The HSP90 Inhibitor Ganetespib Alleviates Disease Progression and Augments Intermittent Cyclophosphamide Therapy in the MRL/lpr Mouse Model of Systemic Lupus Erythematosus

<div><p>Systemic lupus erythematosus (SLE) is a complex, systemic autoimmune disease with a diverse range of immunological and clinical manifestations. The introduction of broad spectrum immunosuppressive therapies and better management of acute disease exacerbations have improved outcomes for lupus patients over recent years. However, these regimens are burdened by substantial toxicities and confer significantly higher risks of infection, thus there remains a significant and unmet medical need for alternative treatment options, particularly those with improved safety profiles. Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone that acts as an important modulator of multiple innate and adaptive inflammatory processes. Of note, accumulating clinical and experimental evidence has implicated a role for HSP90 in the pathogenesis of SLE. Here we evaluated the potential of HSP90 as a therapeutic target for this disease using the selective small molecule inhibitor ganetespib in the well-characterized MRL/lpr autoimmune mouse model. In both the prophylactic and therapeutic dosing settings, ganetespib treatment promoted dramatic symptomatic improvements in multiple disease parameters, including suppression of autoantibody production and the preservation of renal tissue integrity and function. In addition, ganetespib exerted profound inhibitory effects on disease-related lymphadenopathy and splenomegaly, and reduced pathogenic T and B cell lineage populations in the spleen. Ganetespib monotherapy was found to be equally efficacious and tolerable when compared to an effective weekly dosing regimen of the standard-of-care immunosuppressive agent cyclophosphamide. Importantly, co-treatment of ganetespib with a sub-optimal, intermittent dosing schedule of cyclophosphamide resulted in superior therapeutic indices and maximal disease control. These findings highlight the potential of HSP90 inhibition as an alternative, and potentially complementary, strategy for therapeutic intervention in SLE. Such approaches may have important implications for disease management, particularly for limiting or preventing treatment-related toxicities, a major confounding factor in current SLE therapy.</p></div

Ganetespib prevents the development of renal damage in a pilot prophylactic dosing study.

<p>(A) Mean pharmacokinetic plasma concentration-time profile after i.v. administration of ganetespib to MRL/lpr mice. Ganetespib was dosed at 50 mg/kg twice weekly and samples were taken after the second dose. Data represent mean ± SD (n = 3). (B) Average proteinuria scores (±SE) are plotted for vehicle and ganetespib-treated mice over the time course of the study. Average proteinuria scores for non-lupus parental strain (MRL/mp) mice from 8–15 weeks of age are included for comparison. (C) HSP70 induction as a pharmacodynamic marker of ganetespib activity in kidneys from drug-treated animals. Kidneys were harvested at 6 and 24 hours following the second intra-weekly dose of ganetespib (or vehicle) and lysates immunoblotted for HSP70 expression. GAPDH included as a loading control. Each lane represents renal tissue from an individual animal. (D) Representative H&E stained renal histopathology showing extensive chronic inflammation and thickening of the basement membrane with crescent formation in the glomerulus of a vehicle-treated mouse (<i>left panel</i>). In contrast, the kidneys of a ganetespib-treated animal showed no evident pathologic changes (<i>right panel</i>). Original magnification, 40X; scale bar represents 50 μm. (E) To evaluate total lupus-related renal damage 7 primary characteristics of GN (glomerular capillary deposits, hypercellularity, necrosis, tubular atrophy, intratubular casts, interstitial chronic inflammation, and fibrosis) were scored for each animal and summed for a composite disease score. The average summed pathology scores for each treatment group are plotted. (E) Representative IgG immunohistochemical staining of renal tissues harvested from vehicle- and ganetespib-treated animals at 22 weeks of age. Red arrows highlight the marked reduction in glomerular IgG deposition seen following HSP90 inhibitor treatment. Original magnification 20X: scale bar represents 100 μm. (F) Quantification of IgG glomerular immunoreactivity observed in vehicle and drug treated animals.</p

Absence of lymphoproliferation in combination-treated MRL/lpr mice.

<p>(A) Lymph nodes were harvested from mice upon completion of the individual therapeutic dosing regimens. Data are expressed as median pooled lymph node weights for each treatment (± SD). *p<0.05. <i>Gpib</i>, <i>ganetespib; Comb</i>, <i>combination</i>. (B) Representative images of regional lymph nodes harvested from vehicle- and combination (ganetespib + CTX/2)-treated animals at the end of the study. (C) Spleens were harvested at necropsy and weighed. Data are expressed as median spleen weights per treatment group (± SD).*p<0.05. (D) Representative images of spleens taken from mice from all treatment groups.</p

Ganetespib prevents the development of renal damage in a pilot prophylactic dosing study.

<p>(A) Mean pharmacokinetic plasma concentration-time profile after i.v. administration of ganetespib to MRL/lpr mice. Ganetespib was dosed at 50 mg/kg twice weekly and samples were taken after the second dose. Data represent mean ± SD (n = 3). (B) Average proteinuria scores (±SE) are plotted for vehicle and ganetespib-treated mice over the time course of the study. Average proteinuria scores for non-lupus parental strain (MRL/mp) mice from 8–15 weeks of age are included for comparison. (C) HSP70 induction as a pharmacodynamic marker of ganetespib activity in kidneys from drug-treated animals. Kidneys were harvested at 6 and 24 hours following the second intra-weekly dose of ganetespib (or vehicle) and lysates immunoblotted for HSP70 expression. GAPDH included as a loading control. Each lane represents renal tissue from an individual animal. (D) Representative H&E stained renal histopathology showing extensive chronic inflammation and thickening of the basement membrane with crescent formation in the glomerulus of a vehicle-treated mouse (<i>left panel</i>). In contrast, the kidneys of a ganetespib-treated animal showed no evident pathologic changes (<i>right panel</i>). Original magnification, 40X; scale bar represents 50 μm. (E) To evaluate total lupus-related renal damage 7 primary characteristics of GN (glomerular capillary deposits, hypercellularity, necrosis, tubular atrophy, intratubular casts, interstitial chronic inflammation, and fibrosis) were scored for each animal and summed for a composite disease score. The average summed pathology scores for each treatment group are plotted. (E) Representative IgG immunohistochemical staining of renal tissues harvested from vehicle- and ganetespib-treated animals at 22 weeks of age. Red arrows highlight the marked reduction in glomerular IgG deposition seen following HSP90 inhibitor treatment. Original magnification 20X: scale bar represents 100 μm. (F) Quantification of IgG glomerular immunoreactivity observed in vehicle and drug treated animals.</p

Ganetespib-based therapies suppress splenic expansion of activated T cells and abnormal plasma cells.

<p>(A) Plot shows the total number of splenocytes. Data are shown for 7–12 mice per treatment group. (*p<0.007; **p<0.0037; ***p<0.0012 versus vehicle-treated animals). (B) Splenocytes were isolated, counted and analyzed for the total number of CD3⁺CD4^-CD8^- T cells (<i>left panel</i>; *p<0.0001, **p<0.0004) or the number of mature phenotype CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes (<i>right panel</i>; *p = 0.04, **p = 0.002). (C) Numbers of B220^-CD19^-CD38⁺CD138⁺ plasma cells (<i>left panel</i>; *p = 0.03, **p = 0.007, ***p = 0.004) and B220⁺CD19⁺ B cells (<i>right panel</i>).</p

Ganetespib degrades kinases associated with T cell activation and disease pathology.

<p>(A) Splenocytes from MRL/mp mice were incubated for 24 hours with or without activation by plate-bound CD3 + CD28 in the presence or absence of 100nM ganetespib. Cells were lysed and levels of the indicated kinases were determined by Western blot. (B) Relative expression levels for each protein were normalized to GAPDH using densitomery. Culture conditions: <i>rest</i>, resting (unactivated); <i>act</i>, activated; <i>act + g</i>, activated plus ganetespib.</p