Tubulin Hyper-Acetylation In Blood Lymphocytes: Pharmacodynamic (PD) Biomarker For The Selective Histone Deacetylase (HDAC) 6 Inhibitor ACY-1215 In Multiple Myeloma (MM) Patients

HDACs are a family of enzymes that remove acetyl groups from proteins and are involved in many key cellular processes, including apoptosis, cell cycle arrest and angiogenesis. Nonselective HDAC inhibitors has shown promise in treating MM in combination with standard therapies such as proteasome inhibitors (PIs) [1]. However, nonselective HDAC inhibitors have been of limited utility due to adverse effects. HDAC6 is a Class IIb enzyme found in the cytoplasm that is intimately involved in cytoskeletal remodeling and vesicle transport associated with degradation of misfolded protein through the regulation of the acetylation status of α-tubulin. Recent work [2, 3] suggests that selectively inhibiting HDAC6 in MM is an important therapeutic modality as part of a combination therapy with PIs and immunomodulatory agents (IMiDs) and may minimize toxicity due to Class I HDAC inhibition. ACY-1215 is a novel HDAC6-selective small molecule inhibitor that has been shown to be effective in reducing tumor burden in preclinical animal models of MM, and the anti-tumor activities correlate with increased levels of acetyl-tubulin, a HDAC6 specific substrate, in blood and tumor cells. ACY-1215 has now advanced to phase I clinical trials as a combination agent for the treatment of relapsed and refractory MM. A clinical PD biomarker for HDAC6 inhibition is critical to demonstrate that therapeutic plasma levels of ACY-1215 also increase the HDAC6 specific biomarker, acetylated tubulin, versus the Class I HDAC biomarker, acetylated histones. In addition HDAC6 inhibition is expected to be well-tolerated and dose limiting toxicity may not be reached implying continued dose escalation in phase 1 may not be warranted if there is a suitable increase in the PD HDAC6 biomarker. We describe the development of a clinical PD biomarker of HDAC6 inhibition, acetylated α-tubulin, to determine when blood levels of ACY-1215 are reached that give a specific increase in the biomarker versus acetylated histones.

Assay sensitivity, dynamic range and reproducibility have been established using peripheral whole blood from healthy donors and MM patients incubated with ACY-1215 ex vivo. In the clinic, peripheral blood is collected at up to six different PK matched time points after drug administration.Lymphocytes were assessed for tubulin and histone hyper-acetylation by flow cytometry using specific antibodies that recognize acetyl groups in the context of the specific protein targets. ACY-1215 plasma concentration was also determined in the peripheral blood by an appropriate bioanalytical method. More than 50 MM patients have been enrolled in two separate clinical trials and received escalating oral ACY-1215 doses from 40 mg to 240 mg. The ACY-100 study is composed of two parts, ACY-1215 monotherapy and combination therapy with both bortezomib and dexamethasone. A second study, ACE-MM-101, examines ACY-1215 in combination with lenalidomide and dexamethasone.

Patients in ACY-100 and ACE-MM-101 clinical trials have shown elevated tubulin hyper-acetylation in peripheral blood lymphocytes as ACY-1215 dose increases from 40 mg to 240 mg. ACY-1215 plasma concentration reaches a maximal level (C_max) 1 hr after administration. An apparent exposure plateau was reached at dose levels >160 mg where C_max reached 1.2 - 1.8 µM. Correlating to the PK levels, the PD biomarker acetyl-tubulin increases to a maximal level 1 hr after dosing compared to predose, and declines to basal level by >4 hr with an apparent lag period compared to plasma levels of ACY-1215. Acetyl-histone levels were not significantly changed at these doses indicating a predominant HDAC6 selective effect. As plasma levels increased with dose from 40 mg to 160 mg so do the number of patients that have a measurable increase in acetyl-tublin such that at dose levels ≥80 mg all patients have a measurable increase in acetylated tubulin. In conclusion the clinical PK and PD results, comparing to prior preclinical studies [4], suggest that ACY-1215 has reached a pharmacologically relevant level of HDAC6 inhibition at clinical doses ≥80 mg. These results will aid in the determination of the recommend phase 2 dose of ACY-1215 in combination with PIs and IMiDs.

Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Supko:Acetylon: Research Funding.