A sensitive and fast LC–MS/MS method for determination of β-receptor agonist JP-49b: Application to a pharmacokinetic study in rats

• JP-49b, beta agonist, is in preclinical development for diabetic retinopathy.
• We have developed/validated an LC–MS/MS method for JP-49b measurement.
• JP-49b was well distributed into ocular tissues after ocular administration.
• JP-49b demonstrates minimal systemic exposure after ocular administration.

Ocular administration of the beta (β)-adrenergic receptor agonist JP-49b prevents retinopathy-like damage in a preclinical rat model of diabetes. Importantly, JP-49b did not induce characteristic β-adrenergic agonist-related side effects (e.g., left ventricular damage), which led to the hypothesis that JP-49b systemic exposure was minimal following ocular administration. To test this hypothesis, a sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to study the preclinical pharmacokinetics of JP-49b in rats. Animals received either a single periocular or intravenous injection of JP-49b (10 mg/kg) and plasma and tissue samples were obtained. JP-49b and fenoterol hydrobromide (internal standard, IS) were isolated by liquid–liquid extraction and extracts were analyzed by reversed-phase liquid chromatography on a C18 column using a gradient elution (acetic acid in water and methanol). A triple quadrupole mass spectrometer operating in the positive electrospray ionization mode with multiple reaction monitoring was used to detect JP-49b and IS transitions of m/z 346.4 → 195.1 and 304.1 → 134.9. The method was validated for selectivity, linearity, accuracy, and precision in rat vitreous humor, tissue homogenates, and plasma. Following intravenous administration, JP-49b was found to have a rapid clearance (36 ± 5.8 L/h/kg), high volume of distribution (244 ± 51.5 L/kg) and a terminal half-life of 4.8 ± 1.6 h. JP-49b was rapidly absorbed and extensively distributed into ocular tissue following topical administration. However, JP-49b was undetectable in heart tissue 24 h after ocular administration. High local drug concentrations coupled with minimal systemic exposure following ocular administration supports further testing of JP-49b as a localized therapy for diabetic retinopathy.