Small Molecules

Most small molecules developed for the treatment of asthma are formulated for either oral or pulmonary delivery. New drugs for anti-leukotriene therapies have demonstrated oral activity, such as a dual antagonist of cysteinyl-leukotriene receptor 1 (CysLT1) and CysLT2 (Gemilukast) and a 5-lipoxygenase-activating protein inhibitor (GSK2190915). Furthermore, chemokine receptor antagonists (e.g. GW766944), phosphodiesterase (PDE) inhibitors (e.g. Revamilast), and mast cell inhibitors (e.g.

Masitinib) are also administrated orally in clinical trials.

Another typical example of orally administrated drugs is the group of antagonists of chemoattractant receptor- homologous molecule expressed on T helper 2 (Th2) cells (CRTH2), also called D prostanoid receptor 2 (DP2) [271]. Selective antagonists of CRTH2 alleviate allergic inflammation by inhibiting the activation and recruitment of pro-inflammatory cells such as Th2 cells, eosinophils, and basophils [272]. At least 13 different CRTH2 antagonists have been brought to clinical trials for the treatment of asthma, allergic rhinitis, and COPD [271]. Most of the new CRTH2 antagonists are delivered orally. OC000459 is a promising CRTH2 antagonist which is currently studied in clinical trials. In preclinical studies, the ability of OC000459 to displace prostaglandin D₂ (PGD2) from human DP2 was first evaluated using recombinant DP2 expressed on the membrane of CHO cells and native DP2 expressed on Th2 cells. Both of these studies demonstrated the high potency of OC000459 for DP2 (K(i) = 0.013 μM and K(i) = 0.004 μM, respectively). OC000459 had minimal inhibitory activity when assessed using a library of 69 non-related receptors and 19 enzymes, suggesting OC000459 was highly specific for DP2. OC000459 treatment inhibited PGD2 induced chemotaxis of Th2 cells (IC50 = 0.028 μM) and IL-13 production of Th2 cells (IC50= 0.019 μM) as well as competitively antagonized eosinophil shape change in response to PGD2 (pKB = 7.9).

Pharmacokinetics of OC000459 oral administration has been investigated in rats, and the plasma concentration of OC000459 at t1/2 was found to be much higher than its in vitro IC50 evaluated on cells. Additionally, the distribution volume (Vd) was estimated to be 0.5 l/kg, indicating high oral activity of OC000459. The anti-inflammatory effect of OC000459 was evaluated in rats, and oral administration of drug inhibited blood eosinophilia induced by systemic administration of 13, 14- dihydro-15-keto-PGD2

(DK-PGD2) in a dose dependent manner. Furthermore, in guinea pigs, OC000459 treatment inhibited airway eosinophilia induced by inhalation of aerosolized DK-PGD2 [273].

Promising preclinical results have led to the clinical investigation of OC000459 for the treatment of asthma. To determine the efficacy of OC000459, in a phase II clinical trial, patients with mild persistent asthma received 200 mg of OC000459 orally or placebo twice daily for 28 days and the lung function was assessed by measuring the forced expiratory volume in 1 second (FEV1). Results revealed that treatment with OC000459, and not placebo, increased FEV1 while also reducing serum IgE and sputum eosinophil count. Therefore, it was concluded that OC000459 is pharmacologically active in asthma patients [274]. To investigate if OC000459 can reduce lung inflammation in response to an allergic stimulus, another clinical trial was conducted in corticosteroid naïve asthmatic patients. It was reported that treatment with 200 mg OC000459 twice daily for 16 days reduced allergen induced later asthmatic response (LAR) determined by calculations of the area under the curve (AUC) of FEV1. Furthermore, this treatment regimen inhibited the induction of sputum eosinophils 1 day following allergen challenge when compared with placebo. Together, OC000459 was shown to inhibit allergic asthma-related inflammation when administered orally to human patients [275]. To determine the optimal dose of OC000459 for the treatment of asthma, patients with mild to moderate persistent asthma were treated with three doses of OC000459 (25 mg or 200 mg once daily, or 100 mg twice daily) for 12 weeks. Results reflected that the lung function, as determined by change in FEV1, was improvedin all patients who received OC000459 when compared to placebo. Therefore, the lowest dose of 0C000459 tested (25 mg daily) was sufficient to provide therapeutic efficacy. To investigate whether sub-populations would respond to treatment differently, lung function data from asthmatic patients with eosinophilia (blood eosinophils count ≥ 250/μl) and without eosinophilia (blood eosinophils count < 250/μl) were compared.

Patients with eosinophilia showed significant improvement of lung function with OC000459 treatment when compared to placebo. Conversely, lung function was not improved in patients without eosinophilia following OC000459 treatment, suggesting OC000459 was more effective for the treatment of eosinophilic asthma [276]. More clinical trials are ongoing to explore the application of OC000459 in severe eosinophilic asthma or other diseases such as eosinophilic esophagitis, atopic dermatitis, and allergic

rhinitis. The safety of OC000459 is also under investigation in healthy participants because of a potential interaction between OC000459 and cytochrome P450 3A4.

Numerous small molecule asthma drugs have been formulated for pulmonary delivery because inhalation of the drug has the advantage of rapid delivery directly to the effected tissue (i.e. the lung). Small molecule drugs formulated for inhalation in clinical trials include LABAs (e.g. GW642444), long-acting muscarinic antagonists (e.g. Seebri), non-steroidal selective glucocorticoid receptor agonists (e.g. AZD7594), PDE inhibitors (e.g. RPL554), and very late antigen (VLA)-4 inhibitors (e.g. GW559090X).

RPL554, a dual inhibitor of PDE3& PDE4, is administrated trough inhalation to avoid adverse gastrointestinal side effects reported for oral formulations of PDE4 inhibitors [277]. In preclinical studies, RPL554 was shown to potently inhibit the activity of isolated human PDE3 and PDE4 (IC50 = 0.4 nM and IC50 = 1479 nM, respectively).

Inhibition of PDE3 was thought to mediate human airway smooth muscle relaxation, and both an in vitro model and an isolated guinea pig tracheal tissue were used to investigate the ability of RPL554 inhibit the activity of PDE3. RPL554 can relax the reproducible contractile of tracheal smooth muscle elicited by electrical field stimulation in a concentration dependent manner. The ability to inhibit stimulation of immune cells of RPL554 was evaluated in human primary cells. It was shown that RPL554 can inhibit the release of tumor necrosis factor alpha (TNFα) from lipopolysaccharide (LPS) activated human mononuclear cells (IC50 = 0.52 μM) and inhibit the proliferation of mononuclear cells stimulated by phytohemagglutinin (IC50 = 0.46 μM). In guinea pigs, inhalation of RPL554 dry powder (3-5 mg, blended with lactose) can reduce the bronchoconstriction and airway edema in response to i.v.

administration of histamine as well as decrease the infiltration of eosinophils to the airway of ovalbumin-sensitized asthmatic animals, indicating the feasibility to deliver RPL554 through inhalation for anti-inflammatory therapies [278].

Based on the efficient activity of bronchodilation and anti-inflammation, RPL554 was evaluated in clinical trials for asthma therapy. The safety of a single administration of different doses of RPL554 (0.003, 0.009, 0.018 mg/kg) was evaluated in both healthy and asthmatic participants. Inhalation of RPL554 was well tolerated at all doses tested based on adverse event reports, vital signs, and electrocardiograph (ECG) data. RPL554

treatment can improve lung function of asthmatic patients, especially at high dose, as determined by more than 1.5 fold of the concentration of methacholine needed to induce a 20% decrease in FEV1 compared with the original dose. To further evaluate the efficacy as a bronchodilator and the safety of RPL554, asthmatic patients received multiple administrations of inhaled RPL554 at a dose of 0.018 mg/kg, for 6 days. FEV1

was determined for 6 h post-inhalation of RPL554 or placebo on days 1, 3, and 6.

Results showed that RPL554 improved FEV1 when compared to placebo. In another clinical trial, the anti-inflammatory effect of RPL554 was investigated in healthy volunteers challenged by inhaled LPS. Participants received a daily dose of RPL554 of 0.018mg/kg for 6 days before LPS challenge, and their sputum was collected 6 h and 24 h post- challenge. Results showed significantly less macrophages, lymphocytes, neutrophils and eosinophils in the sputum from participants treated with RPL554 when compared with the placebo-treated group. Therefore, inhalation of RPL554 was reported to inhibit the inflammatory response. [279]. Currently, RPL554 is investigated in a clinical trial to compare its efficacy to an active comparator, salbutamol, in asthmatic patients.

Small molecular drugs for asthma are rarely administrated intravenously. However, the novel β2-adrenoceptor agonist, MN-221, was evaluated in clinical trials as an adjunct to standard therapy in patients experiencing an acute exacerbation of asthma. Phase I and II studies demonstrated that MN-221 (dose: 5.25- 1125 µg; rate: 0.35- 60 µg/min) was well tolerated in patients with mild-to-moderate or moderate-to-severe asthma, according to the reported adverse events, laboratory tests, vital signs, and ECG.

Moreover, lung function was improved, as determined by FEV1 measurements, in a dose dependent manner in patients treated with MN-221 [280]. Another clinical trial was conducted to evaluate the efficacy of MN-221 in patients with an acute exacerbation of asthma. In this trial, patients admitted to the emergency room received the standard treatment for acute asthma exacerbation, and those who did not response to standard therapy (FEV1 was less than 50% of predicted) received an i.v. infusion of MN-221 (1200 μg) or placebo, followed by measurements of FEV1 for 3 h. MN-221 treatment in addition to standard therapy failed to significantly improve the lung function determined by AUC0-3h of %FEV1 when compared to standard treatment [281]. Despite the apparent lack of improvement with MN-221 treatment, it is possible that these

patients were resistant to any asthma treatment, because these patients also did not respond well to standard treatment. To better determine the efficacy of MN-221, future clinical trials should include a more heterogeneous population, to avoid a disproportionate number of treatment resistant patients.

In conclusion, small molecules can be effective in several formulations and routes of administration.