An integrated outsourcing practice of nonclinical LC-MS bioanalysis and toxicokinetics at Novartis small molecule drug development.

With decommissioning of internal regulated bioanalytical (BA) and toxicokinetic (TK) capabilities, Novartis has relied on external service providers (ESPs) for all nonclinical LC-MS BA and majority of the associated TK work since 2017. This paper outlines an integrated outsourcing practice of the Novartis nonclinical LC-MS BA/TK group, which covers the roles and responsibilities of Novartis nonclinical LC-MS BA/TK expert scientific monitors, selection of ESPs for Novartis nonclinical LC-MS BA/TK studies, qualification of BA/TK ESPs, study conduct and completion, ESP oversight and evaluation, issue mitigation, and future perspectives.

Compound Property Optimization in Drug Discovery Using Quantitative Surface Sampling Micro Liquid Chromatography with Tandem Mass Spectrometry.

Surface sampling micro liquid chromatography tandem mass spectrometry (SSµLC-MS/MS) was explored as a quantitative tissue distribution technique for probing compound properties in drug discovery. A method was developed for creating standard curves using surrogate tissue sections from blank tissue homogenate spiked with compounds. The resulting standard curves showed good linearity and high sensitivity. The accuracy and precision of standards met acceptance criteria of ±30%. A new approach was proposed based on an experimental and mathematical method for tissue extraction efficiency evaluation by means of consecutively sampling a location on tissue twice by SSµLC-MS/MS. The observed extraction efficiency ranged from 69% to 82% with acceptable variation for the test compounds. Good agreement in extraction efficiency was observed between surrogate tissue sections and incurred tissue sections. This method was successfully applied to two case studies in which tissue distribution was instrumental in advancing project teams' understanding of compound properties.

LC-MS/MS bioanalysis of loratadine (Claritin) in dried blood spot (DBS) samples collected by subjects in a clinical research study.

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of loratadine, an H1 histamine antagonist, in human dried blood spot (DBS) samples following a single self-administered 10 or 20mg oral dose. The samples were produced by spotting approximately 30µl of whole blood onto PE-226 cards. Two 3-mm discs were cut from the DBS samples and extracted using aqueous methanol containing the internal standard. After transfer and drying of the resulting sample extract, the reconstituted residues were chromatographed using a Waters XSelect C18 column and isocratic elution for MS/MS detection. The possible impact due to hematocrit, volume of blood sample spotted, storage temperature, and humidity, on the accuracy of measured DBS results were investigated. The results showed that only spotted blood volume might have an impact; a small volume (10µl) tended to give a larger negative bias in the measured value than the large volume ones (≥20µl). The current method was fully validated over a dynamic range of 0.200-20.0ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.990. The intra-day accuracy and precision at the LLOQ were -11.5 to 0.0% bias and 6.4 to 8.9% CV, respectively. For the other QC samples (0.600, 3.00, 10.0 and 15.0ng/ml), the precision ranged from 4.2 to 9.8% CV and from 6.3 to 8.1% CV, respectively, in the intra-day and inter-day evaluations; the accuracy ranged from -1.7 to 10.0% and 2.7 to 5.3% bias, respectively, in the intra-day and inter-day batches. Loratadine is stable in the DBS samples for at least 271 days at ambient temperature in a desiccator, for at least 24h at 60°C and under 80% relative humidity, followed by re-conditioning at ambient temperature in a desiccator. The current methodology has been applied to determine the loratadine levels in DBS samples collected by subjects in a clinical research study to evaluate pharmacokinetic sampling in point-of-care setting.

Liquid microjunction surface sampling of acetaminophen, terfenadine and their metabolites in thin tissue sections.

BACKGROUND: The aim of this work was to evaluate the analytical performance of a fully automated droplet-based surface-sampling system for determining the distribution of the drugs acetaminophen and terfenadine, and their metabolites, in rat thin tissue sections. RESULTS: The rank order of acetaminophen concentration observed in tissues was stomach > small intestine > liver, while the concentrations of its glucuronide and sulfate metabolites were greatest in the liver and small intestine. Terfenadine was most concentrated in the liver and kidney, while its major metabolite, fexofenadine, was found in the liver and small intestine. CONCLUSION: The spatial distributions of both drugs and their respective metabolites observed in this work were consistent with previous studies using radiolabeled drugs.

Discovery of (R)-N-(3-(7-methyl-1H-indazol-5-yl)-1-(4-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide (BMS-742413): a potent human CGRP antagonist with superior safety profile for the treatment of migraine through intranasal delivery.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.

The synthesis and SAR of calcitonin gene-related peptide (CGRP) receptor antagonists derived from tyrosine surrogates. Part 2.

Various substituted indazole and benzoxazolone amino acids were investigated as d-tyrosine surrogates in highly potent CGRP receptor antagonists. Compound 3, derived from the 7-methylindazole core, afforded a 30-fold increase in CGRP binding potency compared with its unsubstituted indazole analog 1. When dosed at 0.03mg/kg SC, compound 2 (a racemic mixture of 3 and its (S)-enantiomer) demonstrated robust inhibition of CGRP-induced increases in mamoset facial blood flow up to 105min. The compound possesses a favorable predictive in vitro toxicology profile, and good aqueous solubility. When dosed as a nasal spray in rabbits, 3 was rapidly absorbed and showed good intranasal bioavailability (42%).

Pulmonary delivery of a GLP-1 receptor agonist, BMS-686117.

Alternate delivery route of therapeutic peptides is an attractive non-invasive option to patients who must chronically self-administer their medication through injections. In recent years, much attention has centered on pulmonary peptide delivery of peptide drugs such as insulin and GLP-1 mimetic peptides in the treatment of type II diabetes. In this study, we assessed the feasibility of delivering BMS-686117, an 11-mer GLP-1 receptor peptide agonist, to the lung in rats via intratracheal administration. The pharmacokinetic profiles of three spray-dried, prototype inhaled powder formulations, 80/20 BMS-686117/trehalose (I), 100% BMS-686117 (II), and 20/80 BMS-686117/mannitol (III), as well as a lyophilized BMS-686117 powder, were compared with intravenously and subcutaneously administered peptide. The spray-dried formulations were mostly spherical particles with narrow particle size distribution between 2 to 10 microm, which are better suited for inhalation delivery than the lyophilized, irregular shape powder with a wide particle size distribution between 2 to 100 microm. Prototype III exhibited the best physical characteristics and in vivo performance, with bioavailability of 45% relative to subcutaneous administration. The T(max) for lung delivered peptide formulations were almost twice as fast as subcutaneous injection, suggesting potential for rapid absorption and onset of action. This study demonstrated that pulmonary delivery is a promising, non-invasive route for the administration of BMS-686117.

Rat nasal lavage biomarkers to assess preclinical irritation potential of nasal drug formulations and excipients.

The goal of this study was to evaluate biomarkers of nasal mucosal damage for rapid assessment of irritancy potential of formulations in the rat nasal lavage model, a tool to facilitate nasal formulation development prior to histopathology studies. The nasal cavity of anesthetized rats was lavaged with normal saline 20 min pos-tdose. The collected fluid was analyzed for secreted total protein and biomarkers. Solutions tested include: normal saline, buffers, benzalkonium chloride (BAC), lysophosphatidylcholine (LPC), and four marketed nasal products. Total protein, lactate dehydrogenase and interleukin-1alpha biomarkers were secreted to varying degrees. BAC (0.2%) and LPC (0.5%) exhibiting the strongest response with a signal window ranging from 3.4- to 87-fold greater levels than normal saline. Buffer treatments, excipients, and most marketed nasal products yielded levels similar to normal saline. There was a weak correlation between formulation osmolarity and surface tension with any of the biomarkers. Each nasal formulation elicited a unique protein and biomarker profile with total protein secretion correlated with IL-1alpha secretion suggesting the potential for an inflammatory response. Taken together, rapid and potentially mechanistic information on the preclinical acute irritancy potential of formulations was assessed in the rat nasal lavage model by benchmarking treatments relative to controls and marketed nasal products.

Discovery of (R)-4-(8-fluoro-2-oxo-1,2-dihydroquinazolin-3(4H)-yl)-N-(3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-(4-(piperidin-1-yl)piperidin-1-yl)propan-2-yl)piperidine-1-carboxamide (BMS-694153): a potent antagonist of the human calcitonin gene-related peptide receptor for migraine with rapid and efficient intranasal exposure.

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Early chemistry leads suffered from modest potency, significant CYP3A4 inhibition, and poor aqueous solubility. Herein, we describe the optimization of these leads to give 4 (BMS-694153), a molecule with outstanding potency, a favorable predictive toxicology profile, and remarkable aqueous solubility. Compound 4 has good intranasal bioavailability in rabbits and shows dose-dependent activity in validated in vivo and ex vivo migraine models.

Carbopol-mediated paracellular transport enhancement in Calu-3 cell layers.

The interaction of Carbopol polymers with mucus producing Calu-3 human bronchial epithelial cells was evaluated to test for potential paracellular transport enhancement. Using desmopressin (1-deamino-8-arginine-vasopressin, DDAVP) as the model peptide, apical treatment with Carbopol polymer gel formulations resulted in molecular size-dependent permeability enhancement with a concomitant drop in the transepithelial electrical resistance (TEER). Permeability enhancement of DDAVP was dependent on the formulation vehicle composition and polymer concentration, was noncytotoxic, and completely reversible. Carbopol 971P displayed the greatest permeability enhancement across Calu-3 cells compared to other more viscous Carbopol polymers 934P and 974P, and other mucoadhesive cellulosic polymers. The greatest enhancement was observed when C971P formulation was prepared in water at a concentration of 0.25% w/v. Enhancement was confirmed in rabbit dosed with intranasal fluorescent dextran 4400. The C(max) and absorption rate each increased by 48% in C971P formulations compared to control, while the relative exposure increased 30%. In conclusion, Carbopol polymers are potentially useful excipients to enhance intranasal peptide absorption. We hypothesize that the permeation enhancement is related to the chelation of extracellular or tight-junctional Ca(2+) by charged polymer carboxylate groups that leads to temporary disruption of tight-junctions, thereby facilitating paracellular transport.

A rabbit model for sublingual drug delivery: comparison with human pharmacokinetic studies of propranolol, verapamil and captopril.

A rabbit model for investigating sublingual drug absorption was established yielding results consistent with clinical data reported in the literature. Using propranolol as a model compound the effect of formulation and dosing variables was explored as a means to characterize the limiting parameters of this model. In addition, verapamil and captopril were selected as reference compounds to compare this model to sublingual absorption in humans. Rabbits were dosed sublingually and systemic absorption was measured over time. Sublingual absorption of propranolol was dependent on dosing solution pH and volume. Intra-oral spray device did not affect the overall exposure compared to instillation using a syringe. Despite species and dosing regimen differences the relative bioavailabilities of propranolol and verapamil were very similar in rabbits and humans. In contrast, captopril absorption from the sublingual cavity of rabbits was low and did not agree with that observed in man. Here we report a sublingual rabbit model of drug delivery and its potential utility in preclinical development of intra-oral dosage forms.

Retinoic acid and the histone deacetylase inhibitor trichostatin a inhibit the proliferation of human renal cell carcinoma in a xenograft tumor model.

PURPOSE: Therapy for advanced renal cell carcinoma (RCC) is ineffective in the majority of patients. We have previously reported that retinoid-induced up-regulation of retinoic acid receptor beta (RARbeta) correlated with antitumor effects in RCCs. Recent studies show that there is a reduction in the level of RARbeta2 expression in cancer cells due in part to histone hypoacetylation. Therefore, we tested whether combining histone deacetylase inhibitors with retinoic acid (RA) would restore RARbeta2 receptor expression, leading to increased growth inhibition in RCC cells. EXPERIMENTAL DESIGN: Cell proliferation, Western blot, and reverse transcription-PCR analyses of two RA-resistant RCC cell lines, SK-RC-39 and SK-RC-45, were assessed in the presence of all-trans retinoic acid (ATRA), trichostatin A (TSA), or the combination of ATRA and TSA. Analysis of apoptosis was also done on SK-RC-39 cells treated with these combinations. Additionally, a xenograft tumor model (SK-RC-39) was used in this study to investigate the efficacy of a liposome-encapsulated, i.v. form of ATRA (ATRA-IV) plus TSA combination therapy. RESULTS: Enhanced inhibition of the proliferation of RCC cell lines and of tumor growth in a xenograft model was observed with the combination of ATRA plus TSA. Reactivation of RARbeta2 mRNA expression was observed in SK-RC-39 and SK-RC-45 cells treated with TSA alone or TSA in combination with ATRA. A partial G0-G1 arrest and increased apoptosis were observed with SK-RC-39 cells on treatment with ATRA and TSA. CONCLUSIONS: The combination of ATRA and the histone deacetylase inhibitor TSA elicits an additive inhibition of cell proliferation in RCC cell lines. These results indicate that ATRA and histone deacetylase inhibitor therapies should be explored for the treatment of advanced RCC.

pH-dependent dissolution in vitro and absorption in vivo of weakly basic drugs: development of a canine model.

PURPOSE: The aim of this research was to develop a pH-dependent canine absorption model for studying pH effect on both dissolution in vitro and pharmacokinetics in vivo using the weak bases ketoconazole and dipyridamole as model drugs. METHODS: Ketoconazole and dipyridamole pH-dependent dissolution profiles in vitro were determined by dissolution test at different pH values using USP apparatus II and an Opt-Diss Fiber Optic UV System. In vivo absorption studies for ketoconazole and dipyridamole were performed with crossover design in three groups of beagle dogs under control (no treatment), pentagastrin, and famotidine treatments. Ketoconazole and dipyridamole plasma concentrations were quantified by gradient high performance liquid chromatography mass spectroscopy (HPLC MS/MS). Pharmacokinetic parameters were determined from individual plasma concentration vs. time profiles. RESULTS: Ketoconazole and dipyridamole displayed pH-dependent dissolution. Increasing the pH of the dissolution medium from 1.2 to 6.8 reduced the extent of dissolution of ketoconazole and dipyridamole at 1 h by 96% and 92%, respectively. In vivo studies in dogs under control (no treatment), pentagastrin, and famotidine treatments show marked differences in systemic ketoconazole and dipyridamole exposure. Area under the concentration-time curve (AUC) increased more than 4-fold as compared to control group, whereas it increased nearly 30-fold for ketoconazole and 9-fold for dipyridamole with pentagastrin (gastric pH approximately 2-3) as compared to famotidine (gastric pH approximately 5-7.5) treatment. CONCLUSIONS: This work demonstrates a pH-dependent dissolution in vitro and absorption in vivo for the weak bases ketoconazole and dipyridamole independent of food effects. This model is useful to examine pH-dependent effects on oral drug absorption and for screening formulations to overcome the pH dependency.

A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function.

Stably transfected MDCK/hPepT1-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein were established to quantify the relationship between transgene hPepT1 expression levels and its functional kinetics in facilitating peptide and peptide-like drug uptake and transport in vitro. The hPepT1 sequence was amplified from Caco-2 cell mRNA, inserted into the pcDNA3.1 -V5&His TOPO plasmid, and transfected into MDCK cells. Transgene protein levels were quantified by Western Blot analysis utilizing a standard curve generated with a positive control protein containing a V5&His epitope. Three clones expressing different levels of the hPepT1 fusion protein (low, medium, and high) were selected for the functional characterization with [14C]Gly-Sar and [3H]carnosine. The MDCK/hPepT1 cells expressed a novel hPepT1/epitope tag protein with an apparent molecular mass of 110 kDa. The [14C]Gly-Sar uptake in the transfected cells was sodium-independent and pH-dependent, demonstrating enhanced uptake, the rate of which increased significantly from the weakly to strongly expressing hPepT1 MDCK/hPepT1 -V5&His clones as compared to the mock cell line at pH 6.0. The uptake and permeability of [14C]Gly-Sar and [3H]carnosine demonstrated a direct correlation between the hPepT1 level of expression, uptake, and transport capabilities. Molecular and functional characterization of the MDCK/hPepT1-V5&His cell line confirmed a directly proportional relationship between Vmax and Papp versus the molar levels of hPepT1 transgene expression. This stably transfected hPepT1 cell line may serve as a useful in vitro model for screening and quantifying peptide and peptide-like drug transport as a function of hPepT1 expression in drug discovery.