline at stages where ligand diversity is essential.

Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa.

INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.

Atazanavir, Alone or in Combination with Ritonavir, Inhibits SARS-CoV-2 Replication and Proinflammatory Cytokine Production.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.

Impact of phospholipid digests and bile acid pool variations on the crystallization of atazanavir from supersaturated solutions.

Oral delivery of poorly water-soluble drugs (PWSDs), which predominate the development pipeline, poses significant challenges. Weakly basic compounds, such as atazanavir, represent a critical class of PWSDs as even the administration of the crystalline solid may invoke supersaturation during gastric-intestinal transfer. The absorption advantage afforded by this supersaturated state can be offset by inherent metastability and a tendency to revert to the lower energy crystalline state. Therefore, it is important to understand the physiological factors that can affect crystallization to improve in vitro-in vivo predictiveness and to regulate inter-individual responses. The first aim of this study was to elucidate the influence of lyso-phosphatidylcholine (lyso-PC) and sodium oleate on crystallization, as the products of phosphatidylcholine (PC) hydrolysis and the major lipid components of human intestinal fluid (HIF) and updated fasted state simulated intestinal fluid version 3 (FaSSIF-V3). Secondly, as an individual's bile acid pool is unique, dynamic and related to gut microbiome community structure, it was of interest to investigate the impact of bile acid pool variations on crystallization from supersaturated solutions. To study the effect of PC hydrolysis, media with 2.8 mM sodium glycocholate (GCA) and sodium taurocholate (TCA) (1:1) but varying concentrations of PC, lyso-PC or sodium oleate were prepared. To investigate the influence of inter-individual variations in intestinal bile acid pool size and composition, media simulating the profiles of six healthy Western volunteers were prepared based on previously published data. The crystalline and amorphous solubility of atazanavir, a weakly basic antiretroviral drug, was firstly determined in these media. Nucleation-induction time experiments were then performed at an equivalent extent of supersaturation in each medium (corresponding to the amorphous solubility). At a constant level of GCA/TCA, increasing concentrations of both PC and lyso-PC accelerated crystallization onset; however, this was at least 2-fold more pronounced with lyso-PC at a given molar concentration. The addition of sodium oleate was also observed to induce crystallization. Interestingly, substituting GCA/TCA with the bile salt fraction of other biorelevant media partially circumvented the crystallization-inducing effect of phospholipids and their digests. The presence of dihydroxy bile salts was found to be particularly significant in decelerating the crystallization process. Nucleation-induction times in simulated volunteer pools were found to be dependent upon bile salt concentration, with higher bile salt levels generally prolonging supersaturation. Differences of up to 6-fold were observed. This study demonstrates that the choice of biorelevant medium used to evaluate supersaturating formulations can influence the observed crystallization kinetics. While the presence of lyso-PC and sodium oleate in FaSSIF-V3 medium is more physiologically relevant, further attention should be paid to the bile salt fraction when designing a biorelevant medium for supersaturating formulations. In vivo, inter-individual differences in the amount and types of bile acids and phospholipids present may influence the behaviour of supersaturating formulations.

Laser ablation for pharmaceutical nanoformulations: Multi-drug nanoencapsulation and theranostics for HIV.

We introduce the use of laser ablation to develop a multi-drug encapsulating theranostic nanoformulation for HIV-1 antiretroviral therapy. Laser ablated nanoformulations of ritonavir, atazanavir, and curcumin, a natural product that has both optical imaging and pharmacologic properties, were produced in an aqueous media containing Pluronic® F127. Cellular uptake was confirmed with the curcumin fluorescence signal localized in the cytoplasm. Formulations produced with F127 had improved water dispersibility, are ultrasmall in size (20-25 nm), exhibit enhanced cellular uptake in microglia, improve blood-brain barrier (BBB) crossing in an in vitro BBB model, and reduce viral p24 by 36 fold compared to formulations made without F127. This work demonstrates that these ultrasmall femtosecond laser-ablated nanoparticles are effective in delivering drugs across the BBB for brain therapy and show promise as an effective method to formulate nanoparticles for brain theranostics, reducing the need for organic solvents during preparation.

Interplay of Supersaturation and Solubilization: Lack of Correlation between Concentration-Based Supersaturation Measurements and Membrane Transport Rates in Simulated and Aspirated Human Fluids.

Supersaturating formulations are increasingly being used to improve the absorption of orally administered poorly water-soluble drugs. To better predict outcomes in vivo, we must be able to accurately determine the degree of supersaturation in complex media designed to provide a surrogate for the gastrointestinal environment. Herein, we demonstrate that relying on measurements based on consideration of the total dissolved concentration leads to underestimation of supersaturation and consequently membrane transport rates. Crystalline and amorphous solubilities of two compounds, atazanavir and posaconazole, were evaluated in six different media. Concurrently, diffusive flux measurements were performed in a side-by-side diffusion cell to determine the activity-based supersaturation by evaluating membrane transport rates at the crystalline and amorphous solubilities. Solubility values were found to vary in each medium because of different solubilization capacities. Concentration-based supersaturation ratios were also found to vary for the different media. Activity-based measurements, however, were largely independent of the medium, leading to relatively constant values for the estimated supersaturation. These findings have important consequences for modeling and prediction of supersaturation impact on the absorption rate as well as for better defining the thermodynamic driving force for crystallization in complex media.

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.

Modulating cellular autophagy for controlled antiretroviral drug release.

AIM: Pharmacologic agents that affect autophagy were tested for their abilities to enhance macrophage nanoformulated antiretroviral drug (ARV) depots and its slow release. METHODS: These agents included URMC-099, rapamycin, metformin, desmethylclomipramine, 2-hydroxy-ß-cyclodextrin (HBC) and clonidine. Each was administered with nanoformulated atazanavir (ATV) nanoparticles to human monocyte-derived macrophages. ARV retention, antiretroviral activity and nanocrystal autophagosomal formation were evaluated. RESULTS: URMC-099, HBC and clonidine retained ATV. HBC, URMC-099 and rapamycin improved intracellular ATV retention. URMC-099 proved superior among the group in affecting antiretroviral activities. CONCLUSION: Autophagy inducing agents, notably URMC-099, facilitate nanoformulated ARV depots and lead to sustained release and improved antiretroviral responses. As such, they may be considered for development as part of long acting antiretroviral treatment regimens.

Crystallization from Supersaturated Solutions: Role of Lecithin and Composite Simulated Intestinal Fluid.

PURPOSE: The overall purpose of this study was to understand the impact of different biorelevant media types on solubility and crystallization from supersaturated solutions of model compounds (atazanavir, ritonavir, tacrolimus and cilnidipine). The first aim was to understand the influence of the lecithin content in FaSSIF. As the human intestinal fluids (HIFs) contain a variety of bile salts in addition to sodium taurocholate (STC), the second aim was to understand the role of these bile salts (in the presence of lecithin) on solubility and crystallization from supersaturated solutions, METHODS: To study the impact of lecithin, media with 3 mM STC concentration but varying lecithin concentration were prepared. To test the impact of different bile salts, a new biorelevant medium (Composite-SIF) with a composition simulating that found in the fasted HIF was prepared. The crystalline and amorphous solubility was determined in these media. Diffusive flux measurements were performed to determine the true supersaturation ratio at the amorphous solubility of the compounds in various media. Nucleation induction times from supersaturated solutions were measured at an initial concentration equal to the amorphous solubility (equivalent supersaturation) of the compound in the given medium. RESULTS: It was observed that, with an increase in lecithin content at constant STC concentration (3 mM), the amorphous solubility of atazanavir increased and crystallization was accelerated. However, the crystalline solubility remained fairly constant. Solubility values were higher in FaSSIF compared to Composite-SIF. Longer nucleation induction times were observed for atazanavir, ritonavir and tacrolimus in Composite-SIF compared to FaSSIF at equivalent supersaturation ratios. CONCLUSIONS: This study shows that variations in the composition of SIF can lead to differences in the solubility and crystallization tendency of drug molecules, both of which are critical when evaluating supersaturating systems.

Simultaneous Determination of Impurities of Atazanavir and Ritonavir in Tablet Dosage Form by Using Reversed-Phase Ultra Performance Liquid Chromatographic Method.

A simple, rapid, selective and stability indicating reversed phase-ultra performance liquid chromatography method was developed and validated for the simultaneous quantification of process related and degradation impurities present in Atazanavir and Ritonavir tablets. The two proposed drug components and their respective impurities were separated using Acquity BEH C18 (100 mm × 2.1 mm), 1.7 µ column at a flow rate of 0.4 mL/min. Buffer used as Mobile phase-A which consists of 0.01 M monobasic potassium hydrogen phosphate adjusted the pH to 3.6 and acetonitrile is used as organic modifier (mobile phase-B). The detector wavelength of 240 nm was used for quantifying the impurities. Both the drug components along with their impurities were eluted within a runtime of 18 min. The performance of the developed method was checked by validating the method according to the requirements of International Conference on Harmonization for parameters such as specificity, precision, linearity, ruggedness, accuracy, sensitivity (limit of detection (LOD) and limit of quantitation (LOQ)) and robustness. Linearity and range were established from LOQ level to 150% level. Accuracy of the method was demonstrated from LOQ level to 150% level. The developed stability indicating method is capable for determination of impurities of Atazanavir and Ritonavir in combined tablet dosage form as well as individual dosage forms also. The reported method enables lesser solvent consumption and reduces time and cost of the analysis in quality control laboratory.

Antiretroviral unbound concentration during pregnancy: piece of interest in the puzzle?

Atazanavir and darunavir total concentrations (drug bound to plasma proteins plus unbound drug) progressively decrease during pregnancy. This pharmacokinetic variation leads physicians to recommend increasing doses. Conversely, the unbound concentration (Cu), i.e. the pharmacologically active form of the drug, remains unchanged. The explanation of this desynchronization lies in the fact that the clearance of the unbound form, corresponding to the intrinsic metabolic capacity of the hepatocytes, is the only factor driving Cu, and is constant during pregnancy. The attention of HIV physicians should be attracted to this aspect of pharmacokinetics, which is often incompletely understood and could lead to inadequate dose adjustment, which could then cause overexposure of the foetus for many months, with unknown consequences.

Impact of Micellar Surfactant on Supersaturation and Insight into Solubilization Mechanisms in Supersaturated Solutions of Atazanavir.

PURPOSE: The goals of this study were to determine: 1) the impact of surfactants on the "amorphous solubility"; 2) the thermodynamic supersaturation in the presence of surfactant micelles; 3) the mechanism of solute solubilization by surfactant micelles in supersaturated solutions. METHODS: The crystalline and amorphous solubility of atazanavir was determined in the presence of varying concentrations of micellar sodium dodecyl sulfate (SDS). Flux measurements, using a side-by-side diffusion cell, were employed to determine the free and micellar-bound drug concentrations. The solubilization mechanism as a function of atazanavir concentration was probed using fluorescence spectroscopy. Pulsed gradient spin-echo proton nuclear magnetic resonance (PGSE-NMR) spectroscopy was used to determine the change in micelle size with a change in drug concentration. RESULTS: Changes in the micelle/water partition coefficient, K m/w , as a function of atazanavir concentration led to erroneous estimates of the supersaturation when using concentration ratios. In contrast, determining the free drug concentration using flux measurements enabled improved determination of the thermodynamic supersaturation in the presence of micelles. Fluorescence spectroscopic studies suggested that K m/w changed based on the location of atazanavir solubilization which in turn changed with concentration. Thus, at a concentration equivalent to the crystalline solubility, atazanavir is solubilized by adsorption at the micelle corona, whereas in highly supersaturated solutions it is also solubilized in the micellar core. This difference in solubilization mechanism can lead to a breakdown in the prediction of amorphous solubility in the presence of SDS as well as challenges with determining supersaturation. PGSE-NMR suggested that the size of the SDS micelle is not impacted at the crystalline solubility of the drug but increases when the drug concentration reaches the amorphous solubility, in agreement with the proposed changes in solubilization mechanism. CONCLUSIONS: Micellar solubilization of atazanavir is complex, with the solubilization mechanism changing with differences in the degree of (super)saturation. This can result in erroneous predictions of the amorphous solubility and thermodynamic supersaturation in the presence of solubilizing additives. This in turn hinders understanding of the driving force for phase transformations and membrane transport, which is essential to better understand supersaturating dosage forms.

Determination of genotoxic impurity in atazanavir sulphate drug substance by LC-MS.

A sensitive LC-MS method was developed for the determination of tert-butyl 2-[4-(pyridine-2-yl) benzyl] hydrazine carboxylate (GTI-A), a genotoxic impurity in Atazanavir sulphate drug substance. The method was validated as per International Council for Harmonization guidelines, for QL, DL, linearity and accuracy. The QL and DL values obtained were 1.1ppm and 0.3ppm respectively. The Correlation coefficient found for the linearity study was 0.999. The % recovery of the added impurity in the range of 96.4-100.4 ensured the accuracy of the method.

Compromised in vitro dissolution and membrane transport of multidrug amorphous formulations.

Herein, the thermodynamic properties of solutions evolving from the non-sink dissolution of amorphous solid dispersions (ASDs) containing two or more drugs have been evaluated, focusing on the maximum achievable supersaturation and tendency of the system to undergo liquid-liquid phase separation (LLPS). Ritonavir (RTV) and atazanavir (ATV) were co-formulated with polyvinylpyrrolidone to produce ASDs with different molar ratios of each drug, and the dissolution profile of each drug was studied under non-sink conditions. The phase behavior of the supersaturated solutions generated by ASD dissolution was compared to that of supersaturated solutions generated by antisolvent addition. Dissolution of an ASD containing RTV, ATV and lopinavir (LPV) was also investigated. A thermodynamic model was used to predict the maximum achievable supersaturation for ASDs containing two and three drugs. In addition, a transport study with Caco-2 cells was conducted to evaluate the impact of co-addition of drugs on membrane transport. It was found that the formulation containing a 1:1 molar ratio of RTV and ATV achieved only 50% of the supersaturation attained by dissolution of the single drug systems. The maximum achievable concentration of ATV decreased linearly as the mole fraction of ATV in the formulation decreased and a similar trend was observed for RTV. For the dispersion containing a 1:1:1 molar ratio of RTV, ATV and LPV, the maximum concentration of each drug was only one third of that achieved for the single drug formulations. The decrease in the achievable supersaturation was well-predicted by the thermodynamic model for both the binary and ternary drug combinations. These observations can be explained by a decrease in the concentration at which the drugs undergo LLPS in the presence of other miscible drugs, thereby reducing the maximum achievable supersaturation of each drug. The reduced free drug concentration was reflected by a decreased flux across Caco-2 cells for the drug combinations compared to drug alone. This study sheds light on the complex dissolution and solution phase behavior of multicomponent amorphous dosage forms, in particular those containing poorly water soluble drugs, which may undergo supersaturation in vivo.

Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4.

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 µM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 µM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.

Atazanavir-loaded Eudragit RL 100 nanoparticles to improve oral bioavailability: optimization and in vitro/in vivo appraisal.

Atazanavir (ATV) is a HIV protease inhibitor. Due to its intense lipophilicity, the oral delivery of ATV encounters several problems such as poor aqueous solubility, pH-dependent dissolution, rapid first-pass metabolism in liver by CYP3A5, which result in low bioavailability. To overcome afore mentioned limitations, ATV-loaded Eudragit RL100 nanoparticles (ATV NPs) were prepared to enhance oral bioavailability. ATV NPs were prepared by nanoprecipitation method. The ATV NPs were systematically optimized (OPT) using 3(2) central composite design (CCD) and the OPT formulation located using overlay plot. The pharmacokinetic study of OPT formulation was investigated in male Wistar rats, and in-vitro/in-vivo correlation level was established. Intestinal permeability of OPT formulation was determined using in situ single pass perfusion (SPIP) technique. Transmission electron microscopy studies on OPT formulation demonstrated uniform shape and size of particles. Augmentation in the values of Ka (2.35-fold) and AUC0-24 (2.91-fold) indicated significant enhancement in the rate and extent of bioavailability by the OPT formulation compared to pure drug. Successful establishment of in vitro/in vivo correlation (IVIVC) Level A substantiated the judicious choice of the in vitro dissolution milieu for simulating the in vivo conditions. In situ SPIP studies ascribed the significant enhancement in absorptivity and permeability parameters of OPT formulation transport through the Peyer's patches. The studies, therefore, indicate the successful formulation development of NPs with distinctly improved bioavailability potential and can be used as drug carrier for sustained or prolonged drug release.

Prediction of pH dependent absorption using in vitro, in silico, and in vivo rat models: Early liability assessment during lead optimization.

Weakly basic compounds which have pH dependent solubility are liable to exhibit pH dependent absorption. In some cases, a subtle change in gastric pH can significantly modulate the plasma concentration of the drug and can lead to sub-therapeutic exposure of the drug. Evaluating the risk of pH dependent absorption and potential drug-drug interaction with pH modulators are important aspects of drug discovery and development. In order to assess the risk around the extent of decrease in the systemic exposure of drugs co-administered with pH modulators in the clinic, a pH effect study is carried out, typically in higher species, mostly dog. The major limitation of a higher species pH effect study is the resource and material requirement to assess this risk. Hence, these studies are mostly restricted to promising or advanced leads. In our current work, we have used in vitro aqueous solubility, in silico simulations using GastroPlus™ and an in vivo rat pH effect model to provide a qualitative assessment of the pH dependent absorption liability. Here, we evaluate ketoconazole and atazanavir with different pH dependent solubility profiles and based on in vitro, in silico and in vivo results, a different extent of gastric pH effect on absorption is predicted. The prediction is in alignment with higher species and human pH effect study results. This in vitro, in silico and in vivo (IVISIV) correlation is then extended to assess pH absorption mitigation strategy. The IVISIV predicts pH dependent absorption for BMS-582949 whereas its solubility enhancing prodrug, BMS-751324 is predicted to mitigate this liability. Overall, the material requirement for this assessment is substantially low which makes this approach more practical to screen multiple compounds during lead optimization.

Advances and challenges in PBPK modeling--Analysis of factors contributing to the oral absorption of atazanavir, a poorly soluble weak base.

Many active pharmaceutical ingredients (APIs) exhibit a highly variable pharmacokinetic (PK) profile. This behavior may be attributable to pre-absorptive, absorptive and/or post-absorptive factors. Pre-absorptive factors are those related to dosage form disintegration, drug dissolution, supersaturation, precipitation and gastric emptying. Absorptive factors are involved with drug absorption and efflux mechanisms, while drug distribution and clearance are post-absorptive factors. This study aimed to investigate the relative influence of the aforementioned parameters on the pharmacokinetic profile of atazanavir, a poorly soluble weakly basic compound with highly variable pharmacokinetics. The pre-absorptive behavior of the drug was examined by applying biorelevant in vitro tests to reflect upper gastrointestinal behavior in the fasted and fed states. The in vitro results were implemented, along with permeability and post-absorptive data obtained from the literature, into physiologically based pharmacokinetic (PBPK) models. Sensitivity analysis of the resulting plasma profiles revealed that the pharmacokinetic profile of atazanavir is affected by an array of factors rather than one standout factor. According to the in silico model, pre-absorptive and absorptive factors had less impact on atazanavir bioavailability compared to post-absorptive parameters, although active drug efflux and extraction appear to account for the sub-proportional pharmacokinetic response to lower atazanavir doses in the fasted state. From the PBPK models it was concluded that further enhancement of the formulation would bring little improvement in the pharmacokinetic response to atazanavir. This approach may prove useful in assessing the potential benefits of formulation enhancement of other existing drug products on the market.