Identification and Quantification of MIDD0301 Metabolites

Abstract

Background: MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells.

Objective: The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301.

Methods: In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration.

Results: MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma.

Conclusion: MIDD0301 undergoes no phase I and moderate phase II metabolism.

Keywords: GABAA receptor.; MIDD0301; glucosidation; glucuronidation; imaging mass spectrometry; metabolism; taurine conjugate.

Figure 1.

In vitro phase I metabolism of MIDD0301 in the presence of A) human liver S9 fraction; B) beagle dog liver microsomes; C) mouse liver microsomes; D) rat liver microsomes. All assays were performed as two independent assays with n = 3. Data are presented as averages (n = 6) with standard deviations. The percent MIDD0301 remaining at the 120-minute time point is shown on each graph.

Figure 2.

CYP3A4 inhibition assay. Recombinantly expressed CYP3A4 enzyme was incubated in the presence of NADPH, fluorescent substrate, and MIDD0301 (10 μM) or vehicle. Fluorescence intensity was determined over time and depicted as mean with standard deviation and rates determined by non-linear regression (first-order kinetics). % inhibition was defined as (1-((X-B)/(A-B)))*100, A is the rate observed for vehicle, B is the rate observed in the presence of nelfinavir.

Figure 3.

In vitro phase II metabolism of MIDD0301. Glucuronidation of MIDD0301 was determined in the presence of A) human liver S9 and B) and C) mouse liver S9. For C), peak area ratios of internal standard and MIDD0301 glucuronide were plotted versus time and first order kinetic analysis was used to determine rate constant k. Glucosidation of MIDD0301 was determined in the presence of D) mouse liver S9 and E) and F) mouse kidney S9. For F), peak area ratios of internal standard and MIDD0301 glucoside were plotted versus time and first order kinetic analysis was used to determine rate constant k. All assays were performed by LC-MS/MS as two independent assays with n = 3. Data are presented as averages (n = 6) with standard deviations.

Figure 4.

Pharmacokinetic analysis of MIDD0301. A) 25 mg/kg by oral gavage; B) 25 mg/kg by IP injection; C) 1 mg/kg by tail vein injection. Animals were sacrificed at indicated time points and concentrations of MIDD0301 quantified by LC-MS/MS. Data are shown as means (n = 4 for oral and IP and n = 8 for IV). A two-compartment PK model was used for the analysis. Determined elimination rates were used to calculate half-lives (t_1/2).

Figure 5.

Spatial distribution of MIDD0301 in mouse lung. A) IMS image of MIDD0301 in a mouse lung section. MIDD0301 was administered orally and mouse lungs were harvested after 1 h followed by freezing, sectioning, and matrix deposition, which enabled the detection of MIDD0301 using 416 m/z to 372 m/z transition by MALDI-MS/MS. Intensity of selective fragment ions was visualized by a transition from white to black. B) Bright field image lung section at 10x magnification.

Scheme 1.

Synthesis of possible MIDD0301 phase II metabolites.