Hsp90 is an ATP-dependent molecular chaperone, which helps in folding its client proteins, e.g., Bcr-Abl, FLT-3, c-Raf and Akt, into active conformation. Geldanamycin analogue, 17-AAG (Kosan ...Biosciences Inc., Hayward, CA) inhibits the chaperone function of hsp90, which promotes polyubiquitylation and proteasomal degradation of the misfolded hsp90 client proteins. We recently reported that, by inhibiting the activity of histone deacetylase 6, the hydroxamate HDIs such as vorinostat (Merck & Co., Inc.) induce acetylation and inhibition of hsp90, thus also causing the depletion of its client proteins. In the present studies, we determined the anti-leukemia effects of the novel, highly soluble, hsp90 antagonist IPI504 (Infinity Pharmaceuticals), which, in vitro and in vivo, interconverts with 17-AAG, ± vorinostat, against human cultured or primary, wild type or mutant Bcr-Abl or mutant FLT-3 containing acute leukemia cells. Treatment with IPI504 (0.5 to 2.0 μM) for 24 to 48 hours, in a dose dependent manner, induced apoptosis of WT Bcr-Abl-expressing K562 and LAMA-84 cells. This was associated with attenuation of the levels of Bcr-Abl, pCrkL, pSTAT5, c-Raf and pAkt. In a dose dependent manner (50 to 500 nM for 48 hours), IPI504 also induced apoptosis of FLT-3 internal tandem duplication (ITD)-containing human acute leukemia MV4-11 cells, which was associated with attenuation of the levels of FLT-3, pAkt, pSTAT5, pERK1/2. Notably, treatment with IPI504 induced similar level of apoptosis of mouse bone marrow BaF3 cells, which had been transformed and rendered IL-3 independent for growth by ectopic expression of WT Bcr-Abl, its P-loop (Bcr-Abl-E255K) or highly imatinib mesylate (IM) resistant, contact-inhibition (Bcr-Abl-T315I) point mutant. This was also associated with attenuation of the levels of WT and mutant Bcr-Abl-E255K or Bcr-Abl-T315I. In previous studies we had demonstrated that treatment with vorinostat depletes WT and mutant Bcr-Abl levels and induces apoptosis of expressing human leukemia cells. Therefore, we determined the effect of the co-treatment of IPI504 (1.0 μM) and vorinostat (1.0 μM) against cultured or primary human CML cells. Co-treatment with IPI504 and vorinostat induced significantly more apoptosis of K562 and MV4-11 cells, which was associated with more depletion of WT-Bcr-Abl and FLT-3-ITD levels in K562 and MV4-11 cells, respectively. Notably, co-treatment with IPI504 and vorinostat, versus treatment with either agent alone, also induced more apoptosis of primary CML cells (4 samples) derived from patients with IM-resistant CML, including a sample of cells documented to have Bcr-Abl-T315I mutation. Additionally, as compared to treatment with either agent alone, the combination of IPI504 and vorinostat also induced more apoptosis of primary AML cells (4 samples), including two samples that contained FLT-3-ITD. These findings demonstrate that the combination of IPI504 with vorinostat exerts a high level of in vitro activity against FLT-3-ITD-containing acute leukemia, as well as against highly IM-resistant mutant Bcr-Abl-expressing leukemia cells.
IPI-504 is a novel, water-soluble Heat Shock Protein 90 (Hsp90) inhibitor. It is the chemical reduction product of 17-AAG, a well-characterized Hsp90 inhibitor. 17-AAG is currently in clinical ...trials, but suffers from very poor aqueous solubility and must be administered to patients in either suboptimal DMSO formulations, or newer cremophor or emulsion formulations. IPI-504, however, exists as a hydrochloride salt which is soluble in water in excess of 100 mg/mL. It has previously been shown that IPI-504 inter-converts with 17-AAG and exists in a pH and enzyme-mediated dynamic redox equilibrium. When IPI-504 or 17-AAG is administered to mice, rats, or monkeys a dynamic equilibrium occurs in vivo in which both species are present in plasma as well as tissue. Thus, IPI-504 recapitulates all of the Hsp90 inhibitory properties and biological activity of 17-AAG without its formulation liabilities. In preclinical studies IPI-504 has demonstrated in vitro and in vivo anti-tumor effects in a variety of cancers including xenograft and orthotopic models of multiple myeloma. Therefore an open-label phase I dose-escalation trial of IPI-504 infused on days 1, 4, 8 and 11 of a 21 day cycle is being conducted in pts with relapsed/refractory MM. IPI-504 is infused in 250cc of normal saline over thirty minutes. Pts receive IPI-504 for one cycle of treatment. The primary objectives of the study are to determine the maximum tolerated dose (MTD) and characterize the pharmacokinetic (PK) and pharmacodynamic (PD) marker effects of IPI-504. An accelerated titration design was utilized during the first three dose levels. Dose limiting toxicity (DLT) is defined as grade 4 or greater hematologic toxicity and/or grade 3 or greater non-hematologic toxicity within the first 21 days of treatment. Results demonstrate that the in vivo dynamic equilibrium of IPI-504 and 17-AAG is detectable in human plasma PK samples and the water-based formulation is well-tolerated. Data on PK, dose proportionality, safety, tolerability, PD markers, and anti-tumor activity of IPI-504 will be reported. In conclusion, clinical evaluation of this agent is ongoing to define the safety, tolerability and potential activity of IPI-504 to treat pts with advanced MM. These results may help clarify the effects of Hsp90 inhibition using a water-soluble, ansamycin-based Hsp90 inhibitor without the confounding effects of DMSO-based formulations.
IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound ...17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.
Osteoporosis is characterized by low bone mineral density (BMD) and fragility fracture and affects over 200 million people worldwide. Bone quality describes the material properties that contribute to ...strength independently of bone mineral density, and its quantitative analysis is a major priority in osteoporosis research. Tissue mineralization is a fundamental process requiring calcium and phosphate transporters. Here we identify impaired bone quality and strength in Slc20a2-/- mice lacking the phosphate transporter SLC20A2. Juveniles had abnormal endochondral and intramembranous ossification, decreased mineral accrual and short stature. Adults exhibited only small reductions in bone mass and mineralization but a profound impairment of bone strength. Bone quality was severely impaired in Slc20a2-/- mice: yield load (-2.3 SD), maximum load (-1.7 SD), and stiffness (-2.7 SD) were all below values predicted from their bone mineral content as determined in a cohort of 320 wild-type controls. These studies identify Slc20a2 as a physiological regulator of tissue mineralization and highlight its critical role in the determination of bone quality and strength. This article is protected by copyright. All rights reserved
La sous-traitance est une opération économique répandue, qui fait l’objet d’un traitement juridique qui mériterait peut-être d’être repensé et rationalisé. Tout d’abord, la loi relative à la ...sous-traitance a certes posé le cadre général de l’opération juridique. Toutefois, les contextes juridique et économique ont tous deux grandement évolué depuis les années 1975, ce qui conduit à s’interroger sur l’adaptation des règles alors posées. En outre, et plus encore, les obligations se sont largement densifiées, de même que le recours à la sous-traitance s’est accru. Le changement est patent en termes de volume normatif, et la loi Sapin 2 du 9 décembre 2016 en est une illustration, mais cet accroissement n’en est pas le seul reflet. La mutation de la source, avec l’intégration dans le droit dit « dur » d’un droit plus « souple », a également mené au renouvellement du corpus de règles à observer, à leur teneur, ce qui pose des difficultés de mise en œuvre. En outre, la place du risque dans la société contemporaine, qu’il prenne sa source dans le contrat, ou dans le fait juridique, oblige à repenser les techniques qui l’anticipent et permettent sa prévention, ou son traitement.
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