Vaginal Suppositories Containing SHetA2 to Treat Cervical Dysplasia: Pharmacokinetics of Daily Doses and Preliminary Safety Profile.

SHetA2 is a new drug with potential to treat cervical dysplasia, but only 0.02% of the dose is absorbed into the cervix after oral administration. By contrast, 23.9% of the dose is absorbed into the cervix after vaginal administration. This study determines the pharmacokinetic and pharmacodynamic parameters after daily vaginal doses of SHetA2 in suppositories and assesses its safety. Daily dosed mice maintained therapeutic concentrations of SHetA2 in the cervix for 65 h. The steady-state area under the curve concentration versus time (AUCcervix) after the last dose was similar to that after a single dose indicating that there was no drug accumulation in the cervix. By contrast, the maximum drug concentration (Cmax-cervix) was smaller in the daily dosed group (52.19 µg/g) than after a single dose (121.84 µg/g), whereas the half-life (t1/2-cervix) was also shorter in the daily dosed group (9.94 h) than after a single dose (23.32 h). Notably, daily vaginal doses of SHetA2 reduced the levels of cyclin D1 (the pharmacodynamic endpoint) to a larger extent (∼45%) than after the administration of a single dose (∼26%). No adverse effects were observed in the mice for the duration of the study; thus, daily vaginal doses of SHetA2 appear to be safe.

Cryogenic Fabrication of Dry Powders to Enhance the Solubility of a Promising Anticancer Drug, SHetA2, for Oral Administration.

SHetA2 is a novel anticancer drug with poor aqueous solubility. In formal toxicological studies, Kolliphor HS 15 was used as a solubilizing agent to increase the oral bioavailability of SHetA2. The purpose of this study was to formulate SHetA2 and Kolliphor HS 15 as solid powders to facilitate their filling in hard gelatin capsules for clinical trials. Two manufacturing processes, ultra-rapid freeze-drying (URFD) and spray freeze drying (SFD), were employed to fabricate solid powders of SHetA2-Kolliphor HS 15 and trehalose. The morphology, size, flowability, and compressibility of URFD-SHetA2 and SFD-SHetA2 powders were characterized. The crystallinity and apparent maximum solubility of SHetA2 in both powders were also determined. SFD-SHetA2 powders were spherical in shape, small, and with a wide size distribution while the URFD-SHetA2 powders were irregularly shaped and big but with a narrower distribution. DSC and XRD analyses indicated that SHetA2 was mostly amorphous in both powders. The flow of both powders was categorized as "good" (angle of repose < 35°). The uniformity of drug content in URFD-SHetA2 powders was more variable than that in SFD-SHetA2 powders. The solubility profile of SHetA2 in both powders SGF exhibited a transient supersaturation "spring effect" due to the drug's amorphousness followed by extended supersaturation "parachute effect" at approximately 6 µg/ml for both powders compared to 0.02 ± 0.01 µg/ml for unprocessed drug. In conclusion, both URFD and SFD formed solid SHetA2 Kolliphor powders that are possible formulation candidates to be filled in hard gelatin capsules for clinical trials.

Pharmacokinetics and Pharmacodynamics of Escalating Doses of SHetA2 After Vaginal Administration to Mice.

SHetA2 is a novel compound with strong potential to treat cervical dysplasia, but its low aqueous solubility limits its oral bioavailability. A vaginal suppository achieved SHetA2 cervix concentrations that were severalfold above the predicted therapeutic levels. Thus, we aimed at determining the minimum dose that would achieve SHetA2 therapeutic levels while reducing cyclin D1 levels, the pharmacodynamic end point. The disposition of SHetA2 after vaginal administration of escalating SHetA2 doses and the corresponding reduction in cyclin D1 levels was compared to that after the conventional oral treatment. Vaginal administration of a 15-mg/kg dose achieved an area under the cervix concentration versus time curve (AUCcervix) that was ∼120 times larger than that after a 60 mg/kg administered orally. AUCcervix and Cmax-cervix did not increase proportionally with respect to the dose, with the 30-mg/kg dose resulting in higher AUCcervix and Cmax-cervix (1368.53 µg.mL/h and 155.38 µg/g, respectively) compared to the 15 mg/kg (334.98 µg.mL/h and 121.78 µg/g, respectively) or 60 mg/kg (1178.55 µg.mL/h and 410.38 µg/g, respectively). Likewise, the 30-mg/kg dose caused a larger reduction in cyclin D1 levels than the other doses. Thus, the 30-mg/kg dose was selected for future efficacy studies in a mouse model of cervical neoplasia.

Influence of the estrus cycle of the mouse on the disposition of SHetA2 after vaginal administration.

SHetA2 is a novel compound with the potential to treat cervical dysplasia, but has poor water solubility. A vaginal suppository formulation was able to achieve therapeutic concentrations in the cervix of mice, but these concentrations were variable. Histological analysis indicated that mice in the same group were in different stages of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effects of these changes on the pharmacokinetics and pharmacodynamics of SHetA2 when administered vaginally. Mice were synchronized to be either in estrous or diestrus stage for administration of the SHetA2 suppository. Pharmacokinetic parameters were calculated from the SHetA2 concentrations vs. time data. The reduction in the expression of cyclin D1 protein in the cervix was used as pharmacodynamic endpoint. Mice dosed during diestrus had a larger AUCcervix (335 µg mL h-1), higher Cmax (121.8 ±â€¯38.7 µg/g) and longer t1/2-cervix (30.3 h) compared to mice dosed during estrus (120 µg mL h-1, 44.6 ±â€¯29.5 µg/g and 3.6 h respectively). Therapeutic concentrations of SHetA2 were maintained for 48 h in the cervix of mice dosed during diestrus and for only 12 h in the estrus group. The treatment reduced the expression of cyclin D1 protein in the cervix of mice in the estrus to a larger extent. These results indicate that the estrous cycle of mice influences significantly the disposition of SHetA2 after vaginal administration and may also influence its efficacy.

Development of a dietary formulation of the SHetA2 chemoprevention drug for mice.

Development of cancer chemoprevention compounds requires enhanced consideration for toxicity and route of administration because the target population is healthy. The small molecule drug, SHetA2 (NSC 726189), exhibited in vivo chemoprevention activity and lack of toxicity when administered by oral gavage. Our objective was to determine if a dietary formulation of SHetA2 could achieve effective tissue drug levels without toxicity. C57bl/6 J mice were monitored on modified American Institute of Nutrition (AIN)76A diet mixed with SHetA2 in a 3:1 ratio with Kolliphor HS15, a self-emulsifying drug delivery system (SEDDS) to deliver 37.5, 62.5, 125, 187 or 250 mg SHetA2/kg/day. Blood and tissues were evaluated after 1, 3 and 6 weeks. The 187 mg/kg/day dose was identified as optimal based on achievement of maximum blood and tissue drug levels in the effective micromolar range without evidence of toxicity. The 250 mg/kg/day group exhibited lower drug levels and the highest intestinal drug content suggesting that an upper limit of intestinal absorption had been surpassed. Only this highest dose resulted in liver and kidney function tests that were outside of the normal range, and significant reduction of cyclin D1 protein in normal cervical tissue. SHetA2 reduced cyclin D1 to greater extents in cancer compared to non-cancer cell cultures. Given this differential effect, optimal chemoprevention without toxicity would be expected to occur at doses that reduced cyclin D1 in neoplastic, but not in normal tissues. These findings support further development of SHetA2 as a chemoprevention agent and potential food additive.

SHetA2 Dry Powder Aerosols for Tuberculosis: Formulation, Design, and Optimization Using Quality by Design.

Tuberculosis (TB) is a life threatening pulmonary infection caused by Mycobacterium tuberculosis (MTB). Current treatments are complex, lengthy, and associated with severe side effects that decrease patient compliance and increase the probability of the emergence of drug resistant strains. Thus, more effective drugs with little to no side effects are needed to diversify the armamentarium against the global TB epidemic. SHetA2, an anticancer compound with null toxicity at doses much higher than the effective dose, was recently discovered to be active against MTB. In the present study, a dry powder formulation of SHetA2 for pulmonary delivery was developed to overcome its poor aqueous solubility and to maximize its concentration in the lungs, the main site of TB infection. Using quality by design (QbD) methodology, three different formulations of SHetA2 microparticles (MPs) were designed, manufactured, and optimized, SHetA2 alone, SHetA2 PLGA, and SHetA2 mannitol MPs, to maximize the drug dose, target alveolar macrophages, and increase drug solubility, respectively. The resulting three SHetA2 MP formulations had spherical shape with particle size ranging from 1 to 3 µm and a narrow size distribution, suitable for uniform delivery to the alveolar region of the lungs. Upon dispersion with the Aerolizer dry powder inhaler (DPI), all three SHetA2 MP formulations had aerodynamic diameters smaller than 3.3 µm and fine particle fractions (FPF4.46) greater than 77%. SHetA2 remained chemically stable after MP manufacture by spray drying, but the drug transformed from the crystalline to the amorphous form, which significantly enhanced the solubility of SHetA2. Using a custom-made dissolution apparatus, the FPF4.46 of SHetA2 MP dissolved much faster and to a greater extent (21.19 ± 4.40%) than the unprocessed drug (3.51 ± 0.9%). Thus, the physicochemical characteristics, in vitro aerosol performance, and dissolution rate of the optimized SHetA2 MPs appear to be suitable to achieve therapeutic concentrations in the lungs.

Source of cytotoxicity in a colloidal silver nanoparticle suspension.

Silver nanoparticles (AgNPs) are increasingly used in a variety of applications because of their potential antimicrobial activity and their plasmonic and conductivity properties. In this study, we investigated the source of cytotoxicity, genotoxicity, and reactive oxygen species (ROS) production on human dermal fibroblast and human lung cancer (A549) cell lines upon exposure to AgNP colloidal suspensions prepared with the simplest and most commonly used Lee­Meisel method with a variety of reaction times and the concentrations of the reducing agent. The AgNPs synthesized with shorter reaction times were more cytotoxic and genotoxic due to the presence of a few nanometer-sized AgNP seeds. The suspensions prepared with an increased citrate concentration were not cytotoxic, but they induced more ROS generation on A549 cells due to the high citrate concentration. The genotoxicity of the suspension decreased significantly at the higher citrate concentrations. The analysis of both transmission electron microscopy images from the dried droplet areas of the colloidal suspensions and toxicity data indicated that the AgNP seeds were the major source of toxicity. The completion of the nucleation step and the formation of larger AgNPs effectively decreased the toxicity.