SUN-471 - Levoketoconazole: A Potentially Safer Therapeutic Profile for Cushing'S Syndrome via Reduced Toxic Deacetyl Ketoconazole (DAK) Metabolite Formation

Objective:
Levoketoconazole (LKTZ) was approved in 2021 for adults with endogenous Cushing’s syndrome (CS). Because ketoconazole (KTZ)-associated hepatotoxicity is strongly linked to its reactive metabolite, DAK, we evaluated the safety of LKTZ and KTZ by quantifying toxic DAK metabolite formation in primary human hepatocyte cultures (PHH).

Method:
PHH of 9 and 11 donors, respectively were isolated and exposed to Cmax therapeutic concentrations of LKTZ or KTZ (17.7 µg/mL). After single-dose and repeated 72-hour treatments, DAK formation was quantified by LC-MS/SIM and by LC-MS/MS using a >99% pure DAK reference standard. Liver function tests (LFTs), mitochondrial toxicity, reactive oxygen species (ROS), glutathione and intracellular ATP were independently assessed in cohort of 21 donors.

Results:
Daily exposure of PHH for 72 h resulted in intracellular glutathione loss, decreasing statistically significantly by ~44% with LKTZ and ~53% with KTZ. Both treatments lowered intracellular ATP; KTZ achieved statistical significance (p < 0.046). Median LFT were significantly reduced in the LKTZ group, indicating a shift away from amino acid metabolism toward enhanced detoxification. DAK concentrations in PHH corresponded with published human plasma levels observed after a 400 mg oral KTZ dose. Clear treatment-dependent differences emerged: mean DAK concentrations were 2.7-fold lower with LKTZ compared to KTZ following daily treatment of PHH for 72h (1.87 versus 5.01 ng/ml, p=0.003) while repeated KTZ dosing significantly amplified DAK formation from 2.59 (24h) to 5.01 ng/ml (72h), p=0.006). Based on DAK/parent drug ratios, an approximate five-fold significant increase in DAK was observed after daily KTZ treatment compared to LKTZ for 72h (0.18 ± 0.09, LTKZ versus 0.88 ± 0.66 KTZ, p=0.008). Conversely, LKTZ daily dosing for 72h caused an insignificant nearly 20% reduction in this ratio (0.22 ± 0.13 versus 0.18 ± 0.09; p=0.074).

Discussion:
Steroidogenesis inhibitors (SI) remain widely used for CS, with therapy individualized by patient factors and access. SI may prolong QT and cause hypokalemia, and KTZ, LKTZ, and metyrapone inhibit CYP3A4. Published data show DAK inhibits CYP2D6 with 13-fold greater potency than KTZ, underscoring broader safety implications given CYP2D6’s role in metabolizing roughly one-quarter of prescribed drugs. LKTZ shows superior safety due to less glutathione loss and lower, decreasing formation of the hepatotoxic metabolite. DAK drives mitochondrial dysfunction, oxidative stress, toxic protein adducts, CYP inhibition, and hepatocellular necrosis. Experimental data demonstrate that DAK levels correlate with KTZ-induced liver injury, and reducing DAK formation through its stereoselective metabolism provides a mechanistic basis for a better hepatic safety profile of LKTZ.

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