Yukihiro Ikeda

High-throughput cocrystal slurry screening by use of in situ Raman microscopy and multi-well plate

Cocrystal has attracted much attention in order to improve poor physicochemical properties, since cocrystal former crystallize with the ionic drugs as well as nonionic drugs. Cocrystal screening was usually conducted by crystallization, slurry and co-grinding techniques, however sensitivity, cost and time for screening were limited because of issues such as dissociation of cocrystal during crystallization and cost and time required for slurry and co-grinding methods. To overcome these issues, novel high-throughput cocrystal slurry screening was developed by using in situ Raman microscope and a multi-well plate. Cocrystal screening of indomethacin was conducted with 46 cocrystal formers and potential cocrystals were prepared on a large scale for the characterization with powder X-ray diffractometry, thermal analysis, and Raman microscopy and (1)H NMR spectroscopy. Compared with the characterization of scale-up cocrystals, the cocrystal screening indicated that indomethacin structured novel cocrystals with D/L-mandelic acid, nicotinamide, lactamide and benzamide which was not obtained in the screening with crystallization technique previously reported. In addition, the screening provided not only information of cocrystal formation within a day but also information of equilibrium of cocrystal formation and polymorphic transformation in one screening. Information obtained in this screening allows effective solid form selection by saving cost and time for the development.

Characterization and evaluation of miconazole salts and cocrystals for improved physicochemical properties.

Miconazole salts and cocrystals were studied to improve the physicochemical properties of miconazole. Maleate, hemifumarate, and hemisuccinate were prepared and characterized by powder X-ray diffractometry, differential scanning calorimetry, and single crystal X-ray diffractometry. The intrinsic dissolution rate and stability of each miconazole crystal form were compared to those of freebase and nitrate to evaluate the optimal crystal form. Crystal structure analysis indicated that maleate was a salt formed by proton transfer from the acid to the imidazole group of miconazole. Hemifumarate and hemisuccinate were determined to be cocrystals formed by hydrogen bonding between the acids and the base in their crystal lattices. Intrinsic dissolution tests showed that the formation of salts and cocrystals improved the dissolution rate of miconazole. Stability tests of preliminary formulations prepared with each crystal form indicated that maleate and hemifumarate were unstable at 80°C and generated a specific degraded product, i.e., a Michael adduct, between miconazole and the acids. Hemisuccinate had a superior intrinsic dissolution rate and stability, and is thus considered a promising crystal form of miconazole.

Establishment of cocrystal cocktail grinding method for rational screening of pharmaceutical cocrystals.

Cocrystals (CCs) used in the pharmaceutical industry are defined as complex crystals formed by reaction between an API and a cocrystal former (CCF); unlike salts, CCs do not show proton transfer. Recently, pharmaceutical CCs have been used to improve the drug-likeness of APIs, such as solubility and stability. Grinding is more effective for CC synthesis than crystallization from solution because in the former case, the API can predominantly interact with the CCF without being affected by solvents. However, this method is tedious because the API is ground with only one CCF at a time. We developed a cocktail cocrystal grinding (CCG) method, in which a mixture of CCFs having the same functional group was used. No false negatives/positives were observed in CCG when carbamazepine was used as the model compound. This method could be used to obtain CCs of piroxicam and spironolactone. False negatives were observed for only one compound from among three model compounds, indicating that CCG facilitates efficient CC detection and that it has higher throughput than does the conventional method. Further, CCG is fast and suitable for rational CC screening, and it helps identify the partial structure of CCFs that forms synthons with an API.

Pharmacologic profiles of investigational kisspeptin/metastin analogues, TAK-448 and TAK-683, in adult male rats in comparison to the GnRH analogue leuprolide.

Kisspeptin/metastin, a hypothalamic peptide, plays a pivotal role in controlling gonadotropin-releasing hormone (GnRH) neurons, and we have shown that continuous subcutaneous administration of kisspeptin analogues suppresses plasma testosterone in male rats. This study examined pharmacologic profiles of investigational kisspeptin analogues, TAK-448 and TAK-683, in male rats. Both analogues showed high receptor-binding affinity and potent and full agonistic activity for rat KISS1R, which were comparable to natural peptide Kp-10. A daily subcutaneous injection of TAK-448 and TAK-683 (0.008-8μmol/kg) for consecutive 7 days initially induced an increase in plasma luteinizing hormone and testosterone levels; however, after day 7, plasma hormone levels and genital organ weights were reduced. Continuous subcutaneous administrations of TAK-448 (≥10pmol/h, ca. 0.7nmol/kg/day) and TAK-683 (≥30pmol/h, ca. 2.1nmol/kg/day) induced a transient increase in plasma testosterone, followed by abrupt reduction of plasma testosterone to castrate levels within 3-7 days. This profound testosterone-lowering effect was sustained throughout 4-week dosing periods. At those dose levels, the weights of the prostate and seminal vesicles were reduced to castrate levels. These suppressive effects of kisspeptin analogues were more rapid and profound than those induced by the GnRH agonist analogue leuprolide treatment. In addition, TAK-683 reduced plasma prostate specific antigen (PSA) in the JDCaP androgen-dependent prostate cancer rat model. Thus, chronic administration of kisspeptin analogues may hold promise as a novel therapeutic approach for suppressing reproductive functions and hormone-related diseases such as prostate cancer. Further studies are warranted to elucidate clinical significance of TAK-448 and TAK-683.

Design and synthesis of pyrrolo[3,2-d]pyrimidine HER2/EGFR dual inhibitors: improvement of the physicochemical and pharmacokinetic profiles for potent in vivo anti-tumor efficacy.

During the course of our studies on a novel HER2/EGFR dual inhibitor (TAK-285), we found an alternative potent pyrrolo[3,2-d]pyrimidine compound (1a). To enhance the pharmacokinetic (PK) profile of this compound, we conducted chemical modifications into its N-5 side chain and conversion of the chemically modified compounds into their salts. Among them, 2cb, the tosylate salt of compound 2c, showed potent HER2/EGFR kinase inhibitory activity (IC(50): 11/11 nM) and cellular growth inhibitory activity (BT-474 cell GI(50): 56 nM) with a good drug metabolism and PK (DMPK) profile. Furthermore, 2cb exhibited significant in vivo antitumor efficacy in both mouse and rat xenograft models with transplanted 4-1ST gastric cancer cell lines (mouse, T/C=0%, 2cb po bid at 100 mg/kg; rat, T/C: -1%, 2cb po bid at 25 mg/kg).

A novel solubilization technique for poorly soluble drugs through the integration of nanocrystal and cocrystal technologies.

The aim of the present study was to develop a novel solubilization technique consisting of a nano-cocrystal suspension by integrating cocrystal and nanocrystal formulation technologies to maximize solubilization over current solubilizing technologies. Monodisperse carbamazepine-saccharin, indomethacin-saccharin, and furosemide-caffeine nano-cocrystal suspensions, as well as a furosemide-cytosine nano-salt suspension, were successfully prepared with particle sizes of less than 300nm by wet milling with the stabilizers hydroxypropyl methylcellulose and sodium dodecyl sulfate. Interestingly, the properties of resultant nano-cocrystal suspensions were dramatically changed depending on the physicochemical and structural properties of the cocrystals. In the formulation optimization, the concentration and ratio of the stabilizers also influenced the zeta potentials and particles sizes of the resultant nano-cocrystal suspensions. Raman spectroscopic analysis revealed that the crystalline structures of the cocrystals were maintained in the nanosuspensions, and were physically stable for at least one month. Furthermore, their dissolution profiles were significantly improved over current solubilization-enabling technologies, nanocrystals, and cocrystals. In the present study, we demonstrated that nano-cocrystal formulations can be a new promising option for solubilization techniques to improve the absorption of poorly soluble drugs, and can expand the development potential of poorly soluble candidates in the pharmaceutical industry.

Discovery of novel and potent orally active calcium-sensing receptor antagonists that stimulate pulselike parathyroid hormone secretion: synthesis and structure-activity relationships of tetrahydropyrazolopyrimidine derivatives.

As part of our research for novel calcium-sensing receptor (CaSR) antagonists that can function as oral bone anabolic agents, we recently reported the discovery of a tetrahydropyrazolopyrimidine derivative featuring adamantyl group 1b with potent CaSR antagonistic activity. To explore the potential of this calcilytic congener, we introduced the gem-dialkyl benzyl group at the 3-position of the tetrahydropyrazolopyrimidine ring, forming a bioisostere of the adamantyl group by mimicking the adamantyl groups lipophilicity and bulkiness. Optimization directed toward the improvement of solubility and metabolic stability led to the discovery of compound 9e, which stimulated transient PTH secretion when orally administered to normal rats. Further, compound 9e proved to be fully effective in an osteopenic ovariectomized rat model.

Physicochemically and pharmacokinetically stable nonapeptide KISS1 receptor agonists with highly potent testosterone-suppressive activity.

Modifications of metastin(45-54) produced peptide analogues with higher metabolic stability than metastin(45-54). N-terminally truncated nonapeptide 4 ([D-Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46-54)) is a representative compound with both potent agonistic activity and metabolic stability. Although 4 had more potent testosterone-suppressant activity than metastin, it possessed physicochemical instability at pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn48 and Ser49; substitution of Ser49 with Thr49 reduced this instability and maintained KISS1 receptor agonistic activity. Furthermore, [D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54) (14) showed 2-fold greater [Ca2+]i-mobilizing activity than metastin(45-54) and an apparent increase in physicochemical stability. N-terminal acetylation of 14 resulted in the most potent analogue, 22 (Ac-[D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54)). With continuous administration, 22 possessed 10-50-fold more potent testosterone-suppressive activity in rats than 4. These results suggested that a controlled release of short-length KISS1 receptor agonists can suppress the hypothalamic-pituitary-gonadal axis and reduce testosterone levels. Compound 22 was selected for further preclinical evaluation for hormone-dependent diseases.

Enhanced pulmonary absorption of poorly soluble itraconazole by micronized cocrystal dry powder formulations

&NA; Micronized cocrystal powders and amorphous spray‐dried formulations were prepared and evaluated in vivo and in vitro as pulmonary absorption enhancement formulations of poorly soluble itraconazole (ITZ). ITZ cocrystals with succinic acid (SA) or l‐tartaric acid (TA) with a particle size diameter of <2 &mgr;m were successfully micronized using the jet‐milling system. The cocrystal crystalline morphologies observed using scanning electron microscopy (SEM) suggested particle shapes that differed from those of the crystalline or spray‐dried amorphous ITZ. The micronized ITZ cocrystal powders showed better intrinsic dissolution rate (IDR) and pulmonary absorption profile in rats than that of the amorphous spray‐dried formulation and crystalline ITZ with comparable particle sizes. Specifically, in rat pharmacokinetic studies following pulmonary administration, micronized ITZ‐SA and ITZ‐TA cocrystals showed area under the curve from 0 to 8 h (AUC0–8h) values approximately 24‐ and 19‐fold higher than those of the crystalline ITZ and 2.0‐ and 1.6‐fold higher than the spray‐dried ITZ amorphous values, respectively. The amorphous formulation appeared physically instable during the studies due to rapid crystallization of ITZ, which was its disadvantage compared to the crystalline formulations. Therefore, this study demonstrated that micronized cocrystals are promising formulations for enhancing the pulmonary absorption of poorly soluble compounds. Graphical abstract Figure. No caption available.

Novel and potent calcium-sensing receptor antagonists: Discovery of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate (TAK-075) as an orally active bone anabolic agent

The calcium-sensing receptor antagonist (CaSR) has been recognized as a promising target of anabolic agents for treating osteoporosis. In the course of developing a new drug candidate for osteoporosis, we found tetrahydropyrazolopyrimidine derivative 1 to be an orally active CaSR antagonist that stimulated transient PTH secretion in rats. However, compound 1 showed poor physical and chemical stability. In order to work out this compounds chemical stability and further understand its in vivo efficacy, we focused on modifying the 2-position of the tetrahydropyrazolopyrimidine. As a result of chemical modification, we discovered (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate 10m (TAK-075), which showed improved solubility, chemical stability, and in vivo efficacy. Furthermore, we describe that evaluating the active metabolite is important during repeated treatment with short-acting CaSR antagonists.