Supersaturated formulations of poorly soluble weak acid drugs evaluated in rodents; a case study.

In the present study we describe a way of working to overcome oral administration challenges in an early preclinical project. As candidate drugs were obtained, the preclinical delivery route was replaced by the intended route of the product and resources were allocated to optimize the oral absorption. Two main approaches were followed in order to formulate a selected weak acid, AZ'403, for oral administration in large scale toxicological studies and the early clinical phases. Both approaches relies on the suppression of precipitation from obtained supersaturated solutions achieved either by amorphous solid dispersions (using hydroxypropyl methylcellulose acetate succinate, HPMC-AS) or crystalline salts (sodium and potassium salts). In vivo studies in rodents were performed to evaluate oral AZ'403 absorption from amorphous and crystalline formulations, using nano- and micro crystalline particles of the neutral form, as references. The oral absorption of AZ'403 formulated using both approaches was significantly higher compared with the references. The improvements in overall exposures were 7-100 times during the investigated conditions. The pharmacokinetic profiles implied that both solid dispersions and crystalline salts of AZ'403 generated supersaturation in the small intestine in rodents and indicated that both approaches may be ways forward for subsequent late stage product development.

Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of Asthma.

Starting from our previously described PI3Kγ inhibitors, we describe the exploration of structure-activity relationships that led to the discovery of highly potent dual PI3Kγδ inhibitors. We explored changes in two positions of the molecules, including macrocyclization, but ultimately identified a simpler series with the desired potency profile that had suitable physicochemical properties for inhalation. We were able to demonstrate efficacy in a rat ovalbumin challenge model of allergic asthma and in cells derived from asthmatic patients. The optimized compound, AZD8154, has a long duration of action in the lung and low systemic exposure coupled with high selectivity against off-targets.

Uncovering the regional localization of inhaled salmeterol retention in the lung.

Treatment of respiratory disease with a drug delivered via inhalation is generally held as being beneficial as it provides direct access to the lung target site with a minimum systemic exposure. There is however only limited information of the regional localization of drug retention following inhalation. The aim of this study was to investigate the regional and histological localization of salmeterol retention in the lungs after inhalation and to compare it to systemic administration. Lung distribution of salmeterol delivered to rats via nebulization or intravenous (IV) injection was analyzed with high-resolution mass spectrometry imaging (MSI). Salmeterol was widely distributed in the entire section at 5 min after inhalation, by 15 min it was preferentially retained in bronchial tissue. Via a novel dual-isotope study, where salmeterol was delivered via inhalation and d3-salmeterol via IV to the same rat, could the effective gain in drug concentration associated with inhaled delivery relative to IV, expressed as a site-specific lung targeting factor, was 5-, 31-, and 45-fold for the alveolar region, bronchial sub-epithelium and epithelium, respectively. We anticipate that this MSI-based framework for quantifying regional and histological lung targeting by inhalation will accelerate discovery and development of local and more precise treatments of respiratory disease.

A novel in vivo receptor occupancy methodology for the glucocorticoid receptor: toward an improved understanding of lung pharmacokinetic/pharmacodynamic relationships.

Investigation of pharmacokinetic/pharmacodynamic (PK/PD) relationships for inhaled drugs is challenging because of the limited possibilities of measuring tissue exposure and target engagement in the lung. The aim of this study was to develop a methodology for measuring receptor occupancy in vivo in the rat for the glucocorticoid receptor (GR) to allow more informative inhalation PK/PD studies. From AstraZeneca's chemical library of GR binders, compound 1 [N-(2-amino-2-oxo-ethyl)-3-[5-[(1R,2S)-2-(2,2-difluoropropanoylamino)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)propoxy]indazol-1-yl]-N-methyl-benzamide] was identified to have properties that are useful as a tracer for GR in vitro. When given at an appropriate dose (30 nmol/kg) to rats, compound 1 functioned as a tracer in the lung and spleen in vivo using liquid chromatography-tandem mass spectrometry bioanalysis. The methodology was successfully used to show the dose-receptor occupancy relationship measured at 1.5 hours after intravenous administration of fluticasone propionate (20, 150, and 750 nmol/kg) as well as to characterize the time profile for receptor occupancy after a dose of 90 nmol/kg i.v. The dose giving 50% occupancy was estimated as 47 nmol/kg. The methodology is novel in terms of measuring occupancy strictly in vivo and by using an unlabeled tracer. This feature confers key advantages, including occupancy estimation not being influenced by drug particle dissolution or binding/dissociation taking place postmortem. In addition, the tracer may be labeled for use in positron emission tomography imaging, thus enabling occupancy estimation in humans as a translatable biomarker of target engagement.

A novel matrix for the short-term storage of cells: utility in drug metabolism and drug transporter studies with rat, dog and human hepatocytes.

1. The SureTran matrix is a novel method facilitating short-term maintenance of fresh primary hepatocyte cellular function and offers the potential use of primary cells "as fresh" for several days post isolation. In the study presented, the maintenance of several key phase I and II drug metabolizing enzyme and drug transporter activities is demonstrated with rat and dog hepatocytes preserved for up to 7 days after cell isolation. 2. Intrinsic clearance values were determined for 60 new chemical entities using rat hepatocytes freshly isolated at AstraZeneca and rat hepatocytes prepared at the facilities of Abcellute Ltd (SureTran purveyors), stored and incubated 24 hours after isolation. A very good correspondence in the intrinsic clearance values underlines the utility of the cell maintenance matrix. 3. For human hepatocytes many of the enzyme activities assayed were well maintained for 7 days of storage but some declined to below 50% of initial values between day 4 and 7 of storage. Human OATP1B1 activity was only determined with one batch and declined to 51% of the initial test value by day 4 and further down to 35% by day 7.

The Effects of Pregnenolone 16α-Carbonitrile Dosing on Digoxin Pharmacokinetics and Intestinal Absorption in the Rat.

The effect of Pgp induction in rats by pregnenolone 16α-carbonitrile (PCN) (3 days, 35 mg/kg/d, p.o.) on digoxin pharmacokinetics and intestinal transport has been assessed. After intravenous or oral digoxin dosing the arterial and hepatic portal vein (oral) AUC(0-24h) were significantly reduced by PCN pre-treatment. Biliary digoxin clearance increased 2-fold following PCN treatment. PCN significantly increased net digoxin secretion (2.05- and 4.5-fold respectively) in ileum and colon but not in duodenum or jejunum. This increased secretion correlated with increased Pgp protein expression in ileum and colon. Both intestinal and biliary excretion therefore contribute to altered digoxin disposition following PCN.

Effects of nicotinic receptor blockade on the colonic mucosal response to luminal bile acids in anaesthetized rats.

AIM: To test the hypothesis that nicotinic receptor mechanisms mediate the effects of bile acids on the colonic mucosa. METHODS: The epithelial transport response to 4 mm deoxycholic acid (DCA) was studied in vitro and in vivo, in rat colon. In vitro, epithelial resistance was measured by square pulse analysis, and net membrane current was calculated from the transmucosal potential difference (PD) and resistance. In vivo, we measured PD and net fluid transport. RESULTS: In vitro, DCA significantly increased membrane current and induced a progressive decrease in epithelial resistance, which in the distal colon eventually resulted in a significant PD reduction. This response was not significantly affected by hexamethonium. In vivo, DCA reduced PD with a significantly larger response in distal colon, but had no consistent effect on net fluid absorption. Nicotinic receptor blockade per se increased net fluid absorption and slightly reduced PD in proximal colon, and inhibited spontaneous net fluid secretion and markedly reduced PD in distal colon. Nicotinic receptor blockade significantly attenuated the bile-acid induced PD response. CONCLUSION: The data do not support the theory that a bile acid-activated secretory reflex exists in rat colon. The reduced PD response after hexamethonium suggests that a mechanism involving nicotinic receptors may potentiate the permeability response to luminal bile acids.