Objective More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery, and could prove effective for endoscopic signs and symptoms associated with nonacid reflux. The aim herein was to identify small molecule inhibitors of pepsin and test their efficacy to prevent pepsin‐mediated laryngeal damage in vivo. Methods Drug and pepsin binding and inhibition were screened by high‐throughput assays and crystallography. A mouse model of laryngopharyngeal reflux (mechanical laryngeal injury once weekly for 2 weeks and pH 7 solvent/pepsin instillation 3 days/week for 4 weeks) was provided inhibitor by gavage or aerosol (fosamprenavir or darunavir; 5 days/week for 4 weeks; n = 3). Larynges were collected for histopathologic analysis. Results HIV protease inhibitors amprenavir, ritonavir, saquinavir, and darunavir bound and inhibited pepsin with IC50 in the low micromolar range. Gavage and aerosol fosamprenavir prevented pepsin‐mediated laryngeal damage (i.e., reactive epithelia, increased intraepithelial inflammatory cells, and cell apoptosis). Darunavir gavage elicited mild reactivity and no discernable protection; aerosol protected against apoptosis. Conclusions Fosamprenavir and darunavir, FDA‐approved therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin‐mediated laryngeal damage in a laryngopharyngeal reflux mouse model. These drugs target a foreign virus, making them ideal to repurpose. Reformulation for local inhaled delivery could further improve outcomes and limit side effects. Level of evidence NA. Laryngoscope, 133:S1–S11, 2023

Combination treatment with pioglitazone and metformin are utilized clinically in the treatment of type II diabetes. Treatment with this drug combination reduced the development of aerodigestive cancers in this patient population. Our goal is to expand this treatment into clinical lung cancer chemoprevention. We hypothesized that dietary delivery of metformin/pioglitazone would prevent lung adenoma formation in A/J mice in a B[a]P-induced carcinogenesis model while modulating chemoprevention and anti-inflammatory biomarkers in residual adenomas. We found metformin (500 & 850 mg/kg/day) and pioglitazone (15 mg/kg/day) produced statistically significant decreases in lung adenoma formation both as single agent treatments and in combination, compared to untreated controls, after 15 weeks. Treatment with metformin alone and in combination with pioglitazone resulted in statistically significant decreases in lung adenoma formation at both early and late stage interventions. Pioglitazone alone resulted in significant decreases in adenoma formation only at early treatment intervention. We conclude oral metformin is a viable chemopreventive treatment at doses ranging from 500 to 1000 mg/kg/day. Pioglitazone at 15 mg/kg/day is a viable chemopreventive agent at early stage interventions. Combination metformin and pioglitazone performed equal to metformin alone and better than pioglitazone at 15 mg/kg/day. Since the drugs are already FDA approved, rapid movement to human clinical studies is possible.

Furan, a possible human carcinogen, is a product of incomplete combustion and is present in cigarette smoke, engine exhaust, and processed food. Oral administration induces liver toxicity and carcinogenesis in F344 rats and B6C3F1 mice. To assess possible adverse effects from inhalation, A/J mice were nose-only exposed for 3 hours to furan (0, 30, 75, 150, 300, or 600 ppmv) and euthanized after 24 hours, 48 hours, or 1 week. Histopathology evaluation revealed bronchiolar club cell necrosis (diffuse, marked) with airway denudation following exposure to 300 and 600 ppmv furan with evidence of club cell regeneration and partial repair after 1 week. Initial signs of hepatotoxicity were observed in the 150 ppmv furan-exposed group. Acute necrosis and mineralization were observed in livers at 24 and 48 hours with hepatocyte regeneration by 1-week postexposure in mice exposed to 300 and 600 ppmv furan; the 300 ppmv exposed group had multifocal mineralization that evoked a mild granulomatous response. Measurement of urinary furan metabolites confirmed that the mice metabolized furan to the toxic intermediate, cis-2-butene-1,4-dial. These observations indicate that inhaled furan is toxic to lungs with club cells as the target as well as liver.

Pioglitazone is a PPARγ agonist commonly prescribed for the clinical treatment of diabetes. We sought to expand its use to lung cancer prevention in a benzo[a]pyrene (B[a]P) mouse model with direct lung delivery via inhalation. Initially we conducted inhalational toxicity experiments with 0, 15, 50, 150, and 450 µg/kg body weight/day pioglitazone in 40 A/J mice. We examined the animals for any physical toxicity and bronchoalveolar lavage fluids for inflammatory and cytotoxicity markers. Doses up to and including 450 µg/kg bw/day failed to demonstrate toxicity with aerosol pioglitazone. For chemoprevention experiments, A/J mice were randomized to treatment groups of inhaled doses of 0, 50, 150, or 450 µg/kg bw/day pioglitazone one week or eight weeks after the last dose of B[a]P. For the early treatment group we found up to 32% decrease in lung adenoma formation with 450 µg/kg bw/day pioglitazone. We repeated the treatments in a second late stage experiment and found up to 44% decreases in lung adenoma formation in doses of pioglitazone of 150 µg/kg bw/day and 450 µg/kg bw/day. Both the early and the late stage experiment demonstrated biologically relevant and statistically significant decreases in adenoma formation. We conclude aerosol pioglitazone is well tolerated in the A/J mouse model and a promising chemoprevention agent for the lower respiratory tract.

Tobacco smoke is a complex mixture of chemicals, many of which are toxic and carcinogenic. Hazard assessments of tobacco smoke exposure have predominantly focused on either single chemical exposures or the more complex mixtures of tobacco smoke or its fractions. There are fewer studies exploring interactions between specific tobacco smoke chemicals. Aldehydes such as formaldehyde and acetaldehyde were hypothesized to enhance the carcinogenic properties of the human carcinogen, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) through a variety of mechanisms. This hypothesis was tested in the established NNKinduced A/J mouse lung tumor model. A/J mice were exposed to NNK (intraperitoneal injection, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 17 ppmv) for 3 h in a nose-only inhalation chamber, and lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase the severity of NNK-induced lung adenomas. The aldehyde coexposure did not affect the levels of NNK-derived DNA adduct levels. Similar studies tested the ability of a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure to influence lung adenoma formation by NNK. While carbon dioxide alone was not carcinogenic, it significantly increased the number of NNK-derived lung adenomas without affecting NNKderived DNA damage. These studies indicate that the chemicals in tobacco smoke work together to form a potent lung carcinogenic mixture.