Applications of Powder X-Ray Diffraction in Small Molecule Pharmaceuticals: Achievements and Aspirations.

Since the discovery of X-ray diffraction and its potential to elucidate crystal symmetry, powder X-ray diffraction has found diverse applications in the field of pharmaceutical sciences. This review summarizes significant achievements of the technique during various stages of dosage form development. Improved understanding of the principle involved and development of automated hardware and reliable software have led to increased instrumental sensitivity and improved data analysis. These advances continue to expand the applications of powder X-ray diffraction to emerging research fields such as amorphous systems, mechanistic understanding of phase transformations, and "Quality by Design" in formulation development.

Isothermal microcalorimetry to investigate the phase separation for amorphous solid dispersions of AMG 517 with HPMC-AS.

Understanding the crystallization kinetics of an amorphous drug is critical for the development of an amorphous solid dispersion (ASD) formulation. This paper examines the phase separation and crystallization of the drug AMG 517 in ASDs of varying drug load at various conditions of temperature and relative humidity using isothermal microcalorimetry. ASDs of AMG 517 in hydroxypropyl methylcellulose acetate succinate (HPMC-AS) were manufactured using a Buchi 290 mini spray dryer system. ASDs were characterized using modulated differential scanning calorimetry (mDSC) and scanning electron microscopy (SEM) prior to isothermal microcalorimetry evaluation, and crystallinity was measured using (19)F solid state nuclear magnetic resonance spectroscopy (SSNMR), before and after crystallization. The crystallization of ASDs of AMG 517 in HPMC-AS was significantly slowed by the presence of HPMC-AS polymer, indicating enhanced physical stability for the ASD formulations. A two-phase crystallization was observed by isothermal microcalorimetry at temperatures near the glass transition temperature (Tg), indicating a drug-rich phase and a miscible ASD phase. (19)F SSNMR showed that only partial crystallization of the drug occurred for the ASDs, suggesting a third phase which did not crystallize, possibly representing a thermodynamically stable, soluble component. Isothermal microcalorimetry provides important kinetic data for monitoring crystallization of the drug in the ASDs and, together with (19)F SSNMR, suggests a three-phase ASD system for AMG 517 in HPMC-AS.

hERG potency estimates based upon dose solution analysis: What have we learned?

INTRODUCTION: Measurement of drug-induced inhibition of potassium current flow through the hERG channel is used to determine potency at the channel, which is used as an in vitro risk assessment for QTc interval prolongation in vivo. In the hERG assay, test solutions of varying strength are prepared to construct a concentration-response curve based upon the nominal drug concentration (NOM). Dose-solution analysis (DSA) is an analytical approach to confirm the test concentration achieved in an in vitro assay (Herron, Towers, & Templeton, 2004), and can be included as a component of hERG channel study to confirm drug concentration in the assay buffer to determine potency using the "actual" drug level in solution (ACT). Thus, DSA could be helpful in confirming test article concentrations. This study examined whether inclusion of DSA improved the accuracy of potency estimates based upon the ACT compared to the NOM concentration during hERG voltage clamp assays (non-GLP) for 99 diverse agents. METHODS: We examined the correlation of hERG IC(50) derived from NOM with hERG IC(50) derived from ACT, and analyzed potential mechanisms of deviation between ACT and NOM potency values, including solubility, cLogP, PKa, and molecular weights. RESULTS: Seventy-four (74) of 99 agents (73.7%) had NOM- and ACT-derived IC(50) values within 3-fold, 87 of 99 (87.8%) had an IC(50) ratio within 10-fold, and 12 (12.1%) had a >10-fold difference in their NOM IC(50) and ACT IC(50) values. On average, these 12 compounds had less soluble, more lipophilic (high cLogP values), and more basic characters (high pKa values). DISCUSSION: Our investigation indicated that DSA did not alter hERG potency estimation for the majority of compounds in this dataset, i.e., DSA confirmed the NOM concentration within 3-fold. For poorly soluble agents or agents with high cLogP and pKa values, however, DSA did not clarify or improve hERG potency estimates.

Evaluation of a series of naphthamides as potent, orally active vascular endothelial growth factor receptor-2 tyrosine kinase inhibitors.

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.

Naphthamides as novel and potent vascular endothelial growth factor receptor tyrosine kinase inhibitors: design, synthesis, and evaluation.

A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure-activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure-activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.

Kinetics and mechanism of the hydrolytic degradation of indinavir: intramolecular catalysis.

The pH-rate profile of first-order rate constants for the lactonization of Indinavir in aqueous solutions with ionic strength I = 1 (KCl) at 40 degrees C is reported. The lactonization reaction is a subject of strong buffer catalysis with a nonlinear dependence of the first-order rate constants on the concentration of the buffer. The pH-rate profile is more complex than the pH-rate profiles for the hydrolysis of simple peptides and for the intramolecular OH-catalyzed hydrolysis of gamma-hydroxyamides. This complexity appears unique to Indinavir and is a result of the cis-aminoindanol leaving group. The mechanistic pathways for the lactonization are discussed. The buffer catalysis data are consistent with kinetic general acid catalysis. The second-order rate constant for the specific-acid catalyzed hydrolysis of Indinavir at 40 degrees C (k(H) = 2.2 x 10(-4) M(-1) min(-1)) is similar to that for a simple peptide indicating similar interactions in the rate limiting transition state for both reactions.

Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors.

Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. Guided by X-ray crystallography and molecular modeling, a series of 2-aminobenzimidazoles and 2-aminobenzoxazoles were identified as potent inhibitors of VEGFR-2 (KDR) in both enzymatic and HUVEC cellular proliferation assays. In this report we describe the synthesis and structure-activity relationship of a series of 2-aminobenzimidazoles and benzoxazoles, culminating in the identification of benzoxazole 22 as a potent and selective VEGFR-2 inhibitor displaying a good pharmacokinetic profile. Compound 22 demonstrated efficacy in both the murine matrigel model for vascular permeability (79% inhibition observed at 100 mg/kg) and the rat corneal angiogenesis model (ED(50) = 16.3 mg/kg).

High-throughput logP measurement using parallel liquid chromatography/ultraviolet/mass spectrometry and sample-pooling.

A novel approach to high-throughput logP measurement based on liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) is proposed. The logP value is determined by correlation with the logk value, where k is the capacity factor k = (t(r)-t(0))/t(0), with the logP value using a defined set of standards. Since the analyte retention time (t(r)) is determined from the appropriate extracted ion chromatogram (EIC), there are no interferences from impurities and this allows the pooling of multiple compounds into one injection. To ensure the accuracy and instrument robustness in a routine high-throughput environment, a simple and MS-friendly mobile phase consisting of 20 mM ammonium carbonate (pH 8.0) for basic compounds or 20 mM ammonium formate (pH 1.0) for acidic compounds, both in combination with methanol at a ratio of 45:55, is used. This approach has been successfully used on single as well as parallel multi-channel LC/UV/MS systems to screen small to large sets of lead compounds and their analogs. A high-throughput capability to analyze over 1000 compounds per day has been achieved.

Quantification of low levels (<10%) of amorphous content in micronised active batches using dynamic vapour sorption and isothermal microcalorimetry.

During the processing of pharmaceutical solids (e.g. milling, spray drying, tablet compaction, wet granulation and lyophilisation), various degrees of disorder in the form of crystal defects and/or amorphous regions may be generated. Even relatively low levels of amorphous material (<10%) may have a detrimental impact on the stability, manufacturability and dissolution characteristics of the formulated drug product. In this paper an isothermal heat conduction microcalorimetry and dynamic vapour sorption technique have been evaluated for the quantification of low levels (<10%) of amorphous material within a crystalline active. Both techniques were able to detect a 0.5% amorphous content, and in each case the limit of detection may be further lowered by increasing the sample size. The impact of micronisation on the crystallinity of a batch of active was evaluated using the two methods. The isothermal microcalorimetry and dynamic vapour sorption data showed excellent agreement (+/-0.2% amorphous content) and indicated that the amount of amorphous material generated is extremely sensitive to small changes in the operating conditions of the microniser. The techniques described in this paper have been developed at a very early stage of the actives development program such that the impact of small quantities of amorphous material on the quality attributes of the formulation can be fully assessed. The methods can be applied to any active, the only criteria is that the amorphous material will recrystallise on exposure to moisture or solvent vapours, and no hydrates or solvates are formed.