Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Inhalation delivery of nucleic acid gene therapies in preclinical drug development.

INTRODUCTION: Inhaled gene therapy programs targeting diseases of the lung have seen increasing interest in recent years, though as of yet no product has successfully entered the market. Preclinical research to support such programs is critically important in maximizing the chances of developing successful candidates. AREAS COVERED: Aspects of inhalation delivery of gene therapies are reviewed, with a focus on preclinical research in animal models. Various barriers to inhalation delivery of gene therapies are discussed, including aerosolization stresses, aerosol behavior in the respiratory tract, and disposition processes post-deposition. Important aspects of animal models are considered, including determinations of biologically relevant determinations of dose and issues related to translatability. EXPERT OPINION: Development of clinically-efficacious inhaled gene therapies has proven difficult owing to numerous challenges. Fit-for-purpose experimental and analytical methods are necessary for determinations of biologically relevant doses in preclinical animal models. Further developments in disease-specific animal models may aid in improving the translatability of results in future work, and we expect to see accelerated interests in inhalation gene therapies for various diseases. Sponsors, researchers, and regulators are encouraged to engage in early and frequent discussion regarding candidate therapies, and additional dissemination of preclinical methodologies would be of immense value in avoiding common pitfalls.

Composition of aerosols from thermal degradation of flavors used in ENDS and tobacco products.

Aim: The cardiovascular toxicity of unheated and heated flavorants and their products as commonly present in electronic cigarette liquids (e-liquids) was evaluated previously in vitro. Based on the results of in vitro assays, cinnamaldehyde, eugenol, menthol, and vanillin were selected to conduct a detailed chemical analysis of the aerosol generated following heating of each compound both at 250 and 750 °C. Materials and Methods: Each flavoring was heated in a drop-tube furnace within a quartz tube. The combustion atmosphere was captured using different methods to enable analysis of 308 formed compounds. Volatile organic compounds (VOCs) were captured with an evacuated Summa canister and assayed via gas chromatography interfaced with mass spectrometry (GC-MS). Carbonyls (aldehydes and ketones) were captured using a 2,4-dinitrophenylhydrazine (DNPH) cartridge and assayed via a high-performance liquid chromatography-ultra-violet (HPLC-UV) assay. Polyaromatic hydrocarbons (PAHs) were captured using an XAD cartridge and filter, and extracts were assayed using GC-MS/MS. Polar compounds were assayed after derivatization of the XAD/filter extracts and analyzed via GC-MS. Conclusion: At higher temperature, both cinnamaldehyde and menthol combustion significantly increased formaldehyde and acetaldehyde levels. At higher temperature, cinnamaldehyde, eugenol, and menthol resulted in increased benzene concentrations. At low temperature, all four compounds led to higher levels of benzoic acid. These data show that products of thermal degradation of common flavorant compounds vary by flavorant and by temperature and include a wide variety of harmful and potentially harmful constituents (HPHCs).

Adenovirus type 5 SARS-CoV-2 vaccines delivered orally or intranasally reduced disease severity and transmission in a hamster model.

Transmission-blocking strategies that slow the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and protect against coronavirus disease 2019 (COVID-19) are needed. We have developed an orally delivered adenovirus type 5-vectored SARS-CoV-2 vaccine candidate that expresses the spike protein. Here, we demonstrated that hamsters vaccinated by the oral or intranasal route had robust and cross-reactive antibody responses. We then induced a postvaccination infection by inoculating vaccinated hamsters with SARS-CoV-2. Orally or intranasally vaccinated hamsters had decreased viral RNA and infectious virus in the nose and lungs and experienced less lung pathology compared to mock-vaccinated hamsters after SARS-CoV-2 challenge. Naïve hamsters exposed in a unidirectional air flow chamber to mucosally vaccinated, SARS-CoV-2-infected hamsters also had lower nasal swab viral RNA and exhibited fewer clinical symptoms than control animals, suggesting that the mucosal route reduced viral transmission. The same platform encoding the SARS-CoV-2 spike and nucleocapsid proteins elicited mucosal cross-reactive SARS-CoV-2-specific IgA responses in a phase 1 clinical trial (NCT04563702). Our data demonstrate that mucosal immunization is a viable strategy to decrease SARS-CoV-2 disease and airborne transmission.

Inhaled remdesivir reduces viral burden in a nonhuman primate model of SARS-CoV-2 infection.

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.

Local Treatment of Non-small Cell Lung Cancer with a Spray-Dried Bevacizumab Formulation.

Local delivery of biotherapeutics to the lung holds great promise for treatment of lung diseases, but development of physically stable, biologically active dry powder formulations of large molecules for inhalation has remained a challenge. Here, spray drying was used to manufacture a dry powder pulmonary formulation of bevacizumab, a monoclonal antibody approved to treat non-small cell lung cancer (NSCLC) by intravenous infusion. By reformulating bevacizumab for local delivery, reduced side effects, lower doses, and improved patient compliance are possible. The formulation had aerosol properties suitable for delivery to the deep lung, as well as good physical stability at ambient temperature for at least 6 months. Bevacizumab's anti-VEGF bioactivity was not impacted by the manufacturing process. The formulation was efficacious in an in vivo rat model for NSCLC at a 10-fold decrease in dose relative to the intravenous control.

Inhalation delivery dramatically improves the efficacy of topotecan for the treatment of local and distant lung cancer.

Topotecan is potent anti-cancer drug approved for various malignancies but hematopoietic toxicities undermine its wider application and use of its most effective dose. This study aims to improve these limitations through inhalation-delivery. The pharmacokinetics, efficacy, and toxicity of 2-5 times lower inhalation doses of topotecan dry-powder were compared with the standard intravenous (IV) delivery once/twice-a-week. Human-derived EGFR-mutant (H1975), KRAS-mutant (A549), and EGFR/KRAS wild-type (H358) orthotopic and distant lung tumors were evaluated in murine models. Inhalation of 1 mg/kg topotecan significantly improved the half-life and drug exposure (area under the curve, AUC) compared to 5 mg/kg via IV-delivery. AUCs (h*ng/mL) for inhaled/IV topotecan in plasma, lung, liver, and brain were, 831/888, 60,000/1080, 8380/4000, and 297/15, respectively; while the half-life was also greatly increased in these tissues. The average lung tumor burden of H358-derived tumors was reduced from 15.0 g to 8.4 g (44%) in rats treated once-a-week with 2 mg/kg IV and 1.8 g (88%) with 1 mg/kg inhaled topotecan, corroborating previous findings using A549- and H1975-derived orthotopic lung tumors. Importantly, inhaled topotecan showed superior efficacy in suppressing lung tumors at distant sites. The growth of H1975- and H358-derived subcutaneous xenografts were completely arrested and A549-derived tumors were significantly reduced in mice treated twice-a-week with 1 mg/kg inhaled topotecan compared to a minor (H1975 and H358) or no reduction (A549) with twice-a-week 5 mg/kg IV topotecan.

5-Azacytidine inhaled dry powder formulation profoundly improves pharmacokinetics and efficacy for lung cancer therapy through genome reprogramming.

BACKGROUND: Epigenetic therapy through demethylation of 5-methylcytosine has been largely ineffective in treating lung cancer, most likely due to poor tissue distribution with oral or subcutaneous delivery of drugs such as 5-azacytidine (5AZA). An inhalable, stable dry powder formulation of 5AZA was developed. METHODS: Pharmacokinetics of inhaled dry powder and aqueous formulations of 5AZA were compared to an injected formulation. Efficacy studies and effect of therapy on the epigenome were conducted in an orthotopic rat lung cancer model for inhaled formulations. RESULTS: Inhaled dry powder 5AZA showed superior pharmacokinetic properties in lung, liver, brain and blood compared to the injected formulation and for all tissues except lung compared to an inhaled aqueous formulation. Only dry powder 5AZA was detected in brain (~4-h half-life). Inhaled dry powder was superior to inhaled aqueous 5AZA in reducing tumour burden 70-95%. Superiority of inhaled 5AZA dry powder was linked to effectively reprogramming the cancer genome through demethylation and gene expression changes in cancer signalling and immune pathways. CONCLUSIONS: These findings could lead to widespread use of this drug as the first inhaled dry powder therapeutic for treating local and metastatic lung cancer, for adjuvant therapy, and in combination with immunotherapy to improve patient survival.

Comparative effects of parent and heated cinnamaldehyde on the function of human iPSC-derived cardiac myocytes.

Many e-cigarette products contain cinnamaldehyde as a primary constituent of cinnamon flavorings. When used as a food additive, cinnamaldehyde is generally regarded as safe for ingestion. However, little is known about the effects of cinnamaldehyde or its degradation products, generated after heating and inhalation, which may lead to elevated circulatory exposure to the heart. Hence, in this study, we tested the in vitro cardiac toxicity of cinnamaldehyde and its thermal degradation products generated by heating at low (200 ±â€¯50 °C) and high temperatures (700 ±â€¯50 °C) on the contractility, rhythmicity and electrical signaling properties of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs). Cellular impedance measurements on spontaneously beating hiPSC-CMs revealed that cinnamaldehyde significantly alters contraction-dependent signal amplitude, beating rate, and cell morphology. These effects were attenuated after cinnamaldehyde was subjected to heating at low or high temperatures. Current clamp analysis of hiPSC-CM action potentials (APs) showed only modest effects of acute application of 1-100 µM cinnamaldehyde on resting membrane potential, while prolonged (~20 min) application of 100 µM cinnamaldehyde resulted in progressive depolarization and loss of rhythmic AP spiking activity. Collectively, these results suggest that micromolar levels of cinnamaldehyde could alter cardiac excitability, in part by impairing the processes that regulate membrane potential and depolarization. Our results further suggest that heating cinnamaldehyde by itself does not directly lead to the formation of products with greater cardiotoxicity in vitro.

Inhaled Submicron Particle Paclitaxel (NanoPac) Induces Tumor Regression and Immune Cell Infiltration in an Orthotopic Athymic Nude Rat Model of Non-Small Cell Lung Cancer.

Background: This study evaluated the antineoplastic and immunostimulatory effects of inhaled (IH) submicron particle paclitaxel (NanoPac®) in an orthotopic non-small cell lung cancer rodent model. Methods: Male nude rats were whole body irradiated, intratracheally instilled with Calu-3 cancer cells and divided into six treatment arms (n = 20 each): no treatment (Group 1); intravenous nab-paclitaxel at 5.0 mg/kg once weekly for 3 weeks (Group 2); IH NanoPac at 0.5 or 1.0 mg/kg, once weekly for 4 weeks (Groups 3 and 4), or twice weekly for 4 weeks (Groups 5 and 6). Upon necropsy, left lungs were paraffin embedded, serially sectioned, and stained for histopathological examination. A subset was evaluated by immunohistochemistry (IHC), anti-pan cytokeratin staining AE1/AE3+ tumor cells and CD11b+ staining dendritic cells, natural killer lymphocytes, and macrophage immune cells (n = 2, Group 1; n = 3 each for Groups 2-6). BCL-6 staining identified B lymphocytes (n = 1 in Groups 1, 2, and 6). Results: All animals survived to scheduled necropsy, exhibited no adverse clinical observations due to treatment, and gained weight at the same rate throughout the study. Histopathological evaluation of Group 1 lung samples was consistent with unabated tumor growth. Group 2 exhibited regression in 10% of animals (n = 2/20). IH NanoPac-treated groups exhibited significantly higher tumor regression incidence per group (n = 11-13/20; p < 0.05, χ2). IHC subset analysis revealed tumor-nodule cluster separation, irregular borders between tumor and non-neoplastic tissue, and an increased density of infiltrating CD11b+ cells in Group 2 animals (n = 2/3) and in all IH NanoPac-treated animals reviewed (n = 3/3 per group). A single animal in Group 4 and Group 6 exhibited signs of pathological complete response at necropsy with organizing stroma and immune cells replacing areas presumed to have previously contained adenocarcinoma nodules. Conclusion: Tumor regression and immune cell infiltration were observed in all treatment groups, with an increased incidence noted in animals receiving IH submicron particle paclitaxel treatment.

Pharmacokinetic Profile of Inhaled Submicron Particle Paclitaxel (NanoPac®) in a Rodent Model.

BACKGROUND: Inhaled chemotherapeutics may enhance pulmonary drug exposure to malignant lesions in the lung without substantially contributing to systemic toxicities. The pharmacokinetic profile of inhaled submicron particle paclitaxel (NanoPac®) in healthy rodent plasma and lung tissue is evaluated here to determine administration proof-of-principle. METHODS: Healthy male Sprague Dawley rats received paclitaxel in one of three arms: intravenous nab-paclitaxel at 2.9 mg/kg (IVnP), inhaled NanoPac low dose (IHNP-LD) at 0.38 mg/kg, or inhaled NanoPac high dose (IHNP-HD) at 1.18 mg/kg. Plasma and lung tissue paclitaxel concentrations were determined using ultraperformance liquid chromatography tandem mass spectrometry from animals sacrificed at 10 time points ranging up to 2 weeks after administration. Peak concentration (Cmax), apparent residence half-life (T1/2), exposure (AUC(last)), and dose-normalized exposure (AUCD(last)) were determined. Pulmonary histopathology was performed on rats sacrificed at the 336-hour time point. RESULTS: Paclitaxel was detectable and quantifiable in the rat lung for both inhaled NanoPac arms sampled at the final necropsy, 336 hours postadministration. Substantial paclitaxel deposition and retention resulted in an order of magnitude increase in dose-normalized pulmonary exposure over IVnP. Inhaled NanoPac arms had an order of magnitude lower plasma Cmax than IVnP, but followed a similar plasma T1/2 clearance (quantifiable only to 72 hours postadministration). Pulmonary histopathology found all treated animals indistinguishable from treatment-naive rats. CONCLUSION: In the rodent model, inhaled NanoPac demonstrated substantial deposition and retention of paclitaxel in sampled lung tissue. Further research to determine NanoPac's toxicity profile and potential efficacy as lung cancer therapy is underway.

Inhalation delivery of topotecan is superior to intravenous exposure for suppressing lung cancer in a preclinical model.

Intravenous (IV) topotecan is approved for the treatment of various malignancies including lung cancer but its clinical use is greatly undermined by severe hematopoietic toxicity. We hypothesized that inhalation delivery of topotecan would increase local exposure and efficacy against lung cancer while reducing systemic exposure and toxicity. These hypotheses were tested in a preclinical setting using a novel inhalable formulation of topotecan against the standard IV dose. Respirable dry-powder of topotecan was manufactured through spray-drying technology and the pharmacokinetics of 0.14 and 0.79 mg/kg inhalation doses were compared with 0.7 mg/kg IV dose. The efficacy of four weekly treatments with 1 mg/kg inhaled vs. 2 mg/kg IV topotecan were compared to untreated control using an established orthotopic lung cancer model for a fast (H1975) and moderately growing (A549) human lung tumors in the nude rat. Inhalation delivery increased topotecan exposure of lung tissue by approximately 30-fold, lung and plasma half-life by 5- and 4-folds, respectively, and reduced the maximum plasma concentration by 2-fold than the comparable IV dose. Inhaled topotecan improved the survival of rats with the fast-growing lung tumors from 7 to 80% and reduced the tumor burden of the moderately-growing lung tumors over 5- and 10-folds, respectively, than the 2-times higher IV topotecan and untreated control (p < .00001). These results indicate that inhalation delivery increases topotecan exposure of lung tissue and improves its efficacy against lung cancer while also lowering the effective dose and maximum systemic concentration that is responsible for its dose-limiting toxicity.

Use Behaviors, Dependence, and Nicotine Exposure Associated with Ad Libitum Cigar Smoking.

OBJECTIVES: To examine factors important to cigar smoking and subsequent nicotine exposure, we evaluated the impact of cigar type, cigarette smoking history, and inhalation behaviors on nicotine dependence, smoking topography, and biomarkers of exposure in current exclusive cigar smokers. METHODS: Adult, exclusive cigar smokers (N = 77) were recruited based on cigar type, cigarette smoking history, and self-reported inhalation behaviors. Participants smoked their own brand product ad libitum for up to one hour; dependence symptoms, smoking topography, and biomarkers of exposure were assessed. RESULTS: Cigar smokers showed low levels of dependence. Cigar smoking alleviated withdrawal and craving symptoms, increased plasma nicotine concentration, and increased exhaled CO. Multiple regression analyses indicate that inhalation behaviors were associated with increased dependence and greater reductions in withdrawal symptoms upon cigar smoking. Large cigar smokers smoked longer and smoked more tobacco than small cigar and cigarillo smokers. Furthermore, large cigar smokers and self-reported inhalers were exposed to more nicotine than small cigar smokers and non-inhalers. CONCLUSIONS: Our study suggests that cigar type and smoking behaviors affect dependence and nicotine exposure upon cigar smoking. These findings provide additional insight into the substantial risks associated with cigar smoking.

Therapeutic administration of inhaled INS1009, a treprostinil prodrug formulation, inhibits bleomycin-induced pulmonary fibrosis in rats.

Idiopathic pulmonary fibrosis is a progressive and lethal disease and while there are now two approved drugs (Esbriet® and Ofev®) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug hexadecyl-treprostinil (C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycin sulfate (3.5-4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 µg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone (100 mg/kg). Dosing started on day 10 post-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 µg/lung lobe in saline challenged and PBS treated animals to 673 µg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 µg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 µg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content to 522 µg/lung lobe. Histologically, both INS1009 (100 µg/kg) and pirfenidone (100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-ß1 (TGF-ß1)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001-10 µM) inhibited TGF-ß1 (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10-100 µg/kg) inhibited bleomycin-induced pulmonary fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.

Changes in HPBMC markers of immmune function following controlled short-term inhalation exposures of humans to hardwood smoke.

Previous studies have shown that complex mixtures containing particulate matter (PM) and polycyclic aromatic hydrocarbons (PAHs) produce systemic immunotoxicity in animal models following inhalation exposures. While we and others have shown that emissions associated with hardwood smoke (HWS), cigarette smoke and diesel exhaust can suppress the immune systems of animals in vitro and in vivo, there have been few immune function studies on human peripheral blood mononuclear cells (HPBMC) following exposure of humans to HWS. Our work shows that T cells are an important targets of PM and PAH immunotoxicity. These studies were conducted on HPBMC from 14 human volunteers receiving four 2 h nightly exposures to clean air or HWS at a concentration of 500 ug/m(3). We measured anti-CD3/anti-CD28 stimulated T-cell proliferation and HPBMC cytokine production in cell supernatants, including interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), TH1 cytokines γIFN and IL-2, TH2 cytokine IL-4, Th17 cytokine interleukin 17A (IL-17A) and interleukin 10 (IL-10). We analyzed results using analysis of variance (ANOVA), t-tests and Pearson correlation. Results showed that there was significant variation in the amount of T-cell proliferation observed following polyclonal activation with anti-CD3/anti-CD28 antibodies in both the air and HWS-exposed groups. There was not a significant effect of HWS on T-cell proliferation. However, we did find a strong relationship between the presence of proinflammatory cytokines (IL-1ß, TNF-α, IL-6, but not IL-8) and the amount of T-cell proliferation seen in individual donors, demonstrating that brief exposures of humans to HWS can produce changes in systemic immunity that is associated with proinflammatory cytokines.

Preformulation, formulation, and in vivo efficacy of topically applied apomine.

Apomine is a novel compound that inhibits the mevalonate/isoprenoid pathway of cholesterol synthesis and may prove effective as a skin cancer chemoprevention therapy. This research was focused on the development of a new delivery approach for chemoprevention of melanoma using topically delivered apomine. This included evaluating the effect of several factors on the stability of apomine in solution, utilizing these to develop a topical formulation, and conducting efficacy studies with the developed topical formulation in the TPras mouse model. Preformulation included the influence of pH, buffer species, ionic strength, and organic solvents on the stability of apomine at four different temperatures. Apomine was determined to undergo apparent first-order degradation kinetics for all conditions evaluated. Apomine undergoes base-catalyzed degradation. Less than 15% degradation is observed after >200 days under acidic conditions. Long-term stability studies were performed on two different topical cream formulations and indicate that both formulations are chemically stable for over 1 year at both 4 and 23 degrees C. The efficacy of topically applied apomine, from ethanol and developed 1% cream, was evaluated in vivo against the incidence of melanoma. Regardless of delivery vehicle apomine treatment caused a significant reduction in tumor incidence. Ethyl alcohol application of apomine resulted in a greater tumor incidence reduction when compared to the development cream formulation; however, this difference was not significant.

Development of an HPLC method for the analysis of Apomine in a topical cream formulation.

A stability indicating, reversed-phase high performance liquid chromatographic method was developed for the quantification of Apomine, tetraisopropyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-ethyl-1, 1-bisphosphonate, in a topical cream formulation. Analysis of Apomine in the cream formulation was performed through a dilution of the cream base with tetrahydrofuran. This allowed the current method to bypass extraction and/or centrifugation for direct injection and analysis. Separation was achieved using an Alltima C18 5 microm, 150 mm x 2.1 mm column and employed a gradient procedure, beginning with acetonitrile-water (65:35, v/v), at 0.6 mL/min for 9 min, followed by a rinse with isopropyl alcohol for 9 min. The complete gradient method has been optimized to separate Apomine from the nonpolar cream components, wash and equilibrate the column in a 30-min assay. This report demonstrates that this method is effective for quantification of Apomine in a cream formulation.