Medium-throughput hydrate screening using the Crystal 16™.

Hydrates are known to affect the solubility of compounds to a larger extent compared to polymorphic forms and thus, exposure. In addition, hydrates are more likely to form during most pharmaceutical processes. Therefore discovery of the propensity of compounds to form hydrates at an early stage allows for adequate precautions and sometimes, selection of compounds. A medium-throughput screen using the Crystal 16™ with an accelerated thermal cycle in a mixed solvent system was developed and is described. Nine compounds (seven which form known hydrates and two which do not form hydrates) were tested successfully to demonstrate the utility of the method. A preferred cycle (25°C→40°C→5°C→25°C over 6 h) and solvent system (acetonitrile-water) were identified. The method needs approximately 15-20 mg, in most cases, of the compound and finds applicability in early stages of drug development (late lead-development to early candidate selection).

Development of an intravenous formulation of SU010382 (prodrug of SU5416, an anti-angiogenesis agent).

SU5416, the first in a new class of anti-angiogenesis agents, is an insoluble and neutral molecule which requires a formulation containing Cremophor EL, ethanol, and polyethylene glycol. SU010382, a prodrug of SU5416, was designed as N-Mannich base to provide a basic handle that could be exploited to increase the compound's solubility. Though an increase in solubility was obtained, the inherent hydrolytic instability of SU010382 presented a major challenge in formulation development. The aim of this study is to design a stable intravenous formulation of SU010382 at 2 mg/mL equivalent to the 1.5 mg/mL clinical formulation of SU5416 without a high surfactant/co-solvent content. A stable formulation of SU010382 was successfully designed using a combination of adjusted pH and complexation with sulfobutyl-ether-beta-cyclodextrin. This formulation was designed as a lyophilized product to further increase stability. The lyophilized formulation was stable for at least 6 months at 40 degrees C/75% relative humidity, reconstituted completely within 1 min, and was stable for at least 24 h at 25 degrees C following reconstitution.

High-performance liquid chromatographic method for determination of reversible isomers of SU5416.

SU5416 shows light-induced reversible geometric isomerism. A simple, reliable, isocratic HPLC method using an UV-vis detector at lambda(425nm) was developed. The method provides efficient (R(S)=3.5) analysis of the two isomers with retention of the isomeric integrity. Additionally, the method has linearity over a wide range (50-1000microg/mL, r(2)=0.99), is accurate (99-102%, RSD <4%), and reproducible (RSD <0.8%). The method was used for analyzing pharmaceutical samples and understanding the kinetics of SU5416 isomers in methanol. In addition, this method can be used for quantifying the non-isolatable E-isomer.

Discovery of 5-[5-fluoro-2-oxo-1,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase.

To improve the antitumor properties and optimize the pharmaceutical properties including solubility and protein binding of indolin-2-ones, a number of different basic and weakly basic analogues were designed and synthesized. 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (12b or SU11248) has been found to show the best overall profile in terms of potency for the VEGF-R2 and PDGF-Rbeta tyrosine kinase at biochemical and cellular levels, solubility, protein binding, and bioavailability. 12b is currently in phase I clinical trials for the treatment of cancers.

Chemogenetic evaluation of the mitotic kinesin CENP-E reveals a critical role in triple-negative breast cancer.

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E-dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic-pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer.

Optimization of potent, selective, and orally bioavailable pyrrolodinopyrimidine-containing inhibitors of heat shock protein 90. Identification of development candidate 2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide.

A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.

Pharmacokinetics and tissue distribution of liposomal etoposide in rats.

Precipitation of etoposide and adverse events associated with the co-solvents in intravenous solutions can be avoided by using liposomal etoposide (LE). The pharmacokinetics and distribution of the commercial formulation (ETPI) and LE were compared in rats. The pharmacokinetic profiles were biphasic and similar in the initial phase (C(max), Vd, and t(1/2alpha)). However, LE showed a 60% increase in AUC with a 35% decrease in clearance (p < 0.05). This decreased clearance resulted in a 70% increase in the MRT of etoposide. The uptake of etoposide from LE was higher in macrophage-phagocytic endowed tissues indicating that LE is superior to ETPI for targeted delivery of etoposide.

Reversible Z-E isomerism and pharmaceutical implications for SU5416.

SU5416 (Z-isomer), the first in its class of angiogenesis inhibitors, in solution converts to the E-isomer following light exposure and reverts to the Z-isomer in the dark. Kinetics of this Z-E isomerism in pharmaceutical media is reported. Analytical solutions need light protection at 5 degrees C to maintain integrity. While E-isomer in light-exposed product increased to 0.9% in 24 hours, light-protected product showed no change (25 degrees C, 18 months). Infusate studies indicated that < 1.9% E-isomer will be dosed to patients and would likely convert to the Z-isomer, following administration. This report implies Z-E isomerism in SU5416 is controllable with no limitations towards ensuring pharmaceutical product quality.

Powder-in-bottle formulation of SU011248. Enabling rapid progression into human clinical trials.

SU011248 is an oral, multitargeted receptor tyrosine kinase inhibitor (anti PDGFR, VEGFR, Kit, and Flt3) for the treatment of solid tumors. The powder-in-bottle (PIB) approach was used to accelerate development and introduction into Phase I clinical trials. This approach consists of extemporaneously compounding the active pharmaceutical ingredient (API) into a solution or a suspension in the clinic prior to oral administration. The development consisted of physico-chemical assessment, constitution fluid selection, weighing and dosing validation, and stability evaluation of API, before and after constitution with the fluid. Of the oral liquids evaluated, apple juice was selected as the constitution fluid. Particle size of SU011248 had an impact on the weighing validation and the dissolution time. Particle size specifications of breadth d90 < 180 microm and length d90 < 750 microm were set to achieve pharmaceutical acceptability. Dosing validation studies showed complete recovery of SU011248 from the bottle over a dose range of 10 to 2200 mg. SU011248 is stable as the solid API. Following constitution with apple juice, the product is stable through the predicted duration of compounding and dosing at the clinical site. This approach provided a high degree of dosing flexibility during the initial phase of clinical trials. Additionally, the PIB approach reduced the time and API required for clinical development and supplies to < 2 months and < 100 gm, respectively.