Evaluation of Thermal Support during Anesthesia Induction on Body Temperature in C57BL/6 and Nude Mice.

Heat supplementation during surgery is a common practice; however, thermal support is not commonly used during anesthesia induction. Mice lose body temperature quickly, and air movement can exacerbate this, potentially putting mice at a thermal deficit before surgery. Whether the method of warming during induction affects overall heat loss during anesthesia is unknown. We hypothesized that the method of heating would affect body temperature (Tb) during anesthesia induction, maintenance, recovery, and once placed back on the rack. Mice (C57BL/6NHsd-6M/6F [C57BL/6]; Hsd:Athymic Nude-Foxn1nu [Nude]; N = 24;12M/12F) were assigned to a treatment in a factorial design: thermal chamber (TC; ambient temperature [Ta] = 28.8°C); heating pad (HP; induction chamber placed on an electric heating pad;Ta = 28.4°C); and control (Ctrl; Ta = 21.6°C). During induction, one mouse at a time was anesthetized with isoflurane over a 3min period and then maintained under anesthesia for 10min on a hot water heating pad (33 °C). Then isoflurane was stopped and time to ambulation was recorded. Tb and activity were tracked in the home cage on the rack before and after anesthesia. During induction, Ctrl mice lost significantly more heat (-2.8 °C) than did TC (+0.2 °C) and HP mice (+0.1 °C) but TC and HP were not different. During anesthesia maintenance, Ctrl mice regained 1 °C, but their Tb was still lower than that of the treated groups. Nude mice consistently had a lower Tb than C57BL/6 mice, regardless of treatment or anesthesia phase. C57BL/6 Ctrl mice took longer to ambulate than either HP or TC mice, but the method of heating did not differentially affect Nude mice. In general, C57BL/6 as compared with Nude and females as compared with males were comparatively more active and had higher Tb during certain times of day, regardless of the heating methods. Overall, our findings support the provision of heat during anesthesia induction, regardless of method, to reduce overall Tb loss during a short anesthesia event.

EC144 is a potent inhibitor of the heat shock protein 90.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.

Structure-based design of 2,6,7-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines as Aurora kinases inhibitors.

This Letter reports the optimization of a pyrrolopyrimidine series as dual inhibitors of Aurora A/B kinases. This series derived from a pyrazolopyrimidine series previously reported as inhibitors of aurora kinases and CDKs. In an effort to improve the selectivity of this chemotype, we switched to the pyrrolopyrimidine core which allowed functionalization on C-2. In addition, the modeling rationale was based on superimposing the structures of Aurora-A kinase and CDK2 which revealed enough differences leading to a path for selectivity improvement. The synthesis of the new series of pyrrolopyrimidine analogs relied on the development of a different route for the two key intermediates 7 and 19 which led to analogs with both tunable activity against CDK1 and maintained cell potency.

Synthesis, SAR and biological evaluation of 1,6-disubstituted-1H-pyrazolo[3,4-d]pyrimidines as dual inhibitors of Aurora kinases and CDK1.

Since the early 2000s, the Aurora kinases have become major targets of oncology drug discovery particularly Aurora-A and Aurora-B kinases (AKA/AKB) for which the selective inhibition in cells lead to different phenotypes. In addition to targeting these Aurora kinases involved in mitosis, CDK1 has been added as a primary inhibition target in hopes of enhancing the cytotoxicity of our chemotypes harboring the pyrazolopyrimidine core. SAR optimization of this series using the AKA, AKB and CDK1 biochemical assays led to the discovery of the compound 7h which combines strong potency against the 3 kinases with an acceptable microsomal stability. Finally, switching from a primary amide to a two-substituted pyrrolidine amide gave rise to compound 15a which exhibited the desired AKA/CDK1 inhibition phenotype in cells but showed moderate activity in animal models using HCT116 tumor cell lines.

Design, synthesis, and biological evaluation of pyrazolopyrimidine-sulfonamides as potent multiple-mitotic kinase (MMK) inhibitors (part I).

A novel class of pyrazolopyrimidine-sulfonamides was discovered as selective dual inhibitors of aurora kinase A (AKA) and cyclin-dependent kinase 1 (CDK1). These inhibitors were originally designed based on an early lead (compound I). SAR development has led to the discovery of potent inhibitors with single digit nM IC(50)s towards both AKA and CDK1. An exemplary compound 1a has demonstrated good efficacy in an HCT116 colon cancer xenograft model.

ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo.

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate-resistant acid phosphatase (TRAP)-ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP-ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone-resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF-κB, Erk, and Akt signaling compared with wild-type (WT) littermates. This increased bone-resorbing capacity per OCL was associated with increased levels of p-Pyk2 and p-Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF-α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF-α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease.

A SQSTM1/p62 mutation linked to Paget's disease increases the osteoclastogenic potential of the bone microenvironment.

Paget's disease of bone (PDB) is the second most common bone disease and is characterized by focal bone lesions which contain large numbers of abnormal osteoclasts (OCLs) and very active normal osteoblasts in a highly osteoclastogenic marrow microenvironment. The etiology of PDB is not well understood and both environmental and genetic causes have been implicated in its pathogenesis. Mutations in the SQSTM1/p62 gene have been identified in up to 30% of Paget's patients. To determine if p62 mutation is sufficient to induce PDB, we generated mice harboring a mutation causing a P-to-L (proline-to-leucine) substitution at residue 394 (the murine equivalent of human p62(P392L), the most common PDB-associated mutation). Bone marrow cultures from p62(P394L) mice formed increased numbers of OCLs in response to receptor activator of NF-kappaB ligand (RANKL), tumor necrosis factor alpha (TNF-alpha) or 1alpha,25-(OH)(2)D(3), similar to PDB patients. However, purified p62(P394L) OCL precursors depleted of stromal cells were no longer hyper-responsive to 1alpha,25-(OH)(2)D(3), suggesting effects of the p62(P394L) mutation on the marrow microenvironment in addition to direct effects on OCLs. Co-cultures of purified p62(P394L) stromal cells with either wild-type (WT) or p62(P394L) OCL precursors formed more OCLs than co-cultures containing WT stromal cells due to increased RANKL production by the mutant stromal cells. However, despite the enhanced osteoclastogenic potential of both OCL precursors and marrow stromal cells, the p62(P394L) mice had histologically normal bones. These results indicate that this PDB-associated p62 mutation is not sufficient to induce PDB and suggest that additional factors acting together with p62 mutation are necessary for the development of PDB in vivo.