In Vitro, in Silico, and in Vivo Assessments of Intestinal Precipitation and Its Impact on Bioavailability of a BCS Class 2 Basic Compound.

In this study, a multipronged approach of in vitro experiments, in silico simulations, and in vivo studies was developed to evaluate the dissolution, supersaturation, precipitation, and absorption of three formulations of Compound-A, a BCS class 2 weak base with pH-dependent solubility. In in vitro 2-stage dissolution experiments, the solutions were highly supersaturated with no precipitation at the low dose but increasing precipitation at higher doses. No difference in precipitation was observed between the capsules and tablets. The in vitro precipitate was found to be noncrystalline with higher solubility than the crystalline API, and was readily soluble when the drug concentration was lowered by dilution. A gastric transit and biphasic dissolution (GTBD) model was developed to better mimic gastric transfer and intestinal absorption. Precipitation was also observed in GTBD, but the precipitate redissolved and partitioned into the organic phase. In vivo data from the phase 1 clinical trial showed linear and dose proportional PK for the formulations with no evidence of in vivo precipitation. While the in vitro precipitation observed in the 2-stage dissolution appeared to overestimate in vivo precipitation, the GTBD model provided absorption profiles consistent with in vivo data. In silico simulation of plasma concentrations by GastroPlus using biorelevant in vitro dissolution data from the tablets and capsules and assuming negligible precipitation was in line with the observed in vivo profiles of the two formulations. The totality of data generated with Compound-A indicated that the bioavailability differences among the three formulations were better explained by the differences in gastric dissolution than intestinal precipitation. The lack of intestinal precipitation was consistent with several other BCS class 2 basic compounds in the literature for which highly supersaturated concentrations and rapid absorption were also observed.

Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody.