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The present study aimed to develop a practical method for preparing nanosuspension formulations of poorly water-soluble compounds for enhancing oral absorption in toxicology studies in the discovery stage. To obtain a suitable nanosuspension formulation for the intended purpose, formulations were optimized with a focus on the following characteristics: i) containing a high drug concentration, ii) consisting of commonly used excipient types in proper quantities for toxicology studies, iii) having long-term stability, and iv) having versatility for use with diverse compounds. Test compounds were milled with various excipients by wet media milling methods using a mixer mill (10 mg/batch) and a rotation/revolution mixer (0.5 g/batch). As a result, 100 mg/mL nanosuspensions of all 11 test compounds could be prepared with an optimized dispersing agent, 0.5% hydroxypropyl methylcellulose (HPMC) (3 cP)-0.5% Tween 80. Notably, it was found that the molecular weight of HPMC influenced not only particle size but also the stability of nanosuspensions and they were stable for 4 weeks at 5°C. The nanosuspensions increased in vitro dissolution rates and provided 3.9 and 3.0 times higher C max and 4.4 and 1.6 times higher area under the concentration-time curve from 0-24 h (AUC 0-24 h ) in rats (oral dose of 300 mg/kg) for cilostazol and danazol, respectively. In conclusion, applying a wet media milling method with the combination of HPMC of a small molecular weight and Tween 80 as a dispersing agent, nanosuspensions can be practically prepared and conveniently utilized for enhancing the oral absorption of poorly water-soluble compounds in toxicology studies in the discovery stage.Key words nanosuspension; toxicology study; wet media milling; mixer mill; rotation revolution mixer; oral absorptionThe proportion of poorly water-soluble compounds among drug candidates in the discovery stage has been increasing for the past decade.1,2) Because of the poor solubility of these compounds, sufficient oral absorption is not observed in animal studies, and the evaluation of pharmacological and toxicological profiles of drug candidates has therefore become more challenging. In particular, this problem frequently occurs in toxicology studies that require high oral absorption and is now a common problem in pharmaceutical companies.3)The application of formulation technologies such as solubilizing excipients, 4) solid dispersions, 5,6) and nanosuspensions 7,8) is an attractive strategy for improving oral absorption of poorly water-soluble compounds. However, the use of solubilizing excipients and solid dispersions for toxicology studies in the discovery stage is practically limited because of toxicities of excipients and amount of drug substances for formulation studies, respectively. On the other hand, nanosuspensions have the potential to solve these problems. Because nanosuspensions can be prepared as highly drug-loaded formulations with small quantities of excipients, adverse effects caused by excipients can be avoided. 9) In addition, n...

Epidermal growth factor receptor (EGFR) and ErbB2 are currently recognized as validated target molecules in cancer treatment strategies. MP-412 (AV-412) is a potent dual inhibitor of EGFR and ErbB2 tyrosine kinases, including the mutant EGFRL858R,T790M , which is clinically resistant to the EGFR-specific kinase inhibitors erlotinib and gefitinib. In an enzyme assay, MP-412 inhibited the EGFR variants and ErbB2 in the nanomolar range with over 100-fold selectivity compared with other kinases, apart from abl and flt-1, which were both moderately sensitive to the compound. E pidermal growth factor receptor (EGFR) and ErbB2 are members of the ErbB receptor family of type I tyrosine kinases that are overexpressed in various human solid cancers, and are often associated with malignancy as well as poor clinical outcomes.(1,2) Nowadays, both receptors have been recognized as being among the most attractive and common targets for cancer therapy. Regarding small-molecule compounds, the selective EGFR tyrosine kinase inhibitors (TKI) erlotinib and gefitinib showed objective response rates of 9-18% in previously treated or untreated advanced non-small cell lung cancer (NSCLC) in their clinical trials, and have been approved for clinical use. (3,4) Recently, somatic mutations in the EGFR gene were identified in a subset of NSCLC patients and were found to be associated with sensitivity to erlotinib and gefitinib. (5,6) In particular, a deletion in exon 19 and a L858R substitution in exon 21 accounted for approximately 85% of reported sensitizing mutations. Importantly, a T790M point mutation in exon 20 was found in approximately 50% of patients with NSCLC who relapsed after treatment with EGFR TKI.(7) Therefore, examining mutations seems useful for the response prediction of TKI, and at the same time, acquired resistance evokes potential limits of TKI efficacy. There are several reports demonstrating that the irreversible EGFR inhibitors CL-387 785 and HKI-272 are effective against the T790M mutant in vitro. (8,9) Epidermal growth factor receptor forms a heterodimer with ErbB2 to amplify its signal transduction.(10) This crosstalk signaling after heterodimerization elicits synergistic mitogenicity and invasiveness, whereas EGFR homodimerization does not. (11)(12)(13) In addition to this biological implication, there is clinical evidence indicating that cancers overexpressing both receptors have a worse outcome than those overexpressing either receptor alone. (14 -17) These observations suggest that a dual inhibitor of EGFR and ErbB2 should provide benefits to patients suffering from cancers expressing both receptors rather than either one.We have developed a new dual EGFR and ErbB2 inhibitor, MP-412 (AV-412), possessing activity against EGFR L858R,T790M in both enzyme assays and in vitro. This compound showed significant antitumor effects on both EGFR-and ErbB2-overexpressing cancer xenografts, including cells resistant to gefitinib. Materials and MethodsChemicals. MP-412 and gefitinib, synthesized at Mitsubishi Pharma (Y...