Intervention with the Hsp90 Modulator KU-32 Improves Chronic Experimental Diabetic Neuropathy

Abstract

Inducing the heat shock response (HSR) through Hsp90 inhibition augments heat shock protein (Hsp) support and may improve several aspects of neurodegenerative phenotypes. Several Hsps serve as molecular chaperones that assist in the folding of nascent polypeptides (client proteins) into their mature conformations. They also act as intracellular triage units that refold damaged proteins, stabilize protein complexes, solubilize protein aggregates, and help clear irreparable proteins. A confounding issue surrounding Hsp90 inhibitors is their inability to generate therapeutic windows that dissociate cytotoxic client protein degradation from HSR induction. Our novel C-terminal Hsp90 inhibitor, KU-32, induces the HSR while divesting client protein degradation, thus expanding the dose range for neuroprotection. After 16 weeks of streptozotocin (STZ)-induced Type 1 diabetes in Swiss-Webster mice, KU-32 was intraperitoneally injected weekly at a dose of 20 mg/kg KU-32 (~ 43 mM Captisol/saline vehicle) for 10 weeks. Untreated diabetic mice developed significant reductions in motor and sensory nerve conduction velocities and worsening thermal and mechanical hypoalgesia. KU-32 intervention time-dependently restored these deficits back to untreated non-diabetic levels, without adversely affecting non-diabetic mice. Further, untreated diabetic mice exhibited a 31% reduction in hindpaw intraepidermal nerve fiber (iENF) density by 16 weeks, which remained consistent until study completion. KU-32 improved diabetic iENF density to within 11% of non-diabetic levels by 26 weeks. To assess mitochondrial function, a 96-well Extracellular Flux (XF96) Analyzer was used to measure oxygen consumption rates (OCRs) for lumbar (L4-L6) dorsal root ganglia (DRG) isolated and cultured upon study completion at 26 weeks. Treatment with the ATP synthase inhibitor oligomycin reduced diabetic OCRs by ~ 80%, indicating that diabetic DRG devote most of their basal oxygen consumption to ATP synthesis. This is over twice that of untreated non-diabetic DRG at ~ 35%. KU-32 treatment in STZ-diabetes improved OCR reductions to ~ 40%, signifying vast improvements to ATP synthesis efficiency. Upon protonophore injection, DRG from KU-32-treated diabetic mice exhibited a much higher rebound in OCRs compared to diabetes alone, suggesting possible improvements in resiliency to prolonged metabolic stress. Overall, these data suggest that the neuroprotective effects of KU-32 at more chronic stages of diabetic peripheral neuropathy (DPN) may stem from the drug’s ability to improve mitochondrial function and nerve fiber innervation. KU-32 pharmacokinetic (PK) analyses were also performed on DPN-relevant tissues to verify successful drug distribution and elimination from these tissues after intraperitoneal (IP) or oral gavage (OG) treatments. The results showed that KU-32 was rapidly absorbed and distributed to DPN-relevant tissues within 30 minutes of IP treatment and one hour of gavage. Temporal PK analyses suggested that 99.9% of KU-32 distributed to these tissues was eliminated within ~ 30 hours of treatment. This was consistent with findings from an 8 week intervention study, which showed virtually no detectable levels of KU-32 present in diabetic and non-diabetic tissues one week after final treatment. In support of the ongoing hypothesis that inducible Hsp70 is essential for KU-32’s neuroprotective effects in DPN, we showed that drug distribution to DPN-relevant tissues were indistinguishable between wild type C57BL/6 and Hsp70 KO mice. OG also increased drug elimination half-lives for all examined tissues compared to IP treatments, suggesting that OG drug delivery may beneficially increase drug exposure duration.