Characterization of Selective and Potent JAK1 Inhibitors Intended for the Inhaled Treatment of Asthma.

Purpose: Janus kinase 1 (JAK1) is implicated in multiple inflammatory pathways that are critical for the pathogenesis of asthma, including the interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin cytokine signaling pathways, which have previously been targeted to treat allergic asthma. Here, we describe the development of AZD0449 and AZD4604, two novel and highly selective JAK1 inhibitors with promising properties for inhalation. Methods: The effects of AZD0449 and AZD4604 in JAK1 signaling pathways were assessed by measuring phosphorylation of signal transducer and activator of transcription (STAT) proteins and chemokine release using immunoassays of whole blood from healthy human volunteers and rats. Pharmacokinetic studies performed on rats evaluated AZD0449 at a lung deposited dose of 52 µg/kg and AZD4604 at 30 µg/kg. The efficacy of AZD0449 and AZD4604 was assessed by evaluating lung inflammation (cell count and cytokine levels) and the late asthmatic response (average enhanced pause [Penh]). Results: Both compounds inhibited JAK1-dependent cytokine signaling pathways in a dose-dependent manner in human and rat leukocytes. After intratracheal administration in rats, both compounds exhibited low systemic exposures and medium-to-long terminal lung half-lives (AZD0449, 34 hours; AZD4604, 5 hours). Both compounds inhibited STAT3 and STAT5 phosphorylation in lung tissue from ovalbumin (OVA)-challenged rats. AZD0449 and AZD4604 also inhibited eosinophilia in the lung and reduced the late asthmatic response, measured as Penh in the OVA rat model. Conclusion: AZD0449 and AZD4604 show potential as inhibitors of signaling pathways involved in asthmatic immune responses, with target engagement demonstrated locally in the lung. These findings support the clinical development of AZD0449 and AZD4604 for the treatment of patients with asthma.

Characterization of Clinical Absorption, Distribution, Metabolism, and Excretion and Pharmacokinetics of Velsecorat Using an Intravenous Microtracer Combined with an Inhaled Dose in Healthy Subjects.

This open-label, single-period study describes the human absorption, distribution, metabolism, excretion, and pharmacokinetics of velsecorat (AZD7594). Healthy subjects received inhaled velsecorat (non-radiolabeled; 720 µg) followed by intravenous infusion of carbon 14 (14C)-velsecorat (30 µg). Plasma, urine, and feces were collected up to 168 hours post-dose. Objectives included identification and quantification of velsecorat and its metabolites (i.e., drug-related material) in plasma and excreta, and determining the elimination pathways of velsecorat by measuring the rate and route of excretion, plasma half-life (t1/2), clearance, volume of distribution and mean recovery of radioactivity. On average, 76.0% of administered 14C dose was recovered by the end of the sampling period (urine = 24.4%; feces = 51.6%), with no unchanged compound recovered in excreta, suggesting that biliary excretion is the main elimination route. Compared with intravenous 14C-velsecorat, inhaled velsecorat had a longer t1/2 (27 versus 2 hours), confirming that plasma elimination is absorption-rate-limited from the lungs. Following intravenous administration, t1/2 of 14C-drug-related material was longer than for unchanged velsecorat, and 20% of the 14C plasma content was related to unchanged velsecorat. The geometric mean plasma clearance of velsecorat was high (70.7 l/h) and the geometric mean volume of distribution at steady state was 113 l. Velsecorat was substantially metabolized via O-dealkylation of the indazole ether followed by sulfate conjugation, forming the M1 metabolite, the major metabolite in plasma. There were 15 minor metabolites. Velsecorat was well tolerated, and these results support the progression of velsecorat to phase 3 studies. SIGNIFICANCE STATEMENT: This study describes the human pharmacokinetics and metabolism of velsecorat, a selective glucocorticoid receptor modulator, evaluated via co-administration of a radiolabeled intravenous microtracer dose and a non-radiolabeled inhaled dose. This study provides a comprehensive assessment of the disposition of velsecorat in humans. It also highlights a number of complexities associated with determining human absorption, distribution, metabolism, and excretion for velsecorat, related to the inhaled route, the high metabolic clearance, sequential metabolite formation and the low intravenous dose.

Systemic exposure to the metabolites of lesogaberan in humans and animals: a case study of metabolites in safety testing.

During preclinical and early phase clinical studies of drug candidates, exposure to metabolites should be monitored to determine whether safety conclusions drawn from studies in animals can be extrapolated to humans. Metabolites accounting for more than 10% of total exposure to drug-related material (DRM) in humans are of regulatory concern, and for any such metabolites, adequate exposure should be demonstrated in animals before large-scale phase 3 clinical trials are conducted. We have previously identified six metabolites, M1-M6, of the gastroesophageal reflux inhibitor lesogaberan. In this study, we measured exposure in humans, rats, and beagle dogs to lesogaberan and these metabolites. Plasma samples were taken at various time points after lesogaberan dosing in two clinical and three preclinical studies. Concentrations of lesogaberan and its metabolites were measured, and exposures during a single dosing interval were calculated. The parent compound and metabolites M1, M2, M4, and M5 were together shown to constitute all significant exposure to DRM in humans. Only M4 and M5 were present at levels of regulatory concern (10.6% and 18.9% of total exposure to DRM, respectively, at steady state). Absolute exposure to M5 was greater in rats during toxicology studies than the highest absolute exposure observed in humans at steady state (117.0 µmol × h/liter vs. 52.2 µmol × h/liter). In contrast, exposure to M4 in rats was less than 50% of the highest absolute exposure observed in humans. Further safety testing of this metabolite may therefore be required.

Effect of food on the bioavailability of lesogaberan given as an oral solution or as modified-release capsules in healthy male volunteers.

The novel Type B gamma-aminobutyric acid (GABAB)-receptor agonist lesogaberan (AZD3355) has been evaluated as an add-on to proton pump inhibitor treatment for gastroesophageal reflux disease, but the effect of food on the bioavailability of this compound has not been assessed. In this openlabel crossover study, healthy males received single 100 mg doses of lesogaberan (oral solution (A) or oral modified release (MR) capsules with a dissolution rate of 50% (B) or 100% (C) over 4 h) with and without food. Blood plasma concentrations of lesogaberan were assessed over 48 h. A log-transformed geometric mean Cmax and AUC ratio within the 90% confidence interval (CI) range (0.80 - 1.25) was defined as excluding a clinically relevant food effect. Overall, 57 subjects completed the study. Only the oral lesogaberan solution had a fed/fasting Cmax ratio outside the 90% CI range (Cmax ratio: 0.76). AUC ratios were within the 90% CI limits for all three lesogaberan formulations. The only substantial change in tmax associated with food intake was observed for the oral solution (1.0 h without food, 1.8 h with food). In conclusion, a clinically relevant food effect could be excluded for the lesogaberan MR formulations, but not for the oral lesogaberan solution.

(R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action.

Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABA(B) receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABA(B) receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABA(B) receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABA(B) receptor antagonist and absent in GABA(B)-/- mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concentrated in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABA(B) receptors and may offer a potential new approach to treatment of GERD.

Application of a combined effect compartment and binding model for gastric acid inhibition of AR-HO47108: a potassium competitive acid blocker, and its active metabolite AR-HO47116 in the dog.

The effect of AR-HO47108, a potassium competitive acid blocker, and its active metabolite AR-HO47116 was studied in Heidenhain pouch dogs following administration of single oral and intravenous doses of the two compounds. The histamine-stimulated acid secretion was measured in different periods after dose up to 24h. All data obtained in the different studies was pooled and analyzed by non-linear mixed effects modelling. It was found that there is a delay between the plasma concentration-time peak and the maximum inhibitory effect and that the effect persisted longer than anticipated from the plasma concentration half-lives of the compounds. In addition, it was found that the peak effect was reached earlier at higher doses. The effect data was well described by a combined effect compartment and binding model and both distribution to the biophase i.e. the canaliculus of the parietal cell and a rate limiting binding interaction between drug and enzyme appear to contribute to the observed delay. In addition, a secretion rate dependent washout from the biophase may contribute. Furthermore, because the parent compound and metabolite bind to the same enzyme, the effect is determined by competition between the two for the same enzyme. The metabolite was found to be less potent than the parent compound, with Kd values of the combined model of 125 and 11.2 nM for the metabolite and parent compound, respectively. However, the metabolite is generated in high concentrations that rapidly exceed the concentration of parent compound after oral administration of parent compound, and this together with its longer plasma half-life will make its contribution to the overall effect increase with time and slightly prolong the duration of the effect.

Effects of (2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA) on transient lower oesophageal sphincter relaxation in dogs and mechanism of hypothermic effects in mice.

The effects of the novel GABA analogue (2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA) on transient lower oesophageal sphincter relaxations (TLOSRs) were studied in the dog. In addition, the GABA(A)/GABA(B) selectivity was determined in vitro and in vivo, and the pharmacokinetics and the metabolism of the compound were studied in the dog and rat. TLOSRs were reduced by 55 +/- 8% after intragastric administration of AFPSiA at 14 mumol kg(-1) and did not decrease further at higher doses. When evaluated 2 and 4 h after administration, the effect declined to 37 +/- 6 and 16 +/- 9%, respectively. Spontaneous swallowing was only significantly inhibited at 100 micromol kg(-1). The oral availability of AFPSiA was 52 +/- 17 and 71 +/- 4% in the dog and rat, respectively. A fraction of AFPSiA was oxidised to the corresponding sulphonate, (2R)-(3-amino-2-fluoropropyl)sulphonic acid (AFPSoA) after oral administration to the rat and dog. In rat brain membranes, AFPSiA was found to have ten times higher affinity for rat brain GABA(B) (K(i) =47 +/- 4.4 nM) compared to GABA(A) (K(i) = 430 +/- 46 nM) binding sites. The compound was a full agonist at human recombinant GABA(B(1a,2)) receptors (EC(50) = 130 +/- 10 nM). In contrast, the metabolite AFPSoA was considerably more selective for binding to rat brain GABA(A) (K(i) = 37 +/- 3.1 nM) vs GABA(B) (K(i) = 6800 +/- 280 nM) receptors. In the mouse, high doses (1-8 mmol kg(-1)) of AFPSiA induced a rapid and mild hypothermia followed by a profound and sustained hypothermia at the higher doses tested (6 and 8 mmol kg(-1)). This effect was unaffected by the selective GABA(B) receptor antagonist CGP62349. AFPSoA (1 and 2 mmol kg(-1)) produced transient and moderate hypothermia while the hypothermic response was considerably larger at 4 mmol kg(-1).It is concluded that AFPSiA inhibits but does not abolish TLOSRs in the dog. High doses of the compound induce hypothermia in the mouse, which probably is attributable to activation of the GABA(A) receptor. The latter effect may be caused both by AFPSiA and its oxidised sulphonic acid metabolite AFPSoA.