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. 2006 Nov 14;103(46):17408-13.
doi: 10.1073/pnas.0608372103. Epub 2006 Nov 7.

Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90

Jens R Sydor  1 , Emmanuel Normant, Christine S Pien, James R Porter, Jie Ge, Louis Grenier, Roger H Pak, Janid A Ali, Marlene S Dembski, Jebecka Hudak, Jon Patterson, Courtney Penders, Melissa Pink, Margaret A Read, Jim Sang, Caroline Woodward, Yilong Zhang, David S Grayzel, Jim Wright, John A Barrett, Vito J Palombella, Julian Adams, Jeffrey K Tong
Affiliations

Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90

Jens R Sydor et al. Proc Natl Acad Sci U S A. .

Abstract

Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Representative HPLC chromatograms of a 60-min time point of IPI-504 or 17-AAG incubated with human liver microsomes. (A) IPI-504 (50 μg/ml) (dashed line) or 17-AAG (solid line) was incubated in human liver microsomes. The reactions were quenched at 60 min with an equal volume of 2:1 methanol:270 mM citrate (pH 3), 0.5% (wt/vol) EDTA, and 0.5% (wt/vol) ascorbate to preserve IPI-504 and analyzed by HPLC (λ = 230 nm). (B) IPI-504 (50 μg/ml) was incubated with 0.8 mg/ml human liver microsomes for 0 (black line) and 60 min (red line), as well as for 60 min without NADP as a control reaction (blue line). The reaction was quenched by addition of an equal volume of 2:1 methanol:270 mM citrate (pH 3), 0.5% (wt/vol) EDTA, and 0.5% (wt/vol) ascorbate to preserve IPI-504 and analyzed by HPLC (λ = 230 nm).
Fig. 2.
Fig. 2.
Percentage of total peak area of IPI-504 and 17-AAG at different time points of an incubation of IPI-504 (A) or 17-AAG (B) in human liver microsomes. IPI-504 or 17-AAG (50 μg/ml) was incubated with 0.8 mg/ml human liver microsomes and quenched at incremental time points by addition of an equal volume of 2:1 methanol:270 mM citrate (pH 3), 0.5% (wt/vol) EDTA, and 0.5% (wt/vol) ascorbate. The levels of IPI-504 and 17-AAG were analyzed by HPLC and plotted as percentage of total peak area. Each point is the mean ± range for two samples.
Fig. 3.
Fig. 3.
Comparison of pharmacokinetics of IPI-504 and 17-AAG in mice. (A) Plasma Concentration-Time Profiles of IPI-504, 17-AAG, and 17-AG in BALB/c mice after i.v. administration of IPI-504 at 50 mg/kg. (B) Plasma concentration-time profiles of IPI-504, 17-AAG, and 17-AG in BALB/c mice after i.v. administration of 17-AAG at 50 mg/kg. Test articles were administered via the tail vein, plasma was collected at selected time points and quenched to stabilize the hydroquinone IPI-504, and samples were analyzed by online extraction LC-MS/MS (see Materials and Methods).
Fig. 4.
Fig. 4.
Tumor retention and efficacy in MM xenograft models. (A) Selective tumor retention of IPI-504 in RPMI-8226 tumor-bearing mice after i.v. administration of 50 mg/kg IPI-504. RPMI-8226 tumor-bearing mice were administered with test article via the tail vain, and tumor samples were collected at selected time points. Tumor samples were homogenized, and the homogenate supernatants were analyzed by online extraction LC-MS/MS. (B) In vivo efficacy of IPI-504 in an RPMI-8226 xenograft model as a single agent and in combination with bortezomib. Tumor-bearing mice received either (i) IPI-504 and bortezomib vehicle (♦), (ii) IPI-504 at 100 mg/kg (300 mg/m2), twice weekly (▴), (iii) bortezomib 0.3 mg/kg (0.9 mg/m2), twice weekly (○), or (iv) IPI-504 at 75 mg/kg (225 mg/m2) and bortezomib at 0.3 mg/kg (0.9 mg/m2), twice weekly (■), and tumor dimensions were measured twice weekly, as well as λ light chain concentrations 4 h after the last dose. (Inset) Point A, IPI-504 75 mg/kg and bortezomib 0.3 mg/kg; point B, bortezomib 0.3 mg/kg; point C, IPI-504 100 mg/kg; and point D, vehicle).
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