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Physical storage of formulations may result in physical composition changes that affect pharmacokinetics. Dapagliflozin, an oral sodium-glucose cotransporter 2 inhibitor used for type 2 diabetes mellitus, stored under prolonged exposure to heat converts crystalline dapagliflozin to an amorphous form. Bioequivalence of the amorphous to crystalline form and food effects of each form in the 2.5-mg formulation are unknown. Two open-label, crossover, single-dose studies in healthy participants assessed pharmacokinetics for heat-stressed (HS) and non-heat-stressed (NH) dapagliflozin 10-mg (study 1, N=29, fasted+HS food effect) and 2.5-mg (study 2, N=28, fasted+HS and NH food effect) tablets. The 90% confidence intervals for geometric mean ratios of area under the concentration-time curve (AUC) and peak concentration (Cmax) for HS 2.5- and 10-mg tablets were within 80-125%, indicating bioequivalence. In the fed vs. fasted state for 2.5-mg and 10-mg HS tablets, AUCs were similar, time to Cmax was prolonged by 1.25h, and Cmax decreased by approximately 50%. No serious adverse events were reported. Given that dapagliflozin's efficacy is dependent upon AUC, it was concluded that HS and NH dapagliflozin tablets are bioequivalent in 2.5- and 10-mg doses with no clinically meaningful food effect for either form.

The FDA withdrew the draft guidance document for industry “Powder Blends and Finished Dosage Units—Stratified In-Process Dosage Unit Sampling and Assessment.” An FDA’s primary concern was a lack of confidence that the results from USP Uniformity of Dosage Units testing ensure the content uniformity of a batch. ISPE sponsored the Blend Uniformity and Content Uniformity Group (referred to as Group), which was formed in August 2013 to discuss approaches to assess blend and content uniformity. Modifications to the withdrawn draft stratified sampling guidance document are proposed by the Group for the assessment of adequacy of powder mix and content uniformity of the finished product in “Recommendations for the Assessment of Blend and Content Uniformity: Modifications to Withdrawn FDA Draft Stratified Sampling Guidance,” accepted by J Pharm Innov, 2014. Specific statistical methodologies to construct acceptance criteria were not recommended in the paper. This paper provides an example and technical discussion of a statistical approach that can be inserted into the framework described in the Group’s paper. There may be other approaches to sampling plans, statistical analysis, and acceptance criteria that are acceptable to demonstrate content uniformity. ASTM E2709, “Standard Practice for Demonstrating Capability to Comply with an Acceptance Procedure” and E2810, “Standard Practice for Demonstrating Capability to Comply with the Test for Uniformity of Dosage Units” for demonstrating dosage unit uniformity can be used to evaluate content uniformity. These standards are discussed and applied to the sampling plans given in the Group’s paper. The method can provide with a high level of confidence that samples from a lot will have a high probability of passing the USP UDU test. The methodology can be used to evaluate content uniformity data based on a sampling plan that tests either one or more than one dosage unit from each of multiple locations throughout the lot. An acceptance limit table based on sample means and standard deviations can be generated that is easy for the user to apply to content uniformity results. Operating characteristic (OC) curves for process qualification and continued process verification/routine lot release sampling plans are presented and compared along with the USP UDU OC curve. The process qualification criteria/sampling plans are shown to be “conservative” compared to the USP UDU test. A two-stage tiered plan is shown that could be used during continued process monitoring/routine release. The ASTM E2709/E2810 methodology provides acceptance criteria for several sampling plans that could be used for the assessment of content uniformity during process qualification and continued process monitoring/routine release. Passing the acceptance criteria can assure with a high level of confidence that a lot can meet the USP UDU test.

Atomic-level molecular dynamics simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers containing small, amphiphilic, drug-like molecules were carried out to examine the influence of polar functionality on membrane partitioning and transport. Three related molecules (tyramine, phenethylamine, and 4-ethylphenol) were chosen to allow a detailed study of the isolated effects of the amine and hydroxyl functionalities on the preferred solute location, free energies of transfer, and the effect of combining both functional groups in a same molecule. Transfer free energy profiles (from water) generated from molecular dynamics (MD) simulations as a function of bilayer depth compared favorably to comparable experimental results. The simulations allowed the determination of the location of the barrier domain for permeability where the transfer free energy is highest and the preferred binding region at which the free energy is a minimum for each of the three solutes. Comparisons of the free energy profiles reveal that the hydrocarbon chain interior is the region most selective to chemical structure of different solutes because the free energies of transfer in that region vary to a significantly greater extent than in other regions of the bilayer. The contributions of the hydroxyl and amino groups to the free energies of solute transfer from water to the interfacial region were close to zero in both the MD simulations and experimental measurements. This suggests that the free energy decrease observed for solute transfer into the head group region occurs with minimal loss in solvation by hydrogen bonding to polar functional groups on the solute and is largely driven by hydrophobicity. Overall, the joint experimental and simulation studies suggest that the assumption of additivity of free energy contributions from multiple polar functional groups on the same molecule may hold for predictions of passive bilayer permeability coefficients providing that the groups are well isolated. However, this assumption does not hold for predictions of relative liposome-binding affinities.

Computational methods to predict pKa values and partition coefficients of drug molecules based on linear free energy relationships (LFERs) rely largely on the principles of independence and additivity of the functional group contributions in each molecule to the overall free energy. Nonadditivities in functional group contributions are often seen when multiple polar functional groups are in close proximity and in cases where conformational flexibility allows widely separated polar functional groups to interact. The degree to which long-range interactions may alter group contributions in more conformationally constrained molecules such as p-(aminoethyl)phenol and structurally similar analogs is more difficult to predict. In this study, both macroscopic and microscopic ionization constants and decadiene/water partition coefficients as a function of pH at 25°C were obtained for p-(aminoethyl)phenol and six structurally related compounds to explore the reliability of the independence and additivity assumptions necessary in using the LFERs for predictions. A long-range interaction between the phenol and amine groups in the series has been found to affect the pKa values of both groups and to alter species-specific partition coefficients. Pronounced shifts in microscopic ionization constants involving zwitterion forms are clearly indicative of amplified long-range interactions between ionized substituents. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4534–4544, 2009

5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737) is a potent and selective vascular endothelial growth factor receptor-2 antagonist. In this study, liquid chromatography/tandem mass spectrometry and NMR were used to investigate the biotransformation of BMS-645737 in vitro and in the cynomolgus monkey, dog, mouse, and rat. Metabolic pathways for BMS-645737 included multistep processes involving both oxidation and conjugation reactions. For example, the 2-methyl-1H-pyrrolo moiety underwent cytochrome P450-catalyzed hydroxylation followed by oxidation to a carboxylic acid and then conjugation with taurine. Alternatively, the 5-methyl-1,3,4-oxadiazol-2-yl moiety was metabolized by hydroxylation and then conjugation with sulfate. The pyridin-5-yl group underwent direct glucuronidation in hepatocytes (dog, monkey, human) and conjugation with N-acetylglucosamine in the monkey. Conjugation with glutathione and processing along the mercapturic acid pathway was a minor metabolic pathway in vivo, although BMS-645737 did not form conjugates in the presence of glutathione-supplemented liver microsomes. Other minor biotransformation pathways included oxidative dehydrogenation, dihydroxylation, and hydrolytic opening of the oxadiazole ring followed by either deacetylation or hydrolysis of the resulting diacyl hydrazide. Whereas previous studies have shown the formation of N-acetylglucosamine conjugates of alcohols, arylamines, and other small molecules, this report describes the biotransformation of a heterocyclic aromatic amine via direct conjugation with N-acetylglucosamine.

We report herein a series of substituted N-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amines as inhibitors of vascular endothelial growth factor receptor-2 tyrosine kinase. Through structure–activity relationship studies, biochemical potency, pharmacokinetics, and kinase selectivity were optimized to afford BMS-645737 (13), a compound with good preclinical in vivo activity against human tumor xenograft models.

Oral bioavailability of a poorly water-soluble drug was greatly enhanced by using its solid dispersion in a surface-active carrier. The weakly basic drug (pK(a) approximately 5.5) had the highest solubility of 0.1mg/ml at pH 1.5, < 1 microg/ml aqueous solubility between pH 3.5 and 5.5 at 24+/-1 degrees C, and no detectable solubility (< 0.02 microg/ml) at pH greater than 5.5. Two solid dispersion formulations of the drug, one in Gelucire 44/14 and another one in a mixture of polyethylene glycol 3350 (PEG 3350) with polysorbate 80, were prepared by dissolving the drug in the molten carrier (65 degrees C) and filling the melt in hard gelatin capsules. From the two solid dispersion formulations, the PEG 3350-polysorbate 80 was selected for further development. The oral bioavailability of this formulation in dogs was compared with that of a capsule containing micronized drug blended with lactose and microcrystalline cellulose and a liquid solution in a mixture of PEG 400, polysorbate 80 and water. For intravenous administration, a solution in a mixture of propylene glycol, polysorbate 80 and water was used. Absolute oral bioavailability values from the capsule containing micronized drug, the capsule containing solid dispersion and the oral liquid were 1.7+/-1.0%, 35.8+/-5.2% and 59.6+/-21.4%, respectively. Thus, the solid dispersion provided a 21-fold increase in bioavailability of the drug as compared to the capsule containing micronized drug. A capsule formulation containing 25 mg of drug with a total fill weight of 600 mg was subsequently selected for further development. The selected solid dispersion formulation was physically and chemically stable under accelerated storage conditions for at least 6 months. It is hypothesized that polysorbate 80 ensures complete release of drug in a metastable finely dispersed state having a large surface area, which facilitates further solubilization by bile acids in the GI tract and the absorption into the enterocytes. Thus, the bioavailability of this poorly water-soluble drug was greatly enhanced by formulation as a solid dispersion in a surface-active carrier.

Mixtures of poly(ethylene glycols) (PEGs) with polysorbate 80 are often used to dissolve poorly water-soluble drugs in dosage forms, where polysorbate 80 helps either in enhancing dispersion or in inhibiting precipitation of drugs once the solution is mixed with water. Binary phase diagrams of polysorbate 80 with several low molecular weight PEGs and a ternary phase diagram of polysorbate 80 with PEG 400 and water are presented. Two phases were observed in the binary mixtures when the concentration of PEG 200, PEG 300, PEG 400, or PEG 600 was >55%(w/w). The miscibility of the binary mixtures increases with an increase in temperature; the upper consolute temperatures of PEG 200-polysorbate 80, PEG 300-polysorbate 80, PEG 400-polysorbate 80, and PEG 600-polysorbate 80 mixtures were 100, 85, 75, and 40 degrees C, respectively. The upper consolute temperature of PEG 1000-polysorbate 80 could not be determined because the melting temperature of the mixtures is approximately 40 degrees C and the consolute temperature appeared to be less than this temperature. The decrease in upper consolute temperature with an increase in PEG molecular weight indicated a greater miscibility of the two components. In the ternary system, phase separation of polysorbate 80 was observed when the concentration of PEG 400 was >50-60 % (w/w), possibly because of the high exclusion volume of PEG 400.

Molecular interactions and orientations responsible for differences in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer partitioning of three structurally related drug-like molecules (4-ethylphenol, phenethylamine, and tyramine) were investigated. This work is based on previously reported molecular dynamics (MD) simulations that determined their transverse free energy profiles across the bilayer. Previously, the location where the transfer free energy of the three solutes is highest, which defines the barrier domain for permeability, was found to be the bilayer center, while the interfacial region was found to be the preferred binding region. Contributions of the amino (NH2) and hydroxyl (OH) functional groups to the transfer free energies from water to the interfacial region were found to be very small both experimentally and by MD simulation, suggesting that the interfacial binding of these solutes is hydrophobically driven and occurs with minimal loss of hydrogen-bonding interactions of the polar functional groups which can occur with either water or phospholipid head groups. Therefore, interfacial binding is relatively insensitive to the number or type of polar functional groups on the solute. In contrast, the relative solute free energy in the barrier domain is highly sensitive to the number of polar functional groups on the molecule. The number and types of hydrogen bonds formed between the three solutes and polar phospholipid atoms or with water molecules were determined as a function of solute position in the bilayer. Minima were observed in the number of hydrogen bonds formed by each solute at the center of the bilayer, coinciding with a decrease in the number of water molecules in DOPC as a function of distance away from the interfacial region. In all regions, hydrogen bonds with water molecules account for the majority of hydrogen-bonding interactions observed for each solute. Significant orientational preferences for the solutes are evident in certain regions of the bilayer (e.g., within the ordered chain region and near the interfacial region 20-25 Å from the bilayer center). The preferred orientations are those that preserve favorable molecular interactions for each solute, which vary with the solute structure.

Atomic-level molecular dynamics simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers containing small, amphiphilic, drug-like molecules were carried out to examine the influence of polar functionality on membrane partitioning and transport. Three related molecules (tyramine, phenethylamine, and 4-ethylphenol) were chosen to allow a detailed study of the isolated effects of the amine and hydroxyl functionalities on the preferred solute location, free energies of transfer, and the effect of combining both functional groups in a same molecule. Transfer free energy profiles (from water) generated from molecular dynamics (MD) simulations as a function of bilayer depth compared favorably to comparable experimental results. The simulations allowed the determination of the location of the barrier domain for permeability where the transfer free energy is highest and the preferred binding region at which the free energy is a minimum for each of the three solutes. Comparisons of the free energy profiles reveal that the hydrocarbon chain interior is the region most selective to chemical structure of different solutes because the free energies of transfer in that region vary to a significantly greater extent than in other regions of the bilayer. The contributions of the hydroxyl and amino groups to the free energies of solute transfer from water to the interfacial region were close to zero in both the MD simulations and experimental measurements. This suggests that the free energy decrease observed for solute transfer into the head group region occurs with minimal loss in solvation by hydrogen bonding to polar functional groups on the solute and is largely driven by hydrophobicity. Overall, the joint experimental and simulation studies suggest that the assumption of additivity of free energy contributions from multiple polar functional groups on the same molecule may hold for predictions of passive bilayer permeability coefficients providing that the groups are well isolated. However, this assumption does not hold for predictions of relative liposome-binding affinities.

Accurate determination of intrinsic permeability coefficients is critical to the development of structure-permeability relationships and liposomal delivery systems. The apparent release rate of a drug from liposomes may reflect not only its intrinsic permeability coefficient and barrier properties but also a variety of underlying equilibria including drug ionization, membrane binding or complexation, and kinetic processes such as buffer exchange. Additionally, transport of ionizable drugs that are initially at high concentrations in liposomes can generate or dissipate pH gradients across the barrier causing deviations from classical pH-permeability profiles. In this study, the liposomal release of a model amine (tyramine) is determined as a function of drug loading, intravesicular pH, and buffer composition. Kinetic models are derived to study effects of such equilibria (e.g., ionization, membrane binding) and kinetic processes (e.g., pH drift and acid/base carriers). All equilibrium constants needed for the models were independently measured and used. The barrier properties of the lipid bilayers under the experimental conditions were assessed by monitoring the transport of mannitol and bretylium as a function of pH. A corrected intrinsic permeability coefficient of 0.04 cm/s was in close agreement with the value predicted from the barrier domain model for bilayer permeability, suggesting that all perturbing factors were properly addressed.

August 2015 e105 fed state; a low fat breakfast was provided in Part A and a high breakfast in Part B. Results: In both parts, the new formulation substantially increased DRL-17822 exposure in fasted state, characterised by Cmax, AUC0-t and AUC0-∞, compared to the current formulation. Following high fat breakfast, DRL-17822 exposure was significantly less using the new formulation compared to the current formulation (P < 0.01). In addition, the new formulation resulted in similar ratios fed/fasted for AUC0-t in following both types of breakfast while the current formulation had a higher ratio after high fat breakfast compared to a low fat breakfast (Table 1, similar ratios were seen for Cmax and AUC0-∞). Conclusion: The new formulation had favourable pharmacokinetic characteristics compared to the current formulation, showing a food effect of only 3-fold, which may relate in more predictable effect profile.

LFA-1 (leukocyte function-associated antigen-1), is a member of the beta2-integrin family and is expressed on all leukocytes. This letter describes the discovery and preliminary SAR of spirocyclic hydantoin based LFA-1 antagonists that culminated in the identification of analog 8 as a clinical candidate. We also report the first example of the efficacy of a small molecule LFA-1 antagonist in combination with CTLA-4Ig in an animal model of transplant rejection.