Preclinical Safety

Inhaled Enzyme: Pulmozyme®

The first inhaled protein to market was Pulmozyme® (rhDNase) developed to help treat CF. Pulmozyme® is a glycoprotein enzyme (37 kDa) that cleaves deoxyribonucleic acid (DNA) in the mucus in CF patients, making it thinner and easier to clear. Inhalation toxicology studies were conducted in both rats and monkeys for durations of up to 6 months. ⁸ In the 6-month rat study, bronchiolitis was observed at the end of the treatment period but at a somewhat lower incidence than in the 4-week study. These data suggest that the mild lesion was not progressive in rats. In the 6-month monkey study, respiratory rates measured during aerosol exposure to monitor for anaphylactic or irritant responses were unchanged compared to pre-exposure values. Positive serum antibody titers to rhDNase were observed beginning at week 4 and persisted through the treatment period. Serum concentrations of rhDNase at 24 hours postdose indicated that there was no accumulation of rhDNase throughout the 6-month treatment period. Histopathologically, there was increased perivascular lymphocytic cuffing, peribronchial lymphoid hyperplasia, terminal airway-related bronchiolitis/alveolitis with eosinophilic infiltrates, and increased siderophages. There appeared to be a close relationship between the severity of the pulmonary lesions and the antibody titer to rhDNase measured in serum. The lesions were consistent with an allergic or hypersensitivity (type I) response to a foreign protein. This finding is not unexpected, because there are considerable differences between animal and human DNases. ⁸

There is only an 80% homology between the rat and human DNases; monkey DNase, although it has not been completely sequenced, also appears to be highly dissimilar to the human form (S. Shak, personal communication, 1997).

Some of the most persuasive data related to the lack of immune reactions to inhaled therapeutic proteins come from the clinical trials of rhDNase (Pulmozyme®) for the treatment of CF. In the Summary Basis of Approval for Pulmozyme®, ⁹ it was concluded that antibodies to rhDNase were of little consequence to the safety profile of rhDNase in patients because levels were generally low and there was no correlation between antibody levels and clinical responses. Thus, the findings in the animal studies were deemed to be the result of immunological reaction to a non-homologous foreign protein and not relevant to results in people. The clinical data indicate little concern for the homologous protein in humans. ⁴

Inhaled Peptide Hormone: Insulin

Inhaled insulin (5.8 kDa monomer) is an attractive option for the treatment of type 2 diabetes because it avoids the use of needles and may offer better outcomes through improved compliance. A large number of clinical trials have been carried with inhaled insulin by a number of companies as thoroughly reviewed by Siekmeier and Scheuch ¹⁰ and Mastrandea. ¹¹ In controlled clinical trials, more than 13,000 patients have been safely treated with inhaled insulin for an average of 1 year with some patients on Exubera® for up to 8 years. Overall, clinical trials have demonstrated that inhaled insulin is comparable to subcutaneous (sc) insulin for improving glycemic control. Also, pharmacokinetics and pharmacodynamics were roughly similar, with the exception that inhaled regular insulin has faster absorption than sc regular insulin and is comparable to fast acting insulins. The main adverse findings with dry powder insulin formulations were cough and small, reversible decreases in lung function (carbon monoxide diffusion capacity and forced expiratory volume in one second).^11–13

Immunogenicity has been studied extensively. In all major clinical programs, the delivery of insulin by inhalation induced higher antibody levels in some patients than comparators. However, these antibodies were not shown to decrease the effectiveness, safety or tolerability of inhaled insulin over time and did not affect clinical outcomes. ¹⁴

A number of inhaled insulin formulations have been developed; Exubera® was the first to receive marketing authority. Most efforts were discontinued after Pfizer withdrew Exubera® from the market; MannKind, however, continued development of its inhaled insulin powder Afrezza®, and it was approved in the US in June 2014. The major insulin development programs are cited below, but a more complete list can be found in the publication of Siekmeier and Scheuch. ¹⁰

The first inhaled insulin to be approved is Exubera® (insulin human recombinant DNA [rDNA origin]) Inhalation Powder (spray dried dry powder formulation of insulin, sodium citrate, mannitol and glycine)—withdrawn from the market by Pfizer.

Of the major inhaled insulin toxicology programs, four have significant toxicology data available in the public domain.^15–20 In all four cases, no adverse effects on lung were noted.

Pfizer/Nektar (Two 1-month rat, one 6-month rat, one 1-month Cynomolgus monkey, and one 6-month Cynomolgus monkey studies): These studies, in addition to assessing standard toxicological parameters, also evaluated the potential toxicity of both the complete formulation and the excipients alone to the respiratory tract. Investigations included respiratory and pulmonary function, lung cell proliferation indices, insulin antibody titers and histopathology.

There were no toxicological findings relevant to human systemic or pulmonary risk with either the excipients or the complete formulation at doses up to 40 times for rats and 4 times for monkeys compared to the clinical starting dose of 0.15 mg/kg/day. Maximum tolerated doses were limited by hypoglycemia. No evidence of exposure-related inflammatory or immune-mediated hypersensitivity reactions were observed in the respiratory tract. A weak antibody response to the insulin powder was observed in rat serum but anti-insulin antibodies were not detected in monkey serum or bronchoalveolar lavage fluid. Rat insulin differs from human insulin by three amino acids; pig and dog differ by one amino acid; and Cynomolgus monkey insulin is identical to human insulin. A comparable weak antibody response in rats was also observed following sc injection. Assessment of respiratory and pulmonary function parameters revealed no exposure-related effects. No exposure-related histopathological responses were observed in the respiratory tract or in lung-associated lymph nodes in either species. Staining the bronchioles and alveoli with markers for cell proliferation showed no biologically significant differences in proliferation indices attributable to the complete formulation or excipients alone in either species.^15,16

AIR/Lilly (6 months - dogs): The formulation was a dry powder composed of insulin, dipalmitoylphosphatidylcholine (DPPC) and sodium citrate. Dogs were exposed 15 minutes/day to an air control, placebo, maximal placebo (3x the placebo dose), or one of three doses of human insulin inhalation powder (HIIP) (mean inhaled doses of 0.08, 0.24, or 0.70 mg/kg/day for the HIIP-low, HIIP-mid, and HIIP-high dose, respectively). The expected pharmacological effect of insulin was observed with dose-related decreases in serum glucose levels following HIIP administration. There were no toxic effects observed, including no HIIP or placebo treatment related effects on mean body weights, absolute body weight changes, clinical observations, food consumption, respiratory function parameters, ophthalmic examinations, electrocardiograms, heart rates, clinical pathology, or urinalysis. Similarly, there were no HIIP or placebo treatment related effects on pulmonary assessments that included respiratory function parameters, bronchial alveolar lavage assessments, organ weights, or macroscopic and microscopic evaluations, including lung cell proliferation indices. HIIP was considered to have either low or no immunogenic potential in dogs. The no-observed-adverse-effect level (NOAEL) and maximum tolerated dose (MTD) were a mean inhaled dose of 0.70 mg insulin/kg/day, ¹⁷ approximately 5-fold greater than a typical clinical dose.

Aerogen® (1 month - dogs): The inhalation safety of Humulin® R U500 liquid insulin formulation was evaluated in a 28-day repeat dosing study in dogs. In addition to 500 U/ml of human DNA derived insulin, Humulin® R contains glycerin 16 mg/mL, metacresol 2.5 mg/mL and zinc oxide to supplement the endogenous zinc to obtain a total zinc content of 0.017 mg/100 units, and water for injection. The pH was 7.0 to 7.8. Two pulmonary doses of insulin, a low dose and the MTD were evaluated against water and placebo controls. After 28 days of daily exposure, the animals were killed and examined at necropsy. Respiratory tissues were examined microscopically. The mean pulmonary doses achieved were 2.3 and 8.3 U/kg/day for the low and MTD dose levels, respectively. The aerosols were highly respirable with 78% of the insulin aerosols being < 3.8pm. The treatments were well tolerated. No adverse in-life, necropsy or histological findings were detected that were related to insulin or inhalation treatment. ¹⁸

MannKind/Sanofi: (26-week rat, 39-week dogs; carcinogenicity studies: 104-week inhalation - rats; 26-week transgenic mice). In inhalation toxicology studies of Afrezza®, rats exposed to doses up to 1.91 mg/kg/day for 26 weeks and 1.23 mg/kg/day for 104 weeks, and dogs exposed to doses up 1.92 mg/kg/day were well tolerated in all animals. There were no adverse findings, including no microscopic findings in the lungs or evidence of carcinogenicity or proliferation.

The main findings were, in rat 26-week study, goblet cell hyperplasia and eosinophilic globule accumulation of minimal severity in the olfactory/respiratory epithelium; and, in dogs, neutrophil infiltration of minimal severity in lung. The Technosphere® carrier controls at doses of 11.7 mg/kg also had similar lack of findings in the rat 26-week study but also a slight increase in proliferating cell nuclear antigen was observed in the bronchioles. ¹⁹

In a 26-week carcinogenicity study, transgenic mice (Tg-ras-H2) exposed to doses up to 5 mg/kg/day of Afrezza® and to 75 mg/kg/day of Technosphere® carrier had no increased incidence of tumors. Afrezza® was also not genotoxic in Ames bacterial mutagenicity assay and in the chromosome aberration assay, using human peripheral lymphocytes with or without metabolic activation. The Technosphere® carrier alone was not genotoxic in the in vivo mouse micronucleus assay. ²⁰

In a reproduction toxicity study, female rats given sc doses of 10, 30, and 100 mg/kg/day of Technosphere® carrier starting 2 weeks before mating until gestation day 7, had no adverse effects on male fertility at doses up to 100 mg/kg/day, a systemic exposure 14-21 times that following the maximum daily Afrezza® dose of 99 mg based on area under the curve (AUC).

Female rats at 100 mg/kg/day did have increased pre-and post-implantation loss but not at 30 mg/kg/day (14–21 times higher systemic exposure than the maximum daily Afrezza® dose of 99 mg based on AUC). ²⁰

In all of these extensive preclinical studies, at substantial multiples of clinical doses, no adverse effects on lung were found related to either insulin or the excipients. Therefore, the market potential still exists for inhaled insulin since it has been shown to be both safe and effective. Furthermore, its main advantage over sc delivery is convenience, which may result in better compliance. The future for inhaled insulin is certainly brighter now that MannKind's Afrezza® has been approved in the US; Dance Biopharm has also completed Phase 2 clinical trials. ²¹ Another possible approach to treat type 2 diabetes is inhaled glucagon-like peptide 1 (GLP-1) (30 amino acids) and GLP-1 analogs, which have been mainly tested in animals. ²²

Inhaled Peptide Hormone: Human Growth Hormone (hGH)

Inhaled hGH (22 kDa) was under development by Alkermes/Lilly. One-month and 6-month inhalation toxicology studies were carried out in Cynomolgus monkeys with no adverse effects in the lung observed. These studies supported clinical trials in normal volunteers. The trial was a crossover design in 12 young adult subjects, each subject receiving both sc and inhaled hGH on separate days. Inhaled hGH was well tolerated with no coughing or adverse taste issues. Pulmonary function and vital signs were measured with no apparent changes of clinical significance. The pharmacokinetic (PK) profiles for sc injection (4 mg hGH) and inhalation delivery (92 mg hGH in 4 dry powder capsules) were quite similar as seen in Figure 2. Overall delivery efficiency was approximately 5% compared to sc delivery, somewhat less than the approximately 10% values observed for a similar dry powder formulation and delivery system for inhaled insulin. The decreased efficiency with respect to inhaled insulin was expected because of the greater molecular weight of hGH (22 kDa).

OPEN IN VIEWER

FIG. 2.

PK profiles in healthy volunteers following sc or inhalation administration of hGH.

Following the demonstration of safety in normal young adults, a similar crossover study was carried out in pediatric patients. Again, there were no adverse clinical outcomes for inhaled delivery (PK profile was similar to that from sc delivery), but overall delivery efficiency was less than the business development goal of 5% relative to sc delivery. With this result, the program was terminated. ⁵

If improved delivery efficiency can be achieved (or lower costs of hGH manufacture), inhaled delivery of hGH could be considered financially attractive and to improve compliance in children and increase the willingness of parents to start therapy in their children since reluctance to start injections is a negative factor in initiating growth hormone therapy.

Inhaled Cyclic Peptide: Cyclosporin

Cyclosporin is a cyclic peptide (11 amino acids) with potent immunosuppressive properties. Inhaled cyclosporin has been investigated for a number of years to aid in postoperative treatment of lung transplant patients to reduce the incidence of rejection. ²³ Recent data have shown that inhalation of cyclosporin in solution with propylene glycol given in addition to conventional immunosuppression appeared to improve important pulmonary function parameters in lung transplant recipients compared to patients receiving aerosol placebo or conventional immunosuppression alone. The inhaled formulation had no unexpected systemic toxicity or clinically limiting findings in the respiratory tract in 28-day rat and dog studies ²⁴ and 9-month dog studies with exposures 3 days a week. ²⁵ In Phase 2 clinical trials, the inhaled formulation improved overall survival. ²⁶ However, the Phase 3 trial showed no efficacy beyond that of standard of care when used as supplemental targeted therapy. ²⁷ The authors noted that “administering a cyclosporin aerosol to this highly vulnerable patient population is not without challenges and this may have influenced the study outcome.” ²⁷ Other investigators are looking into possible uses of cyclosporin to treat asthma. ¹

Inhaled Protease Inhibitor: Alpha-1 Antitrypsin (AAT)

AAT is a glycoprotein protease inhibitor (54 kDa) that can be used to treat protease-antiprotease imbalance in the lung. For patients with AAT deficiency, low levels of antiproteases in the lungs is a serious issue since domination of proteases will lead to lung inflammation, tissue destruction, and a predisposition to emphysema. Currently, there are three FDA-approved alpha-1 antitrypsins for intravenous (IV) administration: Prolastin® (Grifols), Zemaira™ (AventisBehring), and Aralast™ (Alpha Therapeutic Corporation). All of these products are derived by purification from human serum.

Inhaled AAT has been studied as a potential therapy with generally encouraging results. ²⁸ Siekmeier ²⁹ summarized that “The data demonstrate the feasibility of alpha-1 antitrypsin inhalation for restoration of the impaired protease-antiprotease balance, attenuation of the inflammation and neutralization of the excess activity of neutrophil elastase.” Inhaled AAT may also be useful for treating chronic obstructive pulmonary disease (COPD) and CF as well as pure AAT deficiency since protease antiprotease imbalances have been suggested to contribute to both of these respiratory diseases. Siekmeier ²⁹ also noted that the inhalation route may provide cheaper therapy than that for IV administration since only about 2% of the dose reaches to the lung following IV delivery as compared to possibly 20–30% that could be delivered to the lung following inhalation. The 20–30% aerosol deposition value can only be achieved with optimized devices and formulations.

Kamada ³⁰ is developing inhaled AAT as potential therapy for AAT deficiency and announced results from their European Phase 2/3 clinical study in September 2014. These failed to meet either the primary “time to the first moderate or severe exacerbation event” or secondary exacerbation endpoints in the intention-to-treat (ITT) population. However, there were clinically relevant changes in various lung function measurements in the ITT population, as well as in the most frequent exacerbators population, of which some were statistically significant. Inhaled AAT was also safe and well tolerated in the patients. Based on the strength of the lung function changes, especially in the most frequent exacerbators population, Kamada still plans to file for approval in Europe and the US. The complete data set from the Phase 2/3 clinical study was presented in May 2015. ³⁰ The benefit of inhaled AAT treatment is also being evaluated in other patient populations including cystic fibrosis. ³⁰

One additional opportunity that could enhance inhaled ATT usage would be the availability of a recombinant human protein to reduce immunogenicity potential, increase product reproducibility and potentially reduce costs if an appropriate manufacturing process can be developed.

Inhaled Vaccines: Measles

Inhaled vaccines, particularly measles vaccines, have received considerable attention. The rationale for use of inhaled vaccines is that delivery of the vaccine to the respiratory tract, the natural entry route for the pathogen, could provide an attractive therapy by engaging mucosal immunological mechanisms. Inhaled delivery is attractive for use in developing countries due to lack of suitably trained staff to administer injections and problems in disposal of used needles and syringes. Early work by Albert Sabin ³¹ demonstrated the feasibility followed by a mass vaccination campaign in 4 million children between 1988 and 1990. ³²

In 2002, the Measles Aerosol Vaccine Project was initiated by the World Health Organization (WHO), Centers for Disease Control and Prevention and American Red Cross to develop a practical inhaled measles vaccine using liquid aerosol delivery. ³³ Phase 1 studies showed that the aerosolized vaccine was safe, well tolerated and produced an appropriate immune response. Phase 2/3 studies were completed and showed results equivalent to sc delivery for children ages 10–35 months, but for ages 9–10 months immune response was not as strong as for sc delivery. ³⁴ Challenges with getting good lung deposition in nose-breathing infants likely contributed to these findings. The conclusion by WHO was that aerosol delivery should be effective for children older than 10 months. The conclusion of the final report ³⁵ was that “at the prespecified margin, the aerosolized vaccine was inferior to the subcutaneous vaccine with respect to the rate of seropositivity.” In addition, there have been efforts to develop a dry powder aerosolized vaccine using carbon dioxide assisted bubble drying. ³⁶ This effort was spearheaded by Aktiv-Dry with key support from a Grand Challenges grant from the Gates Foundation. Preclinical studies in monkeys with the dry powder vaccine showed no adverse effects, and the production of an immune response was similar to that from sc delivery. ³⁷ Phase 1 clinical trials have been completed in healthy men with pre-existing immunity to measles. ³⁸ No adverse events were reported and the inhaled dry powder vaccine produced serologic responses generally similar to subcutaneous vaccination, however, these results are difficult to interpret because of the high baseline antibody levels.

A key hope of the inhaled measles vaccine efforts is providing access to therapies in developing countries where infrastructure for needle injection delivery systems is problematic. Another key hope is that ultimately more cost-effective therapies might also be possible. ³⁹ These goals may be challenging but with continued technological innovation appear to be feasible.

Inhaled Gene Therapy

Inhaled gene therapy approaches to treat cystic fibrosis have been largely unsuccessful using approaches such as attenuated adenovirus vectors to deliver the CFTR gene. ⁴⁰ However, newer approaches using lipid base carriers of CFTR DNA are underway. ⁴¹

Inhaled Antibodies: Anti-IgE

In the late 1990s, Genentech investigated using a humanized inhaled monoclonal antibody anti-IgE (150 kDa) to treat asthma. The rationale was that there was evidence for both local and systemic effects of IgE in the etiology of asthma. The hypothesis was that local delivery of anti-IgE to the lung would inhibit IgE-mediated inflammation in the lung and provide improved asthma therapy. Studies in rats and monkeys found aerosol delivery did result in good deposition of the anti-IgE in lungs; however, only < 0.1% of the IgE delivered to lungs was absorbed into blood. ⁴² These data confirmed that local deposition was indeed achieved in the lungs, and the low absorption into blood was consistent with the high molecular weight of anti-IgE (150 kDa), suggesting a potential long-term residence in lung. ⁴² When tested in a 60-day inhalation toxicology study in Cynomolgus monkeys, anti-IgE had no adverse effects. Eosinophil cell infiltration of minimal severity was found in the bronchial mucosa of treated monkeys.^43,44 An antibody response to anti-IgE in the serum was also seen, which was expected due to the differing homology between the humanized monoclonal antibody (anti-IgE) and monkey IgG; however, there were no lymphoid cell infiltrations and aggregates in any tissues or apparent pathologies associated with formation of these immune complexes.^43,44 The definitive data from the clinical trials showed that inhaled anti-IgE was well tolerated; however, “aerosol administration of an anti-IgE monoclonal antibody does not inhibit the airway responses to inhaled allergen in allergic asthmatic subjects.” ⁴⁵ In this case, however, systemic delivery of anti-IgE by sc injection produced superior therapeutic results for treating asthma than local delivery by inhaled administration. ⁴⁵

The results of this program suggest that for new initiatives with inhaled antibodies there needs to be careful consideration of target receptors, receptor affinities, and relative influence of systemic and local effects. Although not discussing inhaled therapies, Catley and coworkers ⁴⁶ have reviewed use of monoclonal antibodies to treat asthma. There has been some conjecture that future targets with relatively high affinity receptors in the lung and high lung specificity might be attractive opportunities. Another factor supporting inhaled use for future therapies would be if there were no systemic delivery alternatives for the proposed therapies.