The in vitro and in vivo investigation of a novel small chamber dry powder inhalation delivery system for preclinical dosing to rats.

PURPOSE: This research describes a novel "minitower" dry powder delivery system for nose-only delivery of dry powder aerosols to spontaneously breathing rats. METHODS: The minitower system forces pressurized air through pre-filled capsules to deliver aerosolized drug to four nose ports; three of which house spontaneously breathing rats, with the fourth used as a control. Within each port are vent filters which capture drug that was not inhaled for further quantitation. These vent filters along with a novel control system referred to as the "artificial rat lung", allow for the theoretical amount of drug delivered and subsequently inhaled by each rat to be calculated. RESULTS: In vitro and in vivo studies have demonstrated this system's ability to deliver aerosolized drug to rats. The in vitro study showed that ∼30% of the starting dose reached the 4 ports and was available for inhalation. During in-vivo studies, rats inhaled ∼34% of the delivered dose. Of the estimated inhaled dose, 12-18% was detectable in the various tissue samples, with over 30% of the recovered dose found in the rat's lungs. CONCLUSION: Results show that this system is capable of reproducibly delivering drug to the lungs of spontaneously breathing rats. Advantages over current delivery methods include being amenable to the administration of multiple doses and using less (milligram) amount of starting material. In addition, this technique avoids anesthesia which is typically required for instillation or insufflation, and thus has the potential as an efficient and noninvasive aerosol delivery method for preclinical drug development.

The in vitro and in vivo investigation of inhaled migraine therapies using a novel aerosol delivery system consisting of an air pressurized capsule device (APCD) in combination with a pMDI spacer for endotracheal dosing into beagle dogs.

CONTEXT: Aerosol delivery to animals in preclinical settings has historically been very challenging, requiring the use of techniques, such as intratracheal instillation and dry powder insufflation, that are somewhat invasive, inefficient and not representative of clinical inhalation. OBJECTIVE: The objective of this work is to develop a system to deliver dry powder to dogs in an efficient and effective manner for the study of new anti-migraine compounds in development. MATERIALS AND METHODS: The new device uses a metered aliquot of a dry gas to force dry powder drug from a pre-filled HPMC capsule into an AeroChamber® spacer for subsequent inhalation by the animal. RESULTS: The delivery of two invesigational migraine drugs via the new device was assessed in vitro using abbreviated Andersen cascade impaction and showed the device is capable of generating a reproducible delivered dose of up to ∼68% with more than 50% of the dose in the respirable range. In vivo studies have also been performed showing that this device effectively delivered the migraine drugs to spontaneously breathing dogs using a proprietary validated dog inhalation model. DISCUSSION: Results confirmed that the air pressurized capsule device (APCD) was effective in delivering the APIs to lungs of the animals. The in vivo data verified the advantages of inhaled delivery over oral delivery for this class of drugs and were used to establish the cardiopulmonary and respiratory side effect liability profile for these compounds. CONCLUSIONS: This work has demonstrated the utility of this device for quick and accurate screening of prospective drug candidates, representing a significant improvement in ease of use and reprodicibility over current delivery methods.

Pharmacological characterization of the late phase reduction in lung functions and correlations with microvascular leakage and lung edema in allergen-challenged Brown Norway rats.

Late phase airflow obstruction and reduction in forced vital capacity are characteristic features of human asthma. Airway microvascular leakage and lung edema are also present in the inflammatory phase of asthma, but the impact of this vascular response on lung functions has not been precisely defined. This study was designed to evaluate the role of increased lung microvascular leakage and edema on the late phase changes in forced vital capacity (FVC) and peak expiratory flow (PEF) in allergen-challenged Brown Norway rats using pharmacological inhibitors of the allergic inflammatory response. Rats were sensitized and challenged with ovalbumin aerosol and forced expiratory lung functions (FVC, PEF) and wet and dry lung weights were measured 48 h after antigen challenge. Ovalbumin challenge reduced FVC (63% reduction) and PEF (33% reduction) and increased wet (65% increase) and dry (51% increase) lung weights. The antigen-induced reduction in FVC and PEF was completely inhibited by oral treatment with betamethasone and partially attenuated by inhibitors of arachidonic acid metabolism including indomethacin (cyclooxygenase inhibitor), 7-TM and MK-7246 (CRTH2 antagonists) and montelukast (CysLT1 receptor antagonist). Antagonists of histamine H1 receptors (mepyramine) and 5-HT receptors (methysergide) had no significant effects indicating that these pre-formed mast cell mediators were not involved. There was a highly significant (P < 0.005) correlation for the inhibition of FVC reduction and increase in wet and dry lung weights by these pharmacological agents. These results strongly support the hypothesis that lung microvascular leakage and the associated lung edema contribute to the reduction in forced expiratory lung functions in antigen-challenged Brown Norway rats and identify an important role for the cyclooxygenase and lipoxygenase products of arachidonic acid metabolism in these responses.

Discovery of oral and inhaled PDE4 inhibitors.

The optimization of oxazole-based PDE4 inhibitor 1 has led to the identification of both oral (compound 16) and inhaled (compound 34) PDE4 inhibitors. Selectivity against PDE10/PDE11, off target screening, and in vivo activity in the rat are discussed.

Steroidal C-21 heteroaryl thioethers (Part 2): discovery of orally bioavailable selective glucocorticoid receptor modulators (dissociated steroids).

The prednisolone C-21 heteroaryl thioethers have been synthesized and evaluated in cell based transrepression and transactivation assays. Most of the compounds demonstrated weak transactivational activity in both human and rat tyrosineaminotransferase functional assay while keeping potent anti-inflammatory activity. The benzimidazole thioether 7 exhibited comparable anti-inflammatory activity and improved safety profile compared to the classical oral steroid prednisolone.

Steroidal C-21 heteroaryl thioethers. Part 3: pregn-4-eno-[3,2-c]pyrazole fused A ring modified steroids as selective glucocorticoid receptor modulators (dissociated steroids).

The introduction of A ring pyrazole modification to the hydrocortisone C-21 heteroaryl thioethers generated compounds with excellent transrepression potency (IL-8 inhibition) compared to their hydrocortisone analogs. However, the transcriptional transactivation activity of these compounds were considerably higher than the corresponding hydrocortisone analogs. Among all the compounds evaluated, a quinoxaline thioether modification demonstrated the best overall in vitro separation.

Ileocolonic-Targeted JAK Inhibitor: A Safer and More Effective Treatment for Inflammatory Bowel Disease.

Janus kinase (JAK) inhibitors, such as tofacitinib (Xeljanz) and filgotinib (Jyseleca), have been approved for treatment of ulcerative colitis with several other JAK inhibitors in late-stage clinical trials for inflammatory bowel disease (IBD). Despite their impressive efficacy, the risk of adverse effects accompanying the use of JAK inhibitors has brought the entire class under scrutiny, leading to them receiving an FDA black box warning. In this study we investigated whether ileocolonic-targeted delivery of a pan-JAK inhibitor, tofacitinib, can lead to increased tissue exposure and reduced systemic exposure compared to untargeted formulations. The stability of tofacitinib in the presence of rat colonic microbiota was first confirmed. Next, in vivo computed tomography imaging was performed in rats to determine the transit time and disintegration site of ileocolonic-targeted capsules compared to gastric release capsules. Pharmacokinetic studies demonstrated that systemic drug exposure was significantly decreased, and colonic tissue exposure increased at 10 mg/kg tofacitinib dosed in ileocolonic-targeted capsules compared to gastric release capsules and an oral solution. Finally, in a rat model of LPS-induced colonic inflammation, targeted tofacitinib capsules significantly reduced concentrations of proinflammatory interleukin 6 in colonic tissue compared to a vehicle-treated control (p = 0.0408), unlike gastric release tofacitinib capsules and orally administered dexamethasone. Overall, these results support further development of ileocolonic-targeted tofacitinib, and potentially other specific JAK inhibitors in pre-clinical and clinical development, for the treatment of IBD.

Effect of mometasone furoate (MF)/formoterol fumarate (F) combination (MF/F) on late-phase responses in allergen-challenged Brown Norway rats.

Mometasone furoate (MF)/formoterol fumarate (F) combination is a new inhaIed corticosteroid/long-acting ß2-adrenergic agonist (ICS/LABA). The purpose of this study was to evaluate the effects of different dose combinations of MF/F on a variety of late-phase responses to aerosolized antigen challenge in ovalbumin sensitized Brown Norway rats. Late-phase responses were assessed by reductions in lung function, measured by forced vital capacity (FVC) and increased numbers of inflammatory cells and pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid of ovalbumin challenged rats. Intratracheal administration of MF/F 5 h before aerosolized ovalbumin challenge inhibited the increase in inflammatory cells, including eosinophils and levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor-α (TNF-α) appearing in the bronchoalveolar lavage fluid 24 h after the antigen challenge. The combination index for inhibition of both inflammatory cells and cytokines was consistently <1 suggesting a synergistic interaction between MF and F. Intratracheal MF/F given 24 h after the aerosolized ovalbumin challenge reversed the reduction in FVC with statistically significant effects seen over a 24 h period after drug whereas MF and F alone reversed the antigen-induced reduction in FVC at selected times only. At 5 h after drug administration, when both MF and F were partially active, the combination index for MF/F was <1 suggesting a synergistic interaction between MF and F for reversal of the lung function. These results demonstrate that MF/F combination inhibits a variety of late-phase responses induced by allergen challenge and it is likely that MF/F will have a significant benefit in clinical asthma to suppress lung inflammation and improve lung function.

Steroidal C-21 mercapto derivatives as dissociated steroids: discovery of an inhaled dissociated steroid.

A series of C-21 mercapto derivatives of hydrocortisone have been synthesized and evaluated in cell based transrepression and transactivation assays. The benzothiazole derivative, compound 6 not only showed a dissociated profile in vitro functional assays but also a pharmacological profile in a Brown-Norway rat therapeutic index model of asthma that dissociated side effects (thymolysis) while maintaining efficacy against pulmonary inflammation and lung function.

Inhaled formulation and device selection: bridging the gap between preclinical species and first-in-human studies.

The factors that influence inhaled first-in-human (FIH) device and formulation selection often differ significantly from the factors that have influenced the preceding preclinical experiments and inhalation toxicology work. In order to minimize the risk of delivery issues negatively impacting a respiratory pipeline program, the preclinical and FIH delivery systems must be considered holistically. This topic will be covered in more detail in this paper. Several examples will be presented that highlight how appropriate scientific strategy can help bridge the gap between delivering to preclinical species and human. Considerations for the FIH device selection (metered dose inhaler, dry powder inhaler and nebulizer) and formulation optimization for small molecules will be discussed in context with the preclinical delivery systems.

Effect of inhaled roflumilast on the prevention and resolution of allergen-induced late phase airflow obstruction in Brown Norway rats.

Orally active phosphodiesterase 4 (PDE4) inhibitors have been developed for the treatment of asthma and chronic obstructive pulmonary disorders (COPD) although their full development has been limited by adverse side effects. Administration of PDE4 inhibitors by inhalation may improve their therapeutic index, but limited information exists on the efficacy of inhaled PDE4 inhibitors to improve lung function. In this study in ovalbumin-sensitized Brown Norway rats, roflumilast was given either intratracheally or by nose-only inhalation and changes in lung function (forced vital capacity, FVC; peak expiratory flow, PEF) and inflammatory cell influx (total cells, eosinophils and neutrophils) into the bronchoalveolar lavage (BAL) fluid were evaluated 24 h after allergen challenge. Intratracheal roflumilast, given 5 h before antigen challenge, inhibited the antigen-induced reductions in FVC (ED50 = 140 microg/kg, i.t.) and total cells appearing in the bronchoalveolar lavage fluid (ED50 = 50 microg/kg, i.t.). By the nose-only inhalation route, roflumilast reduced the bronchoalveolar lavage fluid total cells (ED50 = 10 microg/kg, estimated pulmonary deposition). Intratracheal roflumilast (600 microg/kg, i.t.) was also given to rats 24 h after the antigen challenge and reversed the antigen-induced reductions of FVC by 38% at 1 h, 54% at 5 h and 71% by 16 h. Intratracheal roflumilast also reduced the number of inflammatory cells in the bronchoalveolar lavage fluid and reduced the interstitial airway edema caused by the antigen challenge. These results support the development of inhaled PDE4 inhibitors for the treatment of asthma and COPD, particularly for the improvement of lung function.

Temporal profile of forced expiratory lung function in allergen-challenged Brown-Norway rats.

The Brown-Norway rat is often used to study the allergic pulmonary response. However, relatively little is known about the delayed phase reactions after allergen challenge in this species. To evaluate the temporal changes in lung function and elucidate the mechanisms involved in the delayed phase response, Brown-Norway rats were sensitized and challenged to aerosolized ovalbumin and lung functions were measured by forced expiratory maneuvers and forced oscillation for up to 10 days after a single antigen challenge. Statistically significant (P < 0.05) reductions in inspiratory capacity, forced vital capacity, functional residual capacity, peak expiratory flow and maximum mid-expiratory flow and increases in respiratory system resistance and elastance were seen by 1 to 3 days after ovalbumin challenge that returned to baseline by 10 days. The reductions in lung function after ovalbumin challenge were blocked by the corticosteroid, betamethasone (1 mg/kg, p.o.). Histological evaluation of lung tissue of sensitized rats demonstrated evidence of interstitial pulmonary edema, an increase in tissue eosinophils and an increase in Periodic Acid Schiff-positive cells in the airway epithelium. Bronchoalveolar lavage fluid samples showed large numbers of eosinophils and increased mucin content up to 6 days after antigen challenge. There was also an increase in wet-to-dry lung weight ratio in the lungs of sensitized rats after antigen. These results demonstrate that prolonged reductions in lung function occur after a single antigen challenge in Brown-Norway rats that is probably due to inflammatory processes producing interstitial pulmonary edema, mucus secretion and cellular influx into the lungs.

Antitussive activity of the tachykinin NK1 receptor antagonist, CP-99994, in dogs.

CP-99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] is a selective tachykinin NK(1) receptor antagonist that inhibits cough in guinea pigs and cats. This study examined the antitussive effects of CP-99994 in dogs produced by mechanical stimulation of the intrathoracic trachea. CP-99994 (10 mg/kg, p.o.) inhibited cough frequency by 52% at 2 h, 31% at 6 h and by 21% at 24 h. Cough amplitude was inhibited by 45% at 6 h but unchanged at 2 and 24 h after CP-99994. Plasma levels of CP-99994 were highest at 2 h (75+/-26 ng/ml) and fell to 22+/-6 ng/ml at 6 h. These results demonstrate antitussive activity of CP-99994 in dogs at a dose proven to antagonize tachykinin NK(1) receptors in this species.

Cough reflex in allergic dogs.

This study investigated the effects of antigen challenge on the cough reflex in dogs that were neonatally sensitized to ragweed. Tidal volume (V(T)), respiratory rate (f), pulmonary resistance (R(L)), dynamic lung compliance (C(Dyn)) and the number and amplitude (increase in mean peak expiratory pressure) of coughs induced by mechanical stimulation of the intrathoracic trachea were measured in propofol-anesthetized dogs. Aerosolized ragweed challenge had no effect to induce spontaneous cough but increased f and R(L) and reduced V(T) and C(Dyn). Mechanical stimulation of the intrathoracic trachea at this time produced 19+/-5 coughs with an average increase in cough amplitude of 11+/-1 cm H(2)O which differed significantly from the number (9+/-2 coughs) and amplitude (30+/-5.5 cm H(2)O) of mechanically induced coughs after treatment with aerosolized saline. Both the number and amplitude of mechanically induced coughs returned to baseline values by 24-48 h after the ragweed challenge. Similar results were obtained after challenge with aerosolized histamine (0.3-1% histamine) that did not induce spontaneous coughs but increased f, reduced V(T) and decreased C(Dyn) and increased the number but reduced the amplitude of the mechanically induced coughs. In conclusion, both antigen and histamine bronchoprovocation changed the characteristics of the mechanically induced cough in dogs to a response of increased cough number but reduced mean expiratory cough amplitude.

Pharmacology of a potent and selective inhibitor of PDE4 for inhaled administration.

A strategy to overcome the side effect liabilities of oral PDE4 inhibitors has been to deliver the drugs by inhalation. In this report, we identify 1-[[5-(1(S)-aminoethly)-2-[8-methoxy-2-(triflurormethyl)-5-quinolinyl]-4-oxazolyl] carbonyl]-4(R)-[(cyclopropylcarbonyl)amino]-L-proline, ethyl ester xinafoate salt, (COMPOUND 1) as a potent and selective inhibitor of PDE4 with biological and pharmacokinetic properties suitable for delivery by the inhaled route. COMPOUND 1 potently inhibits human PDE4 (IC(50)=70pM) with little or no activity against other PDEs. It is highly potent against PDE4B and PDE4D which are important isoforms of PDE4 controlling inflammation and airway functions. In an allergen-challenged Brown Norway rat model of asthma, COMPOUND 1 inhibited the late phase influx of inflammatory cells and reductions in lung function following its administration by the intratracheal or nose-only routes of administration. Important differences were seen between intratracheal COMPOUND 1 and our previously published results with the oral PDE4 inhibitor roflumilast (Celly et al., 2005), as COMPOUND 1 rapidly (within 1h) reversed the decline in lung function when it was given therapeutically to rats already challenged with antigen. COMPOUND 1 was weakly active by the oral route which is a finding consistent with results showing this compound has poor oral bioavailability in animals. Positive interactions between COMPOUND 1 and albuterol, and COMPOUND 1 and mometasone furoate were seen on the improvement in lung functions in allergen-challenged rats. These results identify COMPOUND 1 as a potent and selective inhibitor of PDE4 with properties suitable for delivery by inhalation.

Effect of histamine, albuterol and deep inspiration on airway and lung tissue mechanics in cynomolgus monkeys.

Forced oscillation is a technique that has been used to measure airway and lung tissue impedance. To evaluate airway and lung tissue impedance in a colony of cynomolgus monkeys housed at Schering-Plough Research Institute, a forced oscillation technique was used to measure Newtonian resistance (R(N)), tissue damping (G), tissue elastance (H) and lung hysteresivity (eta). Functional residual capacity (FRC) was also measured to correlate the lung impedance data with FRC. There was no difference in R(N), G, H and eta between Ascaris sensitive allergic monkeys (n=25) and a small cohort (n=5) of non-allergic monkeys under baseline conditions. However, a highly significant (p<0.0001) negative correlation (r=0.71) was found between FRC and H. Significant correlations were also found between FRC and G (r=0.53) and FRC and R(N) (r=0.50). Bronchoprovocation with aerosolized histamine increased R(N), G, H and eta and reduced FRC by 29+/-3% (n=30) from baseline. In monkeys that were hyperreactive to the histamine challenge, an exaggerated increase in lung tissue damping was seen whereas monkeys that were less reactive to the histamine showed greater increases in R(N). Aerosolized albuterol (0.003-3mg/ml) produced a concentration-dependent reversal of the increases in R(N), G, H and eta induced by histamine with the greatest reversal seen on R(N). Deep inspiration, performed after the aerosolized albuterol exposure, also reversed the histamine-induced changes in R(N), G, and H with the complete reversal seen on the increase in H. These results demonstrate that significant correlations exist between airway and lung tissue impedance and FRC and that airway and lung tissue mechanics contribute significantly to inherent bronchoconstrictor reactivity and to the bronchodilator response to a beta-adrenergic agonist and deep inspiration in cynomolgus monkeys.

Airway closure after antigen challenge in cynomolgus monkeys: effect of the histamine H1 receptor antagonist, chlorpheniramine maleate.

BACKGROUND: Airway closure is frequently observed in human asthma. However, limited information exists on the factors that cause this condition. In this study, an allergic cynomolgus monkey model was used to characterize the condition of airway closure and assess the contribution of histamine H1 receptors to this response. METHODS: Oscillatory lung mechanics, arterial blood gases during ventilation on 100% O2 and functional residual capacity (FRC) assessed by helium dilution were measured before and then 10 min and 24 h after Ascaris aerosol challenge in 12 male Ascaris-sensitive cynomolgus monkeys. The monkeys were pretreated with intravenous saline or chlorpheniramine maleate (0.3 mg/kg) in a randomized crossover design. RESULTS: Ascaris challenge produced a large increase in airway resistance, an increase in lung tissue damping (G) that measures ventilation inhomogeneity in the lung, a reduction in arterial oxygen tension (PaO2) during ventilation on 100% O2 and a reduction in FRC. These effects were seen 10 min after the Ascaris challenge, but by 24 h, these parameters had returned close to the baseline values. Chlorpheniramine maleate (0.3 mg/kg, i.v.) produced a 12-fold shift in the histamine bronchoconstrictor dose-response curve. Pretreatment of monkeys with chlorpheniramine maleate (0.3 mg/kg, i.v.) attenuated the increase in airway resistance induced by Ascaris challenge, but had only a small effect on the increase in G and the reductions in PaO2 and FRC after antigen. CONCLUSIONS: These results demonstrate that airway closure occurs immediately after the antigen challenge in allergic cynomolgus monkeys and that histamine H1 receptors contribute very minimally to this response.

Role of cholinergic reflexes on the bronchoconstrictor reactivity to neurokinin a in allergic dogs.

Neurokinin A (NKA) potentiates airway cholinergic neurotransmission in several species. In this study, the role of cholinergic reflexes on the bronchoconstrictor response to NKA was evaluated in non-sensitized dogs and in allergic dogs neonatally sensitized to ragweed in which heightened bronchoconstrictor reactivity to NKA has previously been observed. Cardiopulmonary functions, including pulmonary resistance (R(L)) were measured in anesthetized, spontaneously breathing dogs before and after increasing concentrations of aerosolized NKA. The provocative concentrations of NKA increasing R(L) by 25% above the baseline (PC(25)) was measured before and after ( approximately 10 min) aerosolized saline or ipratropium bromide (0.01%). This concentration of ipratropium produced a 250-fold shift in the methacholine dose-response curve. In sensitized dogs, NKA bronchoconstrictor reactivity (PC(25)=0.050+/-0.011%) was 2.5 times more potent than that of non-sensitized controls (PC(25)=0.177+/-0.031%). Ipratropium bromide inhibited the bronchoconstrictor response to NKA in both sensitized and non-sensitized dogs and after ipratropium, NKA reactivity was 5.2-fold less in allergic dogs (PC(25)=0.246+/-0.048%) as compared to 3.5 fold less in non-sensitized controls (PC(25)=0.622+/-0.106%). In conclusion, cholinergic reflexes are important components of the bronchoconstrictor response to NKA in dogs particularly in those sensitized neonatally to ragweed. It is speculated that heightened activity of cholinergic reflexes contributes to the bronchial hyperresponsiveness seen in allergic dogs.