Bronchodilators are a central therapy for symptom relief in respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma, with inhaled β2-adrenoceptor agonists and anticholinergics being the primary treatments available. The present studies evaluated the in vivo pharmacology of (R)-6-[[3-[[4-[5-[[2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino]pent-1-ynyl]phenyl]carbamoyl]phenyl]sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), a novel bifunctional compound with both phosphodiesterase 4 (PDE4) inhibitor and long-acting β2-adrenoceptor agonist (LABA) activity, which has been optimized for inhalation delivery. GS-5759 dose-dependently inhibited pulmonary neutrophilia in a lipopolysaccharide (LPS) aerosol challenge model of inflammation in rats with an ED50 ≤ 10 μg/kg. GS-5759 was also a potent bronchodilator with an ED50 of 0.09 μg/kg in guinea pigs and 3.4 μg/kg in dogs after methylcholine (MCh) and ragweed challenges respectively. In cynomolgus monkeys, GS-5759 was dosed as a fine-particle dry powder and was efficacious in the same dose range in both MCh and LPS challenge models, with an ED50 = 70 μg/kg for bronchodilation and ED50 = 4.9 μg/kg for inhibition of LPS-induced pulmonary neutrophilia. In models to determine therapeutic index (T.I.), efficacy for bronchodilation was evaluated against increased heart rate and GS-5759 had a T.I. of 700 in guinea pigs and >31 in dogs. In a ferret model of emesis, no emesis was seen at doses several orders of magnitude greater than the ED50 observed in the rat LPS inflammation model. GS-5759 is a bifunctional molecule developed for the treatment of COPD, which has both bronchodilator and anti-inflammatory activity and has the potential for combination as a triple therapy with a second compound, within a single inhalation device.

Rationale: GS-5759 is a bi-functional that is phosphodiesterase 4 (PDE4) inhibitor and long-acting β -adrenoceptor agonist (LABA) 2 being developed as a novel therapy for chronic obstructive pulmonary disease (COPD). We evaluated the pharmacology of GS-5759 with respect to its bronchodilator activity and cardiovascular safety profile in guinea pigs. Methods & Results:Bronchoprotection was measured in conscious animals with airway responsiveness (measured as AUC of Penh) to ACh (4 mg/ml) aerosol inhalation was assessed at -24h, 4h, 8h, and 24h post intratracheal instillation (IT) of GS-5759 (7, 70, and 140 nmol/kg) or indacaterol (70 nmol/kg), (n=6/group). GS-5759 demonstrated a dose-and time-dependent protection against ACh induced bronchoconstriction. At the highest dose, GS-5759 (140 nmol/kg) inhibited bronchoconstriction by 77 ± 5% (p<0.01) at 4h, 60 ± 21% (p<0.01) at 8 h and 36 ± 25% at 24h. At equimolar doses of 70 nmol/kg, both GS-5759 and indacaterol had very similar dose-and time-dependent bronchoprotection profiles. At 4h post-dosing GS-463759 had 62 ± 17% (p<0.01) bronchoprotection compared to 55 ± 27% (p<0.05) for indacaterol, at 8h 49 ± 23% (p<0.01) compared to 52 ± 15% (p<0.05) and at 24 h post-dosing 24 ± 27% compared to 14 ± 13%. Studies in anaesthetized animals were conducted to measure airway responsiveness (measured as peak lung resistance) to ACh (60 ug/kg, iv.) challenge at -2h and 2h post IT dosing and heart-rate monitored simultaneously throughout the experiment (n=3-5/ group). A therapeutic β agonist-mediated bronchoprotection (measured as lung resistance) index (TI) was then determined as the dose ratio of the compared to induction of tachycardia GS-5759 and indacaterol demonstrated a dose-dependent inhibition of ACh-induced . bronchoconstriction with ED values of 3.5 nmol/kg and 20 nmol/kg respectively. Both compounds also demonstrated a 80 dose-related increase in heart-rate, however, a much greater dose of GS-5759 (70 nmol/kg) was required to induce a 10% increase in heart-rate compared to indacaterol where only a 0.07 nmol/kg dose elicited the same increase. Indacaterol therefore had a TI of 0.004 whereas GS-5759 had a TI value of 20, which was 5000-fold higher. Conclusion: GS-5759 demonstrates a dose-related, long duration of bronchoprotection in guinea pigs and has equivalent potency with indacaterol on a molar basis. Significantly higher doses of GS-5759 were required to induce tachycardia compared to indacaterol and this translates into a superior TI. GS-5759 is a long acting bronchodilator which may offer a superior safety margin compared to indacaterol in COPD patients.This abstract is funded by: Gilead Sciences Inc.

Rationale: GS-5759 is a bi-functional that is being phosphodiesterase 4 (PDE4) inhibitor and long-acting adrenoceptor agonist (LABA) developed as a novel therapy for chronic obstructive pulmonary disease (COPD). GS-5759 is a potent β -adrenoceptor agonist with low 2 We evaluated the nanomolar activity at cloned human β receptors.2 β agonist 2 pharmacology of GS-5759 at endogenous receptors in guinea pig tracheal smooth muscle with respect to potency, full agonism and association and disassociation kinetics. guinea pig tracheal strips stabilized in oxygenated Krebs buffer containing Methods & Results: Experiments were performed on indomethacin (10 μM) employing a DMT myograph system. The potency of GS-5759 was measured as percentage inhibition of carbachol (0.3 μM) -induced contraction and demonstrated a concentration-dependent inhibition with an IC of 257 nM compared to 356 nM for 50 indacaterol. Incubation of tissue strips with the β 2 receptor antagonist ICI-118551 (10 μM) completely abolished the contractile effects of GS-5759 (300 nM and 1 μM) did not inhibit the ability of isoproterenol (30 nM-10 μM) to mediate relaxation of GS-5759 (1 nM-10 μM). guinea pig tracheal airway smooth muscle, suggesting that it is a full agonist at β -adrenoceptors.2 GS-5759 demonstrated a time-and concentration-dependent relaxation of carbachol pre-contracted strips, at 3 μM the time to 50% association (On t ) was 10 ± 2 min 1/2 compared to indacaterol (3 μM) with an On t of 6 ± 0.3 min. The disassociation rate of GS-5759 was measured by monitoring the 1/2 recovery of functional carbachol-induced contraction following wash-out of compound from tissue baths. After 3h washout GS-5759 and indacaterol (both 300 nM) maintained >90% relaxation compared to t=0, and after 12 h wash-out they retained 60 ± 9% and 49 ± 9% relaxation respectively. These data suggest GS-5759 has a slow functional dissociation rate and are consistent with a long duration of effect at β receptors.2 Conclusion: GS-5759 is a potent, full β 2 -adrenoceptor agonist at endogenous β 2 receptors in guinea pig tracheal smooth muscle strips, with comparable potency to indacaterol. GS-5759 has concentration-dependent association kinetics and has a slow functional dissociation rate comparable to indacaterol. These data are consistent with GS-5759 being a long acting β 2 -adrenoceptor agonist which may have clinical utility in COPD. This abstract is funded by: Gilead Sciences Inc.

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