Development of Formulations of 4-Methoxydiphenylmethane, -a Selective Augmenter of LTA4H and Evaluation of Pharmacokinetics from 3 Different Routes of Administration



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Chronic Obstructive Pulmonary Disease (COPD) is the 3rd leading cause of death in U.S.A. In U.S.A. approximately 120,000 people die from COPD each year. The enzyme leukotriene A4 hydrolase (LTA4H) has been a target for the treatment of the lung inflammation in COPD, since it is responsible for the production of leukotriene B4 (LTB4), a potent chemoattractant for neutrophils. LTA4H is an enzyme of dual activity with both epoxy hydrolytic activity and aminopeptidase activity. The epoxy hydrolytic activity of LTA4H is associated with the production of LTB4. No clinical significance of the aminopeptidase activity of LTA4H was known until recently when it was discovered that Proline-Glycine-Proline (PGP), a peptide which is a potent chemoattractant of neutrophils, is degraded by LTA4H. As a result, LTA4H has both beneficial effect that is by degrading a chemoattractant for neutrophils, PGP and detrimental effect, that is by generating another chemoattractant for neutrophils, LTB4. 4-Methoxydiphenylmethane (4-MDM) is a selective augmenter of the aminopeptidase activity of LTA4H with no effect on the epoxy hydrolytic activity. In previous works, both oral and intraperitoneal administrations of 4-MDM in cigarette smoked mice models resulted in excellent protection of lung damage from emphysema. Before clinical efficacy studies of 4-MDM can be initiated, the development of a stable formulation and characterization of the disposition and pharmacokinetics (PK) are the pre-requisites for the further translational development of the preclinically promising agent. The overall aims in this project were to 1) rationally develop a cosolvent formulation of 4-MDM, 2) identify the metabolites of 4-MDM, 3) develop and validate LC-MS/MS assays for 4-MDM, 4-Benzylphenol (4-BP, active metabolite), and 4-Benzylphenol-Glucuronide (4-BP-G), 4) Evaluate the PK and disposition of 4-MDM, 4-BP, and 4-BP-G in Sprague Dawley rat model. A rational cosolvent based formulation of 4-MDM was developed by the application of Central Composite Design (CCD). The CCD optimized formulation has a 4-MDM concentration of 3 mg/mL with 30% of PEG 400, 22% of propylene glycol, and 15% of glycerol as excipients. The formulation is stable at room temperature for at least 290 days and is non-hemolytic to rat blood, suitable for intravenous administration in addition to the oral administration. Subsequently, the formulation was used to characterize the PK parameters of 4-MDM from oral and IV administrations. Three CYP mediated metabolites of 4-MDM were identified in vitro. Among the metabolites, 4-BP is an active metabolite of 4-MDM. Subsequent in vivo experiments revealed that 4-BP undergoes extensive glucuronidation in liver. Two LC-MS/MS assay methods were developed and validated according to the FDA Guidelines for accurate quantifications of 4-MDM, 4-BP, and 4-BP-G. The quantification ranges of the assays were 5 – 400 ng/mL for 4-MDM, 10 – 800 ng/mL for 4-BP, and ~10 - ~750 ng/mL for 4-BP-G (range varied slightly due to the variation in concentrations of stock solutions prepared from batches of the in-house bio-synthesis of 4-BP-G). Oral administration of 4-MDM in cosolvent formulation in SD rats resulted in a rapid absorption with estimated absorption t1/2 of 0.46 min, and extensive first pass metabolism with an estimated bioavailability of 8.85%. One of the major elimination routes of 4-MDM was identified as CYP mediated conversion of 4-MDM to 4-BP followed by the glucuronidation of 4-BP to 4-BP-G, and subsequent elimination of 4-BP-G by biliary excretion. The individual PK parameters of 4-MDM were derived from IV administration of the cosolvent formulation in SD rats. The rate of elimination (ke) and half-life (t1/2) of 4-MDM were 0.74 ± 0.16 hr-1 and 0.96 ± 0.21 hr, respectively. Furthermore, the clearance of 4-MDM was 0.86 ± 0.04 L/hr/kg. The apparent volume of distribution (Vss) of 4-MDM was 12.01 ± 4.05 L/kg. Apart from those, inhalation administration of 4-MDM from an emulsion formulation was also evaluated in an effort to circumvent the first pass metabolism. The dose normalized exposure (AUC0-6) of 4-MDM and 4-BP in lung from inhalation administration were around 25 and 2.5 times, respectively, higher than those from the oral administration. Finally, three population PK models were developed in Phoenix NLME software to derive the population PK parameters of 4-MDM from oral, IV, and inhalation administrations. These PK parameters can be used for the prediction of future dose and dosing regimen of 4-MDM in preclinical efficacy and toxicokinetic evaluations, as well as clinical trials. In summary, a cosolvent formulation of 4-MDM was rationally developed for oral and IV administrations, and an emulsion of 4-MDM was developed to circumvent the first past metabolism, suitable for inhalation administration in the treatment of COPD. The merit of the inhalation delivery of 4-MDM was demonstrated pharmacokinetically that warrants further efficacy evaluation. The metabolic profiles of 4-MDM with active metabolite of 4-BP and 4-BP-G were characterized for IV, oral and inhalation administrations. Population pharmacokinetic models were established for 4-MDM in the three routes of administration, that enable rational designs of future preclinical efficacy and clinical trials.



4-MDM, Chronic Obstructive Pulmonary Disease (COPD), APPI-LC/MS/MS, Pharmacokinetics, Formulation, LTA4H


Portions of this document appear in: Farhan, Nashid, Sean Fitzpatrick, Yun M. Shim, Mikell Paige, and Diana Shu-Lian Chow. "Ultrapressure liquid chromatography–tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for quantification of 4-methoxydiphenylmethane in pharmacokinetic evaluation." Journal of pharmaceutical and biomedical analysis 128 (2016): 46-52.