Enzyme phenotyping of hydroxyfasudil

ABSTRACT A subarachnoid hemorrhage (SAH) is a devastating neurocritical emergency characterized by the extravasation of blood into the subarachnoid space surrounding the brain. Aside from SAH caused by trauma, most cases of SAH (85%) are caused by a spontaneous rupture of an aneurysm located on a cerebral artery. Approximately 10-15% of individuals who experience an SAH will not survive to reach hospital. At the hospital, the rupture is secured either surgically or endovascularly. However, 2-3 days after the rupture, the spilt blood in the subarachnoid space degrades into inflammatory by-products that cause cerebral arteries to constrict. This constriction is called “cerebral vasospasm,” and is a leading cause of death and disability after SAH. The only treatment ever approved in the US and Europe for cerebral vasospasm is nimodipine, a calcium- channel blocker. However, even with nimodipine, SAH has a cumulative death rate of 18% within the first 48 hours; 22% by the first week; 33% by the first month; and 43% by the first year; with permanent deficits in 75% of the remaining survivors In contrast, Japan has never approved nimodipine. For almost three decades, the Japanese treatment for vasospasm has been a combination of fasudil (a rho-kinase (“ROCK”) inhibitor) and ozagrel (a thromboxane A2 synthesis inhibitor). Neither fasudil nor ozagrel ever have been studied or approved for SAH in the US or EU. A 2022 observational study from China demonstrated that patients who received fasudil in combination with nimodipine demonstrated statistically significant improvements of 120% and 145% respectively over nimodipine alone on well-established measures of cognition (Montreal Cognitive Assessment Score) and functional independence (Barthel Index). However, to establish a causal relationship between the addition of fasudil with the observed improvements, a randomized trial will be required. Before such a randomized trial can begin, both the FDA and EMA require us to identify all metabolizing enzymes responsible for ≥25% of the elimination of both fasudil and any fasudil metabolite expected to account for ≥50% of the parent drug’s in vivo pharmacological effect. The metabolizing enzyme for fasudil already is known, but the metabolizing enzymes for fasudil’s active metabolite (hydroxyfasudil (“HF”)) remain unidentified. In this Phase I SBIR, our Specific Aims will generate the in vitro enzyme-phenotyping data on HF required by the FDA and EMA. Specific Aim 1 quantifies HF’s metabolism from all metabolizing enzymes; Specific Aim 2 characterizes the contribution of Phase I CYP enzymes to HF’s total metabolism; and Specific Aim 3 then characterizes the contribution of Phase II UGT enzymes to HF’s metabolism. Successful conclusion of this program will satisfy the enzyme-phenotyping requirements to begin controlled trials.