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

The SHetA2 suppositories prepared for the present study were yellow due to the natural color of SHetA2, conical in shape to facilitate vaginal insertion, with a 5 mm length and 3 mm diameter of the base. SHetA2 suppositories had a 30 mg/kg dose, a content uniformity of 105.44±0.42%, with a RSD of 3.39% and softening time of less than 8 minutes, meeting the USP specification for suppositories for human use. The SHetA2 cervix concentration versus time profiles after vaginal administration of a single dose (30 mg/kg in suppository) or after vaginal administration of the fifth dose in the multiple dosed group of FVB mice (from a total of five 30 mg/kg doses, in suppositories administered every 24 h, each) are shown in figure 1 . Although single and multiple dosing regimens achieved the maximum SHetA2 concentration in the cervix of treated mice at the same time (t max-cervix =0.5 h), the C max-cervix after a single dose was significantly higher (121.84μg/g) than the C max-cervix after the fifth dose of the multiple dose regimen (52.19 μg/g). Likewise, the SHetA2 cervix concentration at C min-cervix after a single dose was significantly higher (6.38 μg/g) than the C max-cervix after the fifth dose of the multiple dose regimen (0.67 μg/g). Both, single and multiple dosing regimens were able to maintain SHetA2 concentrations in the cervix of treated animals above the predicted therapeutic level (4.0 ± 0.4 μM or 1.6μg/mL) 13 for at least 65 hours. Both cervix concentration versus time profiles exhibited extra peaks at 12 and 48 h, but the differences between the cervix concentration prior to each of these peaks and at the peaks ( figure 1 ) were larger in the profile of mice receiving a single dose. Figure 2 shows the SHetA2 concentration in the uterine horns and fallopian tubes-ovaries versus time profiles after administration of the fifth SHetA2 dose in vaginal suppositories to mice in the multiple dose group. The profiles for both tissues exhibited the similar pattern up to 48 hours when the drug concentration in the uterine horns decreased, whereas the drug concentration increased at a proportional rate in the fallopian tubes-ovaries. Although the magnitude of the initial drug concentrations in these tissues was smaller (0.2 – 20 μg/g) than those in cervix tissue (0.6 – 200 μg/g), they were above the predicted therapeutic levels for at least 50 hours. The pharmacokinetic parameters obtained by the non-compartmental analysis of the SHetA2 cervix concentration versus time data after a single or multiple vaginal doses are listed in table 1 . As expected from the cervix concentration versus time profiles, the AUC cervix after a single dose (1368.53 μg.mL.h −1 ) was larger than that after the fifth dose of the multiple dosing regimen (839.08 μg.mL.h −1 ) and the CL/F Cervix after a single dose was shorter (0.46 mlL/h) than that after the fifth dose (1.24 mL/h). Consequently, the elimination half-life (t 1/2-cervix ) of SHetA2 from the cervix after a single dose was longer (23.32 h) than that after the multiple dose (9.94 h). Likewise, the MRT cervix after a single dose was longer (35.85 h) than after the multiple dose (27.66 h). The accumulation ratio of SHetA2 in the cervix of treated mice after the fifth dose calculated as the ratio of the drug concentration at steady state and the drug concentration at the end of the first dosing interval ( equation 1 ) 28 was 0.03, indicating that there was no significant drug accumulation after five days of consecutive daily dosing. The reduction in the levels of cyclin D1 in the cervix tissue of mice after multiple or single vaginal administration of SHetA2 in suppositories is shown in figure 3 . At the time of maximum SHetA2 concentration (T max = 0.5 h) in the cervix of mice in the multiple dose group, the resulting levels of cyclin D1 expression (5 day C max ) were significantly lower (45%, p<0.05) compared to that of untreated controls and 7% lower compared to that of placebo controls ( figure 3A ). Even though the levels of cyclin D1 expression in the cervices of mice in the multiple dose group remained reduced with respect to that of untreated controls until 72 hours after the fifth dose (5 day C min ), they were not statistically different due to the large variability among mice in the treated group ( figure 3A ). Likewise, there were no significant differences in the levels of cyclin D1 expression between mice treated with multiple doses of SHetA2 suppositories and those treated with multiple placebo suppositories. This suggests that the PD effect is due to SHetA2 and it is not affected by the suppository excipients. A comparison of the levels of cyclin D1 in the cervix tissue of mice after administration of multiple versus a single dose of SHetA2 is shown in figure 3B . Multiple vaginal doses of SHetA2 reduced the levels of cyclin D1 to a larger extent (5 day C max ,~45%) than after administration of a single dose (Single dose C max , ~26%). The levels of cyclin D1 at the end of the study period (C min ) remained reduced in the cervices of mice treated with single or multiple SHetA2 doses, but the variability in the multiple dose group (5 day C min ) was larger than that in the single dosed group (Single dose C min ), which resulted in these reductions being not statistically different. A similar not statistical trend was observed when comparing mice the mice treated with 5 doses of SHetA2 and those treated with placebo suppositories at C max and C min , suggesting that the decrease in the levels of cyclin D1 expression is solely due to SHetA2. Multiple vaginal doses of SHetA2 did not appear to have an effect on the weight gain in the mice of any of the experimental groups during the 5 day- period study, as indicated in figure 4 . Mice treated with the SHetA2 or placebo suppositories appeared to gain slightly more weight than the untreated controls but there was no significant difference between the three study groups. The parameters employed to evaluate the behavior of the mice during the 5 day study period as well as the macroscopic observations of the area of suppository administration and their assigned scores are shown in table 2 . There was no difference between the behavior of mice treated with suppositories (either placebo or containing SHetA2) and the behavior of untreated mice. Only one mouse in the SHetA2-treated group showed aggression (mainly biting) to mice housed in the same cage, which was considered “normal behavior”. A few animals (8 out of 68) exhibited mild itching near the vulvar area immediately after administration of the suppository (shown as self-grooming), but this behavior stopped approximately one hour after administration of the suppository. Thus, a score of 1 was assigned to these mice. Since none of the other parameters listed in table 2 was observed during the period of the study, they were scored as “0”. The cellular composition observed in the hematoxylin/eosin stained slides of the vaginal tissues of mice in the three study groups (multiple SHetA2 or placebo suppository doses and untreated controls collected after the fifth dose) indicated that the majority of mice were in estrus, or between late proestrus and early metestrus. A detailed histological evaluation of the cervix, uterine horns, fallopian tubes and ovaries did not show overt signs of acute toxicity such as immune cell infiltration, mitosis, or apoptosis that may be otherwise observed after repeated administration of a drug or substance that may be an irritant to mucosal tissues. Given that at the end of the safety study mice were in different stages of the estrous cycle, and due to the lack of effect of suppository administration on normal tissue, no figures are included, as they are similar to the ones previously published by our group 24 .

SHetA2 was manufactured by Cayman Chemical Co. under a contract from the Rapid Access to Preventive Intervention Development (RAPID) National Cancer Institute (NCI) program. Cocoa butter was purchased from Nature’s Oils (Streetsboro, OH). Kolliphor HS15 was obtained from BASF (Ludwigshafen, Germany). Sterile saline and isoflurane were obtained from Henry Schein Animal Health Inc. Acetonitrile (HPLC grade ≥99.5%), methanol (HPLC grade≥99.5%), phosphoric acid, hydrochloric acid, crystal violet stain and sodium acetate trihydrate were purchased from Sigma Aldrich (St Louis, MO). Glycerol USP was purchased from VWR International (Radnor, PA). Ultrapure deionized (DI) water was obtained from Pure Lab Ultrapure Water System (ELGA, UK). Heparin (1000 IU/mL) was purchased from Pfizer Injectables (NY, NY). Waters Alliance HPLC System equipped with Waters Xbrigde C 18 , 3.5μm 2.1 × 150mm column and Waters Xbridge BEH C 18 , 3.5μm, 2.1 × 5mm guard column were used to determine drug content in tissues. Captiva® filtration equipment was purchased from Agilent Technologies Inc. for extraction of the drug from tissues. Mouse cyclin D1 Enzyme Linked Immunosorbent Assay (ELISA) kits were purchased from Cedarlane lab (NC). T-PER (tissue protein extraction reagent) was purchased from Thermo-fisher Scientific (Waltham, MA). Protease inhibitor cocktail tablets were purchased from Sigma Aldrich (St Louis, MO). Vaginal suppositories containing a SHetA2 dose of 30 mg/kg body weight were manufactured by the fusion-molding method using a custom-made mold and cocoa butter as the base 19 . The procedure of mold fabrication and optimization of the formulation composition (base and excipients) are described in a previous publication 19 . In addition, “blank” suppositories containing only cocoa butter and Kolliphor were prepared to serve as placebo control in the pharmacodynamic study. The batch of suppositories used in these animal studies were subjected to quality control evaluation as stated by the guidelines specified by the United States Pharmacopoeia (USP) 22 for suppositories of human use. Vaginal suppositories were evaluated for content uniformity (85–115% of intended content), weight variation (no more than two units having a relative standard deviation (RSD) greater than 7.8%) and softening time (less than 30 minutes). All of the animal experiments performed in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (IACUC). The FVB female mice employed in the study were purchased from the NCI Charles River Frederick Research Facility at 7 weeks of age. Mice were housed in a facility that has a constant temperature room at 22 ± 1° C with a 12 h light:12 h dark cycle and provided free access to food and water. Mice were synchronized to be in the diestrus stage of their estrous cycle using a modified Whitten effect 23 to avoid variability in drug absorption from the vaginal cavity 24 . The stages of the estrous cycle were monitored daily by the cell composition in their vaginal lavage. The lavage was performed by first inserting a small volume (30– 50 μl) of saline in the vaginal cavity of the mouse with a pipet tip and then withdrawing it, repeating the procedure three times to ensure adequate sampling of the cells forming the vaginal/cervical epithelia at the time of sampling. The final volume was smeared onto a standard microscope glass slide, air-dried and stained with crystal violet. Vaginal cytology images were taken from the slides with an Olympus FV microscope (Olympus Scientific Solutions Americas Corp., Waltham, MA) to determine the morphologies of the cells that were present in the smear. The stages of the estrus cycle were determined by the differential cell composition, as described by McLean et al . 25 . Mice determined to be in the diestrus stage of their estrous cycle were assigned randomly to different groups to be treated with either 1, 2, 3, 4 or 5 doses of SHetA2 as follows. Mice were treated with the first vaginal suppository containing 30 mg/kg of SHetA2 and this dose was designed as dose 1 (D1). After this, mice were treated similarly with one suppository administered vaginally every 24 h for five days and the doses were designed as dose 2 (D2), dose 3 (D3), dose 4 (D4) or dose 5 (D5). Prior to each suppository administration (dose), mice were anesthetized with isoflurane, placed on its back with the hind quarters slightly elevated. After insertion of the suppository in their vaginal cavity, mice remained anesthetized and in the supine position for 3 minutes and then allowed to recover for 10 minutes under continuous observation before being placed back in their cage. To determine SHetA2 distribution after single or multiple doses, groups of mice (n=5) were euthanized at 0.5, 1, 4, 8, 12, 24, 36, 48, and 72 hours after the first (D1) and after the fifth (D5) dose was administered. The same sample time points were considered for the first dose in the single and multiple dose groups to make sure that the cervix concentration versus time profile in both groups of mice was similar. Having established this similarity, for the 2nd, 3rd and 4th dose, only Cmax and Cmin were considered for the second, third and fourth doses to monitor possible drug accumulation. Lastly, we considered the same number of samples after the fifth dose to determine if the absorption, distribution and elimination of the drug in the cervices of treated mice was similar after the first and fifth dose. Gynecological tissues (cervix, uterine horns, fallopian tubes and ovaries) were collected and thoroughly cleaned with sterile saline before storing them at −80ºC. Concentrations of SHetA2 in tissue homogenates were determined using a validated HPLC method 26 , with a limit of detection of 0.005 μg/mL and limit of quantitation (LOQ) was 0.025 μg/mL. Mice (n=5) treated with 2, 3 and 4 doses were euthanized only after 0.5h (C max ) and 72 hours (C min ) post treatment and their tissues processed as those receiving 5 doses. Additional groups of mice (n=5) were dosed with placebo suppositories every 24 h for 5 days, in the same manner as described above to be used as controls in pharmacodynamics studies. These mice were euthanized at 0.5 h (C max ) and 72 hours (C min ) after the last dose on the fifth day and their cervix tissues collected and washed with sterile saline before storing them at −80ºC. The extent of reduction in the levels of cyclin D1 protein in the cervices of mice elicited after administration of multiple doses of SHetA2 in vaginal suppositories was quantified with respect to that after administration of placebo suppositories and untreated mice. The cervix of each mouse was homogenized in T-PER reagent (10 μL per each mg of tissue) containing protease inhibitor cocktail. Homogenization was performed in an ice bath using an OMNI-GLH general laboratory homogenizer. Homogenates were centrifuged at 4ºC for 5 minutes at 10000 ×g and the supernatants collected. The expression of cyclin D1 protein was determined in the supernatants using an Enzyme Linked Immunosorbent Assay (ELISA) performed according to the manufacturer’s instruction. Each standard blank and test specimen was evaluated in duplicate. The replicates with >15% coefficients of variance were eliminated from the analysis. The average of the test samples was compared to the standard curve to derive the cyclin D1 concentration. Three additional groups of mice (n=5) were employed to evaluate the safety of the proposed regimen: (a) a group dosed with the 30mg/kg SHetA2 suppositories for 5 days; (b) a group dosed with placebo suppositories for 5 days; and (c) a group of untreated mice. The safety of the treatment was evaluated by determining variations in the weight and behavior of each mouse as well as the macroscopic and microscopic changes that may have occurred at the site of the drug administration (vaginal area) and its vicinity. For this purpose, each mouse in the study was weighed once daily, and its weight recorded as weight gain each day whereas their behavior was monitored twice daily. The vaginal region of each mouse was also inspected twice daily for perceived symptoms and any sign of inflammation, excoriation, redness, swelling or irritation in the vaginal region. These observations were scored according to the guidelines recommended by the United States Food and Drug Administration (US FDA) 27 as follows : 0 = none (absent) 1 = mild (slight) 2 = moderate (definitely present) 3 = severe (marked, intense) After euthanizing the mice at the end of the study period, their vagina, cervix, uterine horns, fallopian tubes and ovaries were collected and fixed in 10% neutralized buffered formalin and embedded in a paraffin block. For histological examination, sections of these tissues were cut perpendicularly to the transverse axis from the paraffin blocks so that the lumen and stromal areas were included in each section. Slides of all gynecological tissues were stained with hematoxylin and eosin (H&E) and subjected to a thorough microscopic evaluation performed by a veterinary pathologist who was blind with respect to the treatment of each specimen. The disposition of SHetA2 after multiple vaginal administrations was characterized in terms of its PK parameters, which were determined by non-compartmental analysis using Phoenix WinNonlin® software. The calculated PK parameters included: (C max-cervix = maximum concentration in cervix, T max-cervix = time to achieve maximum concentration in cervix, t 1/2-cervix = half-life in cervix, Vz_F = apparent volume of distribution/F, Cl cervix /F = Clearance/F, MRT cervix = AUMC cervix /AUC cervix ). In addition, the accumulation ratio (AR) was calculated by equation 1 28 : (1) Accumulation Ratio ( AR ) = C ( min − multiple dose ) C ( min − single dose ) = Minimum concentration after multiple dose ( 72 h post 5 th dose ) Concentration after first dosing interval ( 24 hour after 1 st dose ) Statistical analysis of the reduction in the cyclin D1 expression level in the cervix of mice receiving single or multiple dose treatments was performed by GraphPad Prism software. To determine cyclin D1 expression for grouped studies, p values were obtained by two-way ANOVA and multiple comparison Tukey’s test. A p value less than 0.05 was considered to be statistically significant.

Multiple vaginal administration of SHetA2 suppositories was capable of achieving and maintaining predicted therapeutic concentrations for up to 65 hours and to reduce the levels of cyclin D1 in the cervix of treated mice. This regimen also achieved predicted therapeutic concentrations in the uterine horns, fallopian tubes and ovaries of treated mice for up to 48 hours. There were no overt adverse effects observed in the treated mice for the period of the study, but the effects of a longer period of treatment in a non-rodent animal model should be evaluated before any toxicity is ruled out.

For many decades, vaginal formulations such as ointments, creams, and suppositories have been proven to be effective against diseases such as bacterial vaginitis, deep infiltrating endometriosis, and vaginal candidiasis 29 . Previously, we reported that a vaginal suppository containing a 30 mg/kg dose of SHetA2 achieved and maintained predicted therapeutic concentration 13 in the cervices of treated mice, which in turn resulted in the desired pharmacodynamic endpoint of cyclin D1 reduction in the cervix of these mice. The next step in the evaluation of the potential therapeutic effect of SHetA2 suppositories is to assess its efficacy in an animal model of cervical cancer. Upon estrogen treatment, the K14-HPV16 mouse model develops s similar multistage progression from cervical dysplasia towards cervical cancer 6 , which makes it a suitable model for the efficacy study. For consistency, the FVB mouse strain employed in our studies is the background strain for the K14-HPV16 transgenic mouse model of cervical cancer 6 , 30 . To treat any of these local diseases effectively, it is required that the drug formulation achieves therapeutic concentrations and that neither the drug nor the excipients cause adverse effects to the treated area. The goal of the present study was to assess if a dosing regimen consisting of a daily administration of a 30 mg/kg SHetA2 vaginal suppository is capable of maintaining drug concentrations above the predicted therapeutic levels at the site of action, without inducing undesired side effects. The PK study demonstrated that the proposed dosing regimen (a daily administration of 30 mg/kg of SHetA2 in a vaginal suppository) can achieve and maintain predicted therapeutic levels for up to 65 h after administration of the last dose ( figure 1 ). However, most of the PK parameters determined from the multiple dosing data were different than those after a single dose ( table 1 ). Notably, the steady state AUC Cervix after administration of the fifth dose of SHetA2 in the multiple dosing group was almost two-fold smaller than that after a single dose administration; whereas the C max-cervix after multiple vaginal doses was almost three-fold lower than that after administration of a single dose. Likewise, the CL/F Cervix after multiple vaginal doses of SHetA2 was almost three-fold faster than that after a single dose, which resulted in a two-fold shorter t 1/2-cervix after the multiple doses compared to that after a single dose. These differences in the disposition of SHetA2 are likely due to the anatomical and physiological changes in the reproductive organs that female mice undergo during the different stages of their estrous cycle 24 . This cycle lasts for 4–5 days and consists of four stages with different length: proestrus (~12h ), estrus (~12h), metestrus (~21h) and diestrus (~65h) 31 in which the hormonal levels change, and the thickness and cell composition of the stratified squamous epithelium of the gynecological tissues vary. Given that mice in the multiple dose group were treated over a period of 5 days, they must have gone through all 4 stages of the estrus cycles. Previously, we reported that the absorption of SHetA2 in the cervix is maximized after its vaginal administration to mice during the diestrus stage and significantly decreased when the suppository was administered during the estrus stage 24 . Similarly, the SHetA2 concentrations in the cervix of treated mice were maintained above therapeutic levels 13 for at least 48h in mice dosed during diestrus compared to only 12 h when mice were dosed during estrus 24 . These differences were due to the changes in the composition of the vaginal epithelium during the stages of the estrus cycle. During diestrus, the vaginal epithelium is thin and is formed by polygonal, plump epithelial cells due to early mucification; whereas during estrus, the epithelium in the vaginal cavity of mice is thick and the superficial layer is formed by large, anuclear cornified epithelial cells, which are shed (lost) by the end of this stage 32 , 33 . To gain further insight on the effects of the estrous cycle on the disposition of multiple doses of SHetA2, the cervix concentration versus time profiles for the single and multiple dose groups were plotted with respect to the assumed sequence and duration of each estrous stage 32 ( figure 5 ) as follows. Mice in the multiple dose group received the first SHetA2 vaginal dose (D1) when they were in the diestrus stage as well as those mice treated with a single dose ( figure 5 , left plot, arrow #A). Assuming the length of each stage of the estrous cycle as described above, doses 2 and 3 (D2 and D3, at 24 and 48 h, respectively) were also administered to mice in the multiple dose group during diestrus. Then, dose 4 (D4 at 72 h) was administered during proestrus ( figure 5 , left plot, arrow #B), where the cell composition of the vaginal wall would have changed 34 . It is also important to note that at 72 h, the SHetA2 concentrations in the cervix of mice receiving a single dose remained above the predicted therapeutic levels 13 ( figure 5 , left plot, arrow #B), as observed previously by our group 20 . Mice in the multiple dose group went through the estrus stage ( figure 5 , arrow #C) before receiving the fifth dose (D5), which is likely administered when these mice are in late estrus, when the anuclear cornified epithelial cells are shed 31 , or in early metestrus ( figure 5 , right plot, arrow #D). Therefore, it is likely that any drug remaining in the vaginal epithelium from the previous doses (D1-D4) may have been lost at the end of the estrus stage along with the cornified epithelial layer. After delamination of the cornified layer, leukocytes and epithelial cells start to appear in the first part of metestrus 31 and it is plausible that the absorption of SHetA2 after the fifth dose (D5) may have been somehow limited by the appearance of these cells. Hence, it is possible that the lower C max-cervix observed after the fifth dose in the multiple dose group compared to that after a single dose, may be due to the different cell compositions of the vaginal epithelium during late estrus/metestrus (D5, multiple dose) and diestrus (single dose). A few hours after D5 ( figure 5 , right plot, arrow #E), mice in the multiple dose group may have changed from metestrus to diestrus (as per the assumed sequence of the cycle), or estrus/late proestrus/metestrus, as observed by the pathologist, who performed the histological analysis). Regardless of the stage to which these mice change to, the cell composition of the vaginal epithelium changed again 35 . This later change in stage combined with the smaller fraction of drug absorbed after the fifth dose, may account for the lower cervix concentration observed 72 hours after the fifth dose (C min-cervix ) compared to that after a single dose. In conjunction, all of these changes would also explain the differences observed in the pharmacokinetic parameters (AUC cervix , CL/F Cervix , t 1/2-cervix and MRT cervix ) between the single and multiple dose groups. It is also likely that the differences in the magnitude of the extra peaks observed at 12 and 48 h in the cervix concentration versus time profiles were due to the fact that mice in the single dose group were in diestrus; whereas mice in the multiple dose group may have gone through 2 or 3 different stages. We have identified these additional peaks as the equilibrium between supersaturation, precipitation and re-solubilization of the drug that is established in the vaginal epithelium after vaginal administration of the drug 20 . This phenomenon has been observed after oral administration of danazol 36 and other water insoluble drugs 37 . Despite the lower C max-cervix observed after the fifth dose of the multiple dosing regimen compared to that after a single dose, the reduction in the levels of cyclin D1 ( figure 3B ) was greater (~45%) after multiple dosing than after a single dose (~26%). This suggests that the extent of reduction in the levels of cyclin D1 is not directly proportional to the C max-cervix observed at the same time point. Thus, the PK/PD relationship between these two parameters appears to be more complex and may be influenced by the multiple administrations of SHetA2 or the anatomical and physiological changes of the cervix during the estrous cycle. Nevertheless, the reduction in the levels of cyclin D1 is still a good PD endpoint for our study, as the elevated expression of cyclin D1 is associated with poor prognosis in cervical cancer patients and the increased risk of recurrence 12 . Moreover, a reduction in cyclin D1 levels in the transgenic mouse model of small intestinal polyps showed a direct correlation with reduced polyp formation 38 and has been considered as a pharmacologic target for the development of chemo-preventive drugs 39 – 41 . The safety of the multiple dose regimen using vaginal SHetA2 suppositories was supported by the lack of adverse side effects observed during the period of the study ( table 2 and figure 4 ). Assessment of the weight gain/loss is often used in efficacy studies with different cancer types to determine the humane endpoint of the study 42 , but is also used as a measure of toxicity after the chronic administration of drugs. Untreated mice and those treated vaginally with SHetA2 or placebo suppositories all gained weight ( figure 4 ), indicating the lack of effect of the multiple doses of SHetA2 on the weight of the mice. Although in the present study there was no effect of the multiple vaginal doses of SHetA2 on the weight of mice, future studies should evaluate this parameter for a longer treatment period, such as in other studies with SHetA2. A chemoprevention study in the small intestinal tumorigenesis mouse model showed that oral administration of SHetA2 in suspension for 4 weeks did not result in weight loss during the period of treatment 38 . In the draft guidance entitled “Skin Irritation and Sensitization Testing of Generic Transdermal Drug Products”, the US FDA outlined a scoring system similar to the one employed in the present study to monitor local irritation after topical administration of drug candidates 27 . The daily behavioral observations and the macroscopic evaluation of the area of suppository administration (placebo and those containing SHetA2) using this guidance did not find any significant adverse effects as indicated by most of the parameters evaluated being scored at “0” ( table 2 ). There were no other signs of irritation in the vaginal region (either redness, inflammation or wounds) in any of the mice, confirming previous reports that SHetA2 did not cause skin irritation after topical administration to mice 13 . This outcome is noteworthy given that irritation is often associated with vaginal administration of drugs 29 , 43 , such as that observed with terconazole suppositories 44 and some formulations of miconazole nitrate 45 – 47 . The histological evaluation of the cervix, uterine horns, fallopian tubes and ovaries also supported the safety of repeated vaginal administration of SHetA2 suppositories. Thus, it is plausible to assume that the multiple dose regimen proposed in the present study would be safe for efficacy studies to be performed in the K14-HPV16 transgenic mouse model of cervical cancer. Given that the efficacy studies would last several weeks, and it is reasonable to assume that mice would pass through the different stages of the estrus cycle several times during this period. Consequently, the safety profile of the treatment could potentially be similar to that of the present study. It is important to note that usually the toxicity of compounds to tissues is evaluated microscopically by the presence of leukocytes, mitosis, and cellular apoptosis/necrosis 48 . However, these cells and events are also observed during the changes occurring in the vaginal epithelium due to the variation in the stages of the estrous cycle 49 , 50 . Hence, it may be plausible that the normal anatomical and physiological changes observed within the vagina and uterus of cycling female mice 25 , 32 could be masking the effects of multiple suppository administrations. Therefore, the safety of the multiple dosing regimen proposed for the SHetA2 vaginal suppositories should be performed in non-cycling animals such as rabbits 51 or in animals that exhibit similar mucus secretion/clearance in the cervico-vaginal tract such as pigs 52 , 53 or sheep 54 , 55 before any toxicity can be ruled out.

The implementation of vaccination programs and stronger recommendations of regular Pap smear tests has reduced the incidence and mortality of cervical cancer in the United States in the last few years 1 , 2 . Unfortunately, cervical cancer continues to be one of the major health concerns in low and middle-income countries due to the lack of accessibility to screening and affordable treatment 3 – 5 . Persistent human papilloma virus (HPV) infection causes precancerous changes in the uterine cervix resulting in cervical dysplasia and multistage progression leading to cervical cancer 6 . Instead of only treating cervical cancer, we hypothesize that treating cervical dysplasia caused by HPV would prevent its progression to cervical cancer and this would have a bigger impact in reducing the global incidence of cervical cancer. Given that at the initial stages of cervical dysplasia the lesions are highly localized in the stratified squamous epithelium, alternative topical chemotherapies were investigated as alternative treatment options. Yet, topical chemotherapies with Imiquimod and Tretinoin were abandoned due to local irritation and the lack of patient acceptability 7 – 9 . Thus, until this date, there is no topical chemotherapy approved for cervical dysplasia. SHetA2 is a flexible heteroarotinoid that induces G1 arrest and differential apoptosis of a number of cancer cell lines 10 , 11 . The pathway that causes G1 cell cycle arrest by degradation of cyclin D1 is particularly dysregulated in cervical cancer 12 . In vitro cytotoxicity assays in 4 different cervical carcinoma cell lines, the HT-3, SiHa, CC-1 and C33a human cervical cell lines determined that the therapeutic dose of SHetA2 would be (4.0 ± 0.4 μM or 1.6μg/mL) 13 . Although derived from retinoids, SHetA2 does not need to activate the retinoic acid receptors to exert its anticancer activity 14 , 15 . Consequently, SHetA2 does not replace retinoic acid in murine fetal development 15 nor induce teratogenicity, fetal malformations, or induce classical retinoid toxicities, such as skin irritation 13 . Formal toxicological studies also demonstrated that administration of SHetA2 at doses (25 – 2,000 mg/kg/day) that were up to 1000-fold higher than the therapeutic doses (1.6 μ/ml) to rats or dogs were not mutagenic or genotoxic 16 , 17 . However, SHetA2 has a poor aqueous solubility 18 , which limits its oral bioavailability to approximately 10 % 17 . Using Quality by Design (QbD) methodology, we developed and optimized a vaginal suppository formulation for SHetA2 with the objective to maximize drug concentrations at the target site for cervical dysplasia, the cervix 19 . Initial studies demonstrated that vaginal administration of a suppository containing a 15-mg/kg dose of SHetA2 achieved an area under the cervix concentration versus time curve (AUC cervix ) that was ~120 times larger than that after an oral administration of a 60 mg/kg dose 20 . A dose-range finding study using mouse-sized suppositories containing 15, 30 and 60 mg/kg doses, determined that 30 mg/kg was the dose of SHetA2 that achieved and maintained a predicted therapeutic concentration (> 4 μM) at the cervix tissue of Friend Leukemia Virus B (FVB) mice for the longest period of time (72 h) 20 . This dose was also capable of inducing and maintaining a reduction in the levels of cyclin D1 protein, which is the desired pharmacodynamic endpoint 12 . Thus, the next logical step in the process to bring this promising treatment to the clinic was to determine the dosing regimen to be used in efficacy studies performed in a mouse model of cervical dysplasia. For this purpose, the present work evaluates the effect that multiple vaginal administrations of SHetA2 suppositories have on drug levels in the cervix of treated mice and on the resulting levels of cyclin D1. Often, drugs fail to make the transition from successful preclinical studies to clinical practice due to unanticipated adverse side effects 7 , 8 ; thus, determination of the safety of the proposed treatment should be assessed as part of the preclinical evaluation. Many of the women carrying HPV DNA and having cervical intraepithelial neoplasia (CIN) lesions remain asymptomatic and virtually healthy 21 . Therefore, we evaluated the effect of multiple doses of SHetA2 vaginal suppositories in healthy animals. Therefore, the objectives of the present study were: (1) to determine the effect of daily doses of SHetA2 using the optimized vaginal suppository formulation on drug distribution, local concentrations in target tissues and their resulting pharmacokinetic (PK) parameters and pharmacodynamic (PD) endpoints; (2) to evaluate the safety of the proposed treatment in terms of drug accumulation in the tissue and the macroscopic or microscopic gross sign of toxicity.