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BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019182
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BIOLOGY OF REPRODUCTION 69, 1843–1851 (2003)
DOI: 10.1095/biolreprod.103.019182
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Development and Evaluation of a Thermoreversible Ovule Formulation of Stampidine, a Novel Nonspermicidal Broad-Spectrum Anti-Human Immunodeficiency Virus Microbicide1

Osmond J. D'Cruz2,3,7, Peter Samuel4,7, Barbara Waurzyniak5,7, and Fatih M. Uckun6

Drug Discovery Program, Department of Reproductive Biology,3 Pharmaceutical Science,4 Experimental Pathology,5 Virology6 Parker Hughes Institute, St. Paul, Minnesota 55113 Paradigm Pharmaceuticals, LLC,7 St. Paul, Minnesota 55113


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5–2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.

assisted reproductive technology, female reproductive tract, vagina


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterosexual transmission is the main mode of HIV-1 transmission worldwide and accounts for nearly 90% of all HIV infections in women [1–4]. Currently an estimated 19.2 million women worldwide are living with HIV/AIDS, accounting for 50% of the 38.6 million adults [5]. Women are 4–16 times more likely to contract HIV from infected males than vice versa, and young women are especially vulnerable [6]. In addition, the incidence of AIDS has increased rapidly among younger persons. Eighty-five percent of women with AIDS are between the ages of 15 and 44 [2, 4, 6]. Considering that the AIDS epidemic is still in its infancy on a global scale, this evolving demographic situation warrants urgent attention for the adolescent population. Therefore, effective strategies are needed to reduce heterosexual and perinatal HIV transmission.

In the absence of an effective prophylactic anti-HIV therapy or vaccine, new emphasis has been placed on the development of mechanism-based nontoxic microbicidal agents capable of reducing the heterosexual transmission of HIV [7, 8]. Because semen is an important vehicle for sexual transmission of HIV-1, microbicides would provide protection by directly inactivating HIV or preventing HIV from attaching, entering, or replicating in susceptible target cells as well as dissemination from target cells present in semen or the host cells that line the vaginal/rectal wall. Thus, ideally, anti-HIV microbicides should be capable of attacking HIV from different angles. Microbicides that are currently being investigated are directed mainly at preventing pregnancy as well as protection against sexually transmissible diseases, especially HIV [7–16]. The availability of a nonspermicidal microbicide is equally important for allowing sexually active women to have children while protecting both mother and child from HIV and for prevention of transmission of HIV via semen, especially for HIV-1 serodiscordant couples, prior to assisted reproductive technology (ART) procedures.

In a systematic effort to identify a broad-spectrum anti-HIV microbicide potentially capable of preventing the sexual transmission of HIV without affecting fertility, we have synthesized a series of aryl phosphate derivatives of stavudine [STV/d4T] (2',3'-didehydro-2',3'-dideoxythymidine) and identified STV-5'-(p-bromophenyl methoxyalaninyl phosphate) [stampidine] as the lead compound [17, 18]. Stavudine is a pyrimidine nucleoside analogue used in the treatment of HIV infection [19]. It inhibits viral reverse transcriptase [RT] as do zidovudine (ZDV/AZT), didanosine, zalcitabine, and lamivudine, which comprise the family of NRTI [20, 21]. The 5'-triphosphate of STV, generated intracellularly by the action of nucleoside and nucleotide kinases, are potent inhibitors of HIV-1 RT [22, 23]. The rate-limiting step for the generation of the bioactive metabolite, STV-triphosphate, is the conversion of STV to its monophosphate derivative. Stampidine was synthesized in an attempt to overcome the dependence of STV on intracellular nucleoside kinase activation. The para-bromine group in the phenyl moiety of stampidine was shown to contribute to its ability to undergo rapid hydrolysis yielding the key active metabolite alaninyl-STV-monophosphate (Ala-STV-MP) in a thymidine kinase-independent fashion [24]. The successful intracellular delivery of bioactive nucleotides by the prodrug in blood mononuclear cells and CEM T cells despite low or absent thymidine kinase activity is particularly promising for curbing the sexual transmission of HIV by leukocytes and sperm [25, 26].

Stampidine was 100 times more active than stavudine and twice as active as zidovudine (ZDV/AZT) against nine clinical HIV-1 isolates of non-B envelope subtypes (A, C, F, and G) originating from South America, Asia, and sub-Saharan Africa [27]. Further, stampidine was effective against 20 genotypically and phenotypically nucleoside analogue reverse transcriptase inhibitor (NRTI)-resistant and six nonnucleoside inhibitor (NNRTI)-resistant HIV-1 isolates at subnanomolar to low nanomolar concentrations [28]. Stampidine was active against NRTI-resistant HIV-1 isolates with five thymidine analogue mutations at subnanomolar concentrations [27, 28]. Orally administered stampidine exhibited significant and dose-dependent in vivo anti-HIV activity against a NRTI-resistant clinical HIV-1 isolate BR/92/019 in a humanized severe combined immunodeficient (Hu-SCID) mouse model for AIDS [29]. In the feline immunodeficiency virus (FIV)-infected domestic cat model for AIDS, orally administered stampidine showed a dose-dependent antiretroviral effect in chronically FIV-infected cats [30]. Stampidine therapy was not associated with any clinical or laboratory evidence of toxicity at dose levels as high as 500 mg/kg or at cumulative dose levels as high as 8.4 g/kg [31]. Stampidine exhibited favorable pharmacokinetic behavior in mice, rats, dogs, and cats following oral administration [29–33].

We have examined the potential utility of stampidine as a nonspermicidal microbicide for prevention of sexual transmission of HIV. Pretreatment of human semen with stampidine even at a concentration 106 times higher than its IC50 value against HTLVIIIB strain had no adverse effect on functional sperm motility or sperm transport within cervical mucus [34]. Pretreatment of rabbit semen before artificial insemination of ovulated rabbits had no adverse effect on pregnancy outcome [34]. Stampidine did not affect the viability of normal human vaginal and cervical epithelial cells even at millimolar concentrations. The documented in vitro potency of stampidine against primary clinical HIV-1 isolates with genotypic and/or phenotypic NRTI or NNRTI resistance as well as non-B envelope subtype together with its in vivo antiretroviral activity in HIV-infected Hu-PBL SCID mouse and FIV/cat models warrants the further development of this promising new NRTI compound as a noncontraceptive anti-HIV microbicide.

As part of an effort to develop an acceptable formulation of stampidine for clinical use as an intravaginal microbicide, we developed a thermoreversible ovule formulation of stampidine. We examined the in vitro dissolution profile of formulated stampidine in simulated vaginal fluid and its potential to cause mucosal toxicity in the New Zealand White (NZW) rabbit model. The marked stability of stampidine at acidic pH as well as the rapid spreadability and the lack of significant mucosal toxicity of stampidine in a thermoreversible ovule formulation may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of Stampidine

Stampidine (2',3'-didehydro-2',3-dideoxythymidine 5'-(p-bromophenyl methoxyalaninyl phosphate)) was synthesized by condensing 2',3'-didehydro-2',3-dideoxythymidine (STV/d4T) with para-bromophenyl alaninyl phosphochloridate in the anhydrous tetrahydrofuran solvent according to our published procedure [17]. Stampidine differs from STV by a phenyl phosphate group with an alanine side chain and a bromo substitutent on the C-5 position of phenyl ring (Fig. 1). The structure of stampidine was confirmed using standard analytical techniques. Purity of the compound was >99%. All other chemicals, including buffer components, were reagent grade from Aldrich (Milwaukee, WI), Sigma Chemical Co. (St. Louis, MO), or Fisher Scientific (Pittsburgh, PA) and were used without purification. Organic solvents were HPLC grade.



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  		FIG. 1. Chemical structure of stampidine and its major in vivo metabolites. Stampidine (2',3-dideoxythymidine 5'-(p-bromophenyl methoxyalaninyl phosphate)) differs from stavudine (STV/d4T) by the presence of a phenyl phosphate group with an alanine side chain and a bromo substitutent on the C-5 position of phenyl ring. The prodrug forms three major metabolites in vivo, namely, alaninyl-STV-monophosphate (Ala-STV-MP), STV [2',3-dideoxythymidine 5'-(alaninyl phosphate)], and p-bromophenyl sulfate (p-Br-Phe-S)

HPLC Analysis of Stampidine

Chromatographic analysis of stampidine and its major metabolites, alanine-STV-monophosphate (Ala-STV-MP) and STV, were carried out using a previously established and validated HPLC method [30, 32, 33]. The HPLC system used for these studies was a Hewlett Packard (Agilent Technologies, Palo Alto, CA) series 1100 instrument equipped with a quaternary pump, an autosampler, an automatic electronic degasser, an automatic thermostatic column compartment, a diode array detector, and a computer with Chemstation software for data analysis. The analytical column used was Zorbax SB-Phenyl (5 µm, Agilent Technologies, Kenner, LA) attached to a guard column (Agilent Technologies). The column was equilibrated prior to data collection. The employed linear gradient mobile phase (flow rate = 1.0 ml/min) was 100% A/0% B at 0 min, 88% A/12% B at 20 min, 8% and A/92% B at 30 min (A: 10 mM ammonium phosphate buffer, pH 3.7; B: acetonitrile [Burdick & Jackson, Muskegon, MI]). The detection wavelength was 268 nm, and the peak width, response time, and slit were set at >0.03 min, 0.5 sec, and 4 nm, respectively.

Aqueous Stability Studies

The kinetics of the degradation of stampidine in aqueous solution was studied over the pH range 2–12 at 25°C (ionic strength, 0.2 M). The amounts of stampidine remaining in citrate buffer solution were followed as a function of time by a reversed-phase HPLC as described previously [32, 33]. The rate of stampidine degradation was fit to a first-order exponential decay to obtain an observed rate constant (kobs).

Preparation of Thermoreversible Ovule Formulation

A comprehensive preformulation study of stampidine was performed for its further preclinical and clinical development. An effective drug solubilization method for the vaginal bioavailability in a clinically applicable ovule formulation was developed. Stampidine was solubilized at 0.5%, 1%, and 2% concentration in a thermoreversible ovule formulation composed of polyethylene glycol 400 (Union Carbide, Danbury, CT) and polyethylene glycol fatty acid esters (Solutol HS; BASF, Mount Olive, NJ) as carriers and Tween-80 (polyoxyethylene sorbitanmonooleate; ICI Americas, Wilmington, DE) as the surfactant. The pH of the formulation was 6.1. The formulation providing sol-gel characteristics was prepared by varying the ratios of polyethylene glycol and polyethylene glycol fatty acid esters such that the formulation is a solid at 25°C while it transforms at 37°C to a semisolid for rapid dispersibility of the drug at the point of application. The formulation was designed for encapsulation in hard gelatin capsules that dissolve readily in aqueous environment at 37°C. The ovule formulations of stampidine were highly stable at 5°C or at ambient temperature. The proposed mechanism of the release of stampidine is by diffusion of the active material through the ovule formulation [35].

In Vitro Dissolution Test

The in vitro dissolution test was performed using the apparatus I method (basket, USP) on a Vankel 750 dissolution apparatus (USP, 2000). Stampidine formulation was prepared at 1% (w/w) concentration, warmed at 37°C to produce a flowable liquid, and loaded in size 00 hard gelatin capsules (Capsugel Corp., Greenwood, SC). The dissolution conditions were temperature 37 ± 0.5°C, volume 500 ml, spindle speed 100 rpm. The amount of stampidine released into synthetic vaginal fluid (pH = 4.2) [36] as a function of time was determined by analytical HPLC. The analytical column was Lichrosphere RP (5 µM). The mobile phase was composed of acetonitrile and water (containing 0.1% trifluoroacetic acid and 0.1% triethylamine) in a ratio of 35:65 (v/v). The column was equilibrated and eluted under isocratic conditions utilizing a flow rate of 1 ml/min at 20°C before injection of 20 µl of samples. The detection wavelength was set at 265 nm (reference 400 nm), and the run time was 15 min. Peak width, response time, and slit were set at >0.03 min, 0.5 sec, and 4 nm, respectively. During dissolution, 1-ml samples were collected at 5, 15, 30, 60, 90, and 120 min; filtered through a 0.2-µM filter; and assayed directly by HPLC. The retention time for stampidine was between 10 and 11 min. Three replicate runs were carried out for each time point.

Animals

Thirty female, sexually mature (>7 mo old; >4.0 kg), specific-pathogen free, NZW rabbits were obtained from Charles River Laboratories (Wilmington, MA). All rabbits were identified with specific metal ear tags and housed in single cages. Tap water and rabbit food pellets (Teklad 7015; Harlan Teklad, Madison, WI) were available ad libitum. They were maintained in rooms that were kept at 20 ± 2°C with relative humidity of 50 ± 10% and a 12-h fluorescence light cycle. The rabbits were isolated for a minimum of 3 wk before the intravaginal study. Animal studies were approved by the Parker Hughes Institute Animal Use and Care Committee, and all animal care procedures were conducted according to the current USDA Guidelines.

Rabbit Vaginal Irritation Test

For the vaginal irritation study, 12 female rabbits in subgroups of three were administered intravaginally with 1 ml of ovule formulation with and without (placebo control) 0.5%, 1%, or 2% stampidine for 14 consecutive days. As a positive control, three rabbits were administered a gel formulation containing 4% nonoxynol-9 (N-9). Body weights were obtained before and after completion of 14 days' dosing. Animals were killed on Day 15, and their genital tracts were examined grossly (swelling, redness as well as bleeding) and microscopically after completion of the study [37]. The vaginal tissues were rapidly removed and parts of the upper (cervico-vagina), middle, and lower (uro-vagina) regions of each vagina were fixed in 10% neutral-buffered formalin.

Fixed vaginal tissues were embedded in paraffin, sectioned at a thickness of 4–6 µm, and stained with hematoxylin and eosin (H & E) and examined under 200x and 400x magnification using a Leica light microscope (Milton Keynes, Buckinghamshire, UK) interfaced with an image analysis system. The images were transferred to Adobe Photoshop 6.0 software (Adobe Systems Inc., San Jose, CA) for observation and analysis. Each of the three regions of vagina were examined for epithelial exfoliation, vascular congestion, leukocyte infiltration, and lamina propria thickness (edema). The irritation scores were assigned based on the general semiquantitative scoring system for inflammation [37], which was as follows: individual score: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, and 4 = intense irritation. The cumulative score for the epithelium, leukocytes, congestion, and edema were as follows: minimal irritation 1–4, mild irritation 5–8, moderate irritation 9–11, and marked irritation 12–16. This scoring system has been correlated to human vaginal irritation potential as follows: Scores of 0–8 are acceptable, scores of 9–10 indicate borderline irritation potential, and scores >=11 are indicative of significant irritation potential.

The systemic absorption of stampidine from intravaginally applied 2% stampidine-ovule formulation was monitored by analytical HPLC of plasma extracts. Fifteen rabbits in subgroups of three were administered intravaginally 2 ml of a 2% stampidine-containing ovule formulation (12 mg/kg), and blood samples (~2 ml) were drawn from the median ear artery at 0, 15, 30, 60, and 120 min. Heparinized blood samples were immediately centrifuged at 7000 x g for 2 min to separate the plasma fraction from the whole blood. The plasma levels of stampidine and its major metabolites, Ala-STV-MP and STV, were determined by using a previously established and validated HPLC method [27, 29, 30]. In brief, each plasma sample (200 µl) was mixed 1:4 with acetone (800 µl) and vortexed for at least 30 sec. Following centrifugation, the supernatant was transferred into a clean tube and dried under nitrogen. A 50-µl solution of 50% methanol in 200 mM HCl was used to reconstitute the extraction residue, and 40 µl of the reconstituted sample were subjected to analytical HPLC. The HPLC chromatograms were compared with control plasma extracts spiked with known amounts of stampidine and its known major metabolites. The chromatographic retention times measured for stampidine and its metabolites in spiked samples were 28.9 ± 0.02 min (n = 13), 15.3 ± 0.2 min (Ala-STV-MP; n = 30), and 18.5 ± 0.1 min (STV; n = 30). The plasma extraction recovery rates were >98% for stampidine, 57% for Ala-STV-MP, and 96% for STV. The lowest limit of detection was 0.25 µM at signal-to-noise ratio of ~4. Good linearity (r > 0.995) was observed between concentrations ranging from 0.5 to 12.5 µM and from 12.5 to 100 µM in 200 µl of plasma. Intra- and interassay variabilities were less than 8%.

Evaluation of Cell Proliferation by Proliferating Cell Nuclear Antigen (PCNA) Staining

PCNA immunostaining was performed to evaluate the vaginal tissues for inflammation. Immunostaining of sections (4 µm) was performed using the Zymed PCNA kit (Zymed Laboratories, South San Francisco, CA). Briefly, deparaffinized tissue sections were treated to remove endogenous peroxidase activity, blocked with goat serum for 30 min at room temperature, and then incubated with a biotinylated anti-PCNA monoclonal antibody (clone PC10). Sections were washed in phosphate-buffered saline (PBS), 0.1% Tween-20, and incubated with streptavidin-peroxidase for 30 min at room temperature and washed in PBS. The bound horseradish peroxidase complexes were developed using diaminobenzidine tetrahydrochloride according to the manufacturer's instructions. The sections were counterstained with Harris's hematoxylin, dehydrated, and mounted with glass coverslips. Positive controls consisted of sections of mouse intestine known to express PCNA. Negative controls consisted of sections stained without the primary antibody. Sections were then observed with an Olympus BH-40 light microscope (Olympus Corporation, Lake Success, NY). The nature of staining and the distribution of PCNA immunoreactivity were determined by scoring a minimum of 300 cells in the vaginal epithelium and the stromal region in several random fields, and the percentage of PCNA-positive cells for each region of the tissue section was calculated.

Statistical Analysis

Data are presented as mean ± SD or SEM. Statistical significance of the differences between the mean values of scores based on the histologic grading system for vaginal irritation was determined with the Kruskall-Wallis nonparametric ANOVA test, followed by Dunnett's multiple comparisons test to determine difference between control and test groups. P values of <0.05 were indicative of significant differences. The pH-rate profiles and product-time curves were fit to the general nonlinear curve fitting model using GraphPad Prism version 3.0 Software (San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Degradation Kinetics of Stampidine as a Function of pH

The prodrugs must have adequate chemical stability in any formulation, especially in ready-to-use intravaginal dosage forms. Therefore, the kinetics of degradation of stampidine in aqueous solution as a function of pH was studied. The amount of stampidine remaining in the citrate buffer solution over a pH range of 2.0–12.0 at ambient temperature (ionic strength, 0.2 M) was measured for up to 100 days by a reversed-phase HPLC (Fig. 2). The pH of maximum stability (>100 days) occurred within the acidic pH range of 4.0–6.0, which is ideal for formulation from a physiological viewpoint. The observed constants (kobs) for the total degradation of stampidine, calculated from the slopes of the logarithmic concentration-time plots, are shown in Figure 3. The logarithmic plot of the percentage remaining amount of intact stampidine versus time at various pH values shows that the pH affected the overall degradation of stampidine, and the reaction rates followed pseudo-first-order kinetics.



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  		FIG. 2. Chemical stability of stampidine in aqueous buffer as a function of pH at 25°C. The influence of pH on the stability of stampidine was tested in standard citrate buffer (200 mM) solutions at pH 2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0 as well as deionized distilled water (pH range 6.0–6.5) purified via the Millipore Milli-Q system. The amount of stampidine remaining was determined by analytical HPLC as described in Materials and Methods. The data are normalized to a value of 100 at zero time. Points represent the experimental data, and the solid lines were drawn using the nonlinear least squares regression analysis



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  		FIG. 3. Plot of the observed degradation rate constant (kobs) for stampidine as a function of pH at 25°C. The degradation rate constants were calculated from the linear relationship between the logarithm of the remaining drug concentration and time. The solid line is the curve fit to the experimental data points. The U-shaped profile shows that maximum stability is obtained in the 4–6 pH range

Thermoreversible Ovule Formulation

A comprehensive preformulation study of stampidine was conducted for its further preclinical and clinical development. The developed ovule formulation contains polyethylene glycol 400 and polyethylene glycol fatty acid esters as carrier and polysorbate 80 as the surfactant. Solubility of stampidine was 6.8 mg/ml in 10% aqueous polyethylene glycol 300/400, 5.8 mg/ml in 10% aqueous propylene glycol, and 8.7 mg/ml in 10% aqueous polysorbate 80. The solubility values for stampidine with admixtures of excipients and water showed an exponential rather than a linear relationship with increasing amounts of excipients. These solubility studies indicated that formulations of stampidine at 1–2% are feasible by adjusting the ratios of the excipients for preclinical and clinical studies. The thermoreversible formulation was designed by varying the ratios of polyethylene glycol and polyethylene glycol fatty acid esters such that the formulation is in a solid state at 25°C, while it transforms at 37°C to a semisolid state for rapid dispersibility of the drug at the point of application. The sol-gel formulation was developed for encapsulation in hard gelatin capsules that dissolve readily in aqueous environment at 37°C. Stampidine was solubilized at 0.5%, 1%, and 2% concentration. The pH of the formulation was 6.1.

Dissolution Profile of Stampidine-Ovule in Simulated Vaginal Fluid

The dissolution testing of stampidine was carried out under near physiological conditions using synthetic vaginal fluid (pH = 4.2) [37] allowing interpretation of the dissolution data with regard to in vivo performance of the product. The dissolution of stampidine was followed in gelatin-encapsulated ovules using the Basket apparatus and assayed using the HPLC technique. The appearance of stampidine from the ovules submerged in 500 ml of simulated vaginal fluid was followed over a period of 120 min at 37°C. The release of stampidine from the ovule was rapid and complete within 15 min (Fig. 4). The solubility of stampidine in vaginal fluid was >1 mg/ml. At 1% drug loading, under sink conditions, the maximum drug concentration at 100% release was <0.01 mg/ml (well below the sink conditions of 0.1 mg/ml).



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  		FIG. 4. In vitro dissolution profile of stampidine from ovules into synthetic vaginal fluid at 37°C. Stampidine formulation was prepared at 1% (w/w) concentration, warmed at 37°C to produce a flowable liquid, and loaded in size 00 hard gelatin capsules. The dissolution test was performed using the apparatus I method on a Vankel 750 Dissolution apparatus. The dissolution conditions were temperature 37 ± 0.5°C, volume 500 ml, spindle speed 100 rpm. The amount of stampidine released into simulated vaginal fluid (pH = 4.2) as a function of time was determined by analytical HPLC. Samples (1 ml) were withdrawn at 5, 15, 30, 60, 90, and 120 min for analysis. Three replicate runs were carried out for each time point. The data are normalized to a value of 100 at zero time

Lack of Vaginal Irritation from Stampidine in the Rabbit Model

In the 14-day repeated intravaginal dose study, no adverse clinical signs of toxicity were seen. Body-weight profiles were unaffected by intravaginal dosing (data not shown). Table 1 summarizes the combined scores of histological changes in three different regions of the rabbit vaginal tissue after 14 days of intravaginal application of ovule formulation with or without 0.5%, 1%, or 2% stampidine. Intravaginal administration of 0.5–2% stampidine, which corresponds to approximately 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value against HTLVIIIB, did not result in significant microscopic abnormalities. The morphological alterations noted in the vaginal epithelium and submucosa were basically similar to those seen in the placebo formulation. The mean irritation scores for epithelial exofoliation, vascular congestion, leukocyte infiltration, and submucosal edema were within the acceptable range for a clinical trial (mean individual scores 0–2 out of 4; total score 4–6 out of 16). The intensity of histopathological changes observed for stampidine-treated group were considerably lower than that for N-9-treated group, which consistently showed mild to moderate vaginal irritation (mean individual scores 1–3; total score 9).


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  		TABLE 1. Mean vaginal irritation scores for NZW rabbits given ovule formulation with and without stampidine intravaginally for 14 consecutive days.a

The left panels in Figure 5 show representative vaginal tissue sections from rabbits treated with ovule formulation containing 0%, 0.5%, 1%, or 2% stampidine. Light-microscopic examination revealed intact vaginal epithelium and no leukocyte influx following daily intravaginal administration of ovule formulation alone (Fig. 5A; total score 3) or ovule formulation containing 0.5% (Fig. 5B; total score 4), 1% (Fig. 5C; total score 6), or 2% stampidine (Fig. 5D; total score 5) for 14 consecutive days. Following 2% stampidine (Fig. 5D) treatment, only minimal to mild irritation (total scores 2–7 out of 16) was apparent.



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  		FIG. 5. Light-microscopy images of stampidine-treated rabbit vaginal sections. Left panels: Representative hematoxylin- and eosin-stained, paraffin-embedded sections of the midvaginal region of a rabbit treated intravaginally with a thermoreversible ovule formulation containing 0% (A), 0.5% (B), 1.0% (C), or 2.0% (D) stampidine for 14 consecutive days. Note the intactness of vaginal epithelium (E) and lack of leukocyte influx in the submucosa (SM) in stampidine-treated rabbit tissues. Right panels: Representative PCNA-positive and hematoxylin-counterstained, paraffin-embedded sections of the midvaginal region of a rabbit treated intravaginally with a thermoreversible ovule formulation containing 0% (A'), 0.5% (B'), 1.0% (C'), or 2.0% (D') stampidine for 14 consecutive days. Scattered epithelial and stromal cell nuclei stain brown (arrow), indicating PCNA expression. Original magnification x400

Intravaginal Stampidine Causes No Cellular Inflammation or Hyperplasia

Since immunohistochemical detection of PCNA is closely related to the cell cycle, this method was used to visualize proliferative activity of inflammatory cells or vaginal epithelial cells in paraffin-embedded vaginal tissue sections of control and stampidine-treated rabbits. PCNA-positive cells identified by their darkly stained nuclei were detected in both vaginal epithelial and stromal cell nuclei of all vaginal tissues examined. Table 2 summarizes the percentage of PCNA-positive cells in the vaginal epithelium and stromal cells of tissue sections from rabbits given ovule formulation with and without 0.5%, 1%, or 2% stampidine. Nearly 67% of vaginal epithelial cells and 30% of stromal cells showed nuclear staining for PCNA in control tissues. No significant differences in PCNA positivity were noted in the epithelium or stromal cells of placebo control and stampidine-treated tissues.


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  		TABLE 2. Measurement of cell proliferation in the NZW rabbit vaginal tissue after intravaginal application of stampidine-containing ovule formulation for 14 days.a

The right panels in Figure 5 show the PCNA staining pattern of representative vaginal sections of rabbits given ovule formulation with and without 0.5%, 1%, or 2% stampidine. Light-microscopic examination PCNA-stained vaginal tissues of rabbit following daily intravaginal administration of ovule formulation alone (Fig. 5A') or ovule formulation containing 0.5% (Fig. 5B'), 1% (Fig. 5C'), or 2% stampidine (Fig. 5D') for 14 consecutive days showed comparable reactivity. Thus, repeated intravaginal stampidine exposure does not cause cellular inflammation or hyperplasia in vaginal epithelium.

Lack of Vaginal Absorption of Stampidine

Intravaginally administered microbicides should not result in significant systemic exposure levels to antiviral drugs. Therefore, the systemic absorption of stampidine from 2% stampidine in ovule formulation applied intravaginally was studied in vivo in NZW rabbits. Following intravaginal application of 2% stampidine (12 mg/kg), blood was collected at 15-, 30-, 60-, and 120-min intervals. Blood plasma extracts were subjected to analytical HPLC. Stampidine and its major metabolites, Ala-STV-MP and STV, can be clearly separated using established HPLC conditions with retention times of 28.9, 15.3, and 18.5 min, respectively (Fig. 6A). The retention times of stampidine and its major metabolites are compared to the peaks detected in the HPLC chromatograms of plasma extracts from rabbits following 15 min (Fig. 6B) and 120 min (Fig. 6C) after intravaginal administration of 2% stampidine-containing ovule formulation. No in vivo metabolite peaks matching the retention times of stampidine or its metabolites were detectable at both time points. All blood samples had undetectable stampidine concentrations throughout the 2-h sampling period. These results indicated that stampidine is not absorbed through the vaginal epithelium to a significant degree.



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  		FIG. 6. Representative HPLC chromatograms of stampidine and its major in vivo metabolites in blood plasma. A) HPLC profile of control rabbit plasma spiked with purified stampidine, Ala-STV-MP, and STV prior to extraction. B and C) HPLC profiles of rabbit blood plasma extract prepared 15 min (B) and 120 min (C) following intravenous administration of 2 ml of 2% stampidine (12 mg/kg) in a thermoreversible ovule formulation. The flow rate was 1 ml/min. Stampidine and its major in vivo metabolites were undetectable in the HPLC chromatograms of blood plasma extracts following intravaginal administration. Arrows indicate the retention times for stampidine (28.9 ± 0.02 min), Ala-STV-MP (15.3 ± 0.2 min), and STV (18.5 ± 0.1 min)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study was to investigate the clinical potential of a thermoreversible ovule formulation of the nonspermicidal broad-spectrum anti-HIV agent stampidine as an intravaginal microbicide. Novel vaginal formulations are under development to reduce the increasing incidence of sexually transmitted diseases, especially AIDS. Currently marketed vaginal formulations are available in different dosage forms (aerosols, creams, foams, films, gels, ovules, suppositories, tablets, tampons, and so on) [38–40]. A thermoreversible ovule is attractive for intravaginal drug delivery since it is in a solid state at ambient temperature and forms a bioadhesive semisolid gel following intravaginal insertion, thereby increasing the rapid spreadability and local bioavailability of the drug required to prevent the mucosal transmission of HIV. In the present study, repeated intravaginal administration of stampidine via a novel thermoreversible ovule formulation even at concentrations as high as 4 x 107-fold higher than its in vitro anti-HIV IC50 value did not cause significant vaginal irritation in the rabbit model. Stampidine was highly stable in aqueous solution over the pH range of 2.0–6.0 and was rapidly bioavailable in ovule formulation.

The potential of thermoreversible gels as drug delivery vehicles has been widely studied. Gels formed by the oxyethylene-oxypropylene-oxyethylene triblock polymer and the detergent Pluronic F127 have been investigated for use as drug delivery systems for ophthalmic [41], rectal [42], intraperitoneal [43, 44], nasal [45], dermatological [46], and more recently vaginal [47, 48] administration. However, the high concentration of these nonbiodegradable block copolymers (20–30% w/w) needed to achieve gelation can induce toxicity especially when intended for drug delivery to a mucosal body cavity. In contrast, the biodegradable components of the thermoreversible ovule formulation of stampidine including polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80 are pharmaceutically, pharmacologically, and pharmacokinetically acceptable nontoxic excipients used in the preparation of a variety of topical and/or oral medications [49–51]. Among the various surfactants currently used in cosmetic products, Tween-80 was found to be the least cytotoxic [52–54]. The in vitro cytotoxicity data correlated with ocular or skin irritation tests. This is in contrast to the currently used surfactant spermicide/microbicide N-9, which is cytotoxic at low micromolar concentrations [9–15, 55, 56]. Therefore, unlike the currently used nonionic and cationic detergent-type microbicides, the thermoreversible ovule formulation of stampidine is not likely to cause harmful side effects following repetitive intravaginal application.

Repeated intravaginal treatment with stampidine ovule formulation did not cause inflammation or hyperplasia in the rabbit vaginal epithelium and submucosa. The rabbit vaginal mucosa is more sensitive than of the rat and remains the animal model of choice for measuring the potential irritancy of new intravaginal microbicides since comparisons can be made with products of known irritancy. Rabbits have a simple cuboidal or columnar epithelium that is highly sensitive to mucosal irritants when compared to the stratified squamous epithelium of human vagina. A correlation exists between rabbits and humans with respect to the irritation potential of vaginal formulations. This correlation is well known and used extensively in the pharmaceutical industry. However, it is now becoming apparent that the "soft" preclinical endpoints established for the clinical development of vaginal spermicides may not be rigorous enough for the development of safe vaginal or rectal microbicides. In the light of currently marketed detergent spermicides having no significant adverse effects in trials conducted before 1998 and being judged safe, there is still the possibility of nonclinical irritation and inflammatory responses increasing mucosal HIV transmission. Thus, the criteria for mucosal safety that will lead to the clinical advancement of potential microbicides needs steadfast guidelines in view of irritative genitourinary symptoms reported for the first-generation microbicides that have advanced to clinical testing [57]. Furthermore, alternative in vitro models designed to evaluate the mucosal toxicity without in vivo testing have severe limitations since these in vitro cell/tissue models lack a network of mechanisms operative in tissues such as the presence of chemoattracting cytokines needed for the recruitment and infiltration of leukocytes.

Stampidine was consistently and significantly more effective than other NRTIs such as ZDV/AZT, STV/d4T or lamivudine (LMV) [17, 18, 27]. Stampidine was 100-fold more potent than STV and 2-fold more potent than ZDV against clinical HIV-1 isolates of non-B envelope subtype originating from South America, Asia, and sub-Saharan Africa [27, 28]. Stampidine inhibited the in vitro replication of 20 genotypically and phenotypically NRTI-resistant HIV-1 isolates carrying two to five thymidine analogue mutations associated with NRTI resistance at nanomolar concentrations [28]. Stampidine inhibited the replication of NNRTI-resistant HIV strains with mutations involving K103N, V106A, Y181C, or Y188L with subnanomolar to nanomolar IC50 values. In particular, the Y181C and K103N mutants may be the most difficult to treat because they are resistant to most of the NNRTIs that have been examined. The potency of stampidine against genotypically and phenotypically NRTI-resistant HIV-1 isolates may be due to the rapid kinetics of the generation of its active triphosphate metabolite yielding much higher inhibitor concentrations at the catalytic site sufficient to overcome the binding restrictions imposed by the NRTI resistance-associated RT mutations [58, 59]. Furthermore, the presence of an alaninyl side chain may promote the binding and/or incorporation of the triphosphate metabolite of stampidine. It has also been proposed that aryl phosphate derivatives of STV enter target cells easier than STV [59], which could also contribute to higher inhibitor concentrations at the catalytic site.

We have investigated the in vivo pharmacokinetics, metabolism, toxicity, and antiretroviral activity of this promising new anti-HIV agent in rodent and nonrodent species [30–33]. In mice and rats, stampidine was found to form two active metabolites with favorable pharmacokinetics after systemic administration, namely, Ala-STV-MP and STV [33]. Stampidine was very well tolerated in mice and rats without any detectable acute or subacute toxicity at single intraperitoneal or oral bolus dose levels as high as 500 mg/kg. Notably, daily administration of stampidine intraperitoneally or orally for up to 8 consecutive weeks was not associated with any detectable toxicity at cumulative dose levels as high as 6.4 g/kg. Stampidine exhibited dose-dependent and potent in vivo anti-HIV activity in Hu-PBL-SCID mice against a genotypically and phenotypically NRTI-resistant clinical HIV-1 isolate at nontoxic dose levels [29]. In accordance with its safety profile in rodent species, a 4-wk stampidine treatment course with twice-daily administration of hard gelatin capsules containing 25–100 mg/kg stampidine was very well tolerated by dogs and cats at cumulative dose levels as high as 8.4 g/kg [31–33]. Stampidine therapy was not associated with any clinical or laboratory evidence of toxicity. No stampidine-related toxic lesions were found in any of the organs from stampidine-treated cats or dogs. Notably, the results of our pharmacokinetic studies provided direct evidence that therapeutic concentrations of stampidine >4 logs higher than its IC50 value can be achieved after its per os administration to dogs as well as cats at the 50- or 100-mg/kg nontoxic dose levels. A 4-wk treatment course with stampidine administered in gelatin capsules twice daily showed a dose-dependent antiretroviral effect in chronically FIV-infected cats [30].

The documented in vitro potency of stampidine against primary clinical HIV-1 isolates with genotypic and/or phenotypic NRTI or NNRTI resistance as well as non-B envelope subtype together with its in vivo antiretroviral activity in HIV-infected Hu-PBL SCID mouse and FIV/cat models warrant the further development of this promising new NRTI compound for possible clinical use in both treatment-naive and treatment-experienced HIV-1-infected persons harboring highly drug-resistant strains of HIV-1. Furthermore, pretreatment of human sperm with stampidine even at a concentration 106 times higher than its in vitro anti-HIV-1 IC50 value had no adverse effects on sperm motility, kinematics, or cervical mucus penetrability [34]. Stampidine was noncytotoxic to female genital tract epithelial cells. Pretreatment of rabbit semen with stampidine had no adverse effects on pregnancy outcome in the rabbit model [34]. Taken together, the results of the present study indicate that stampidine, when delivered via a thermoreversible ovule, may be useful as a broad-spectrum vaginal anti-HIV microbicide without contraceptive activity.


   FOOTNOTES
 
1 This work was supported in part by National Institute of Health grants HD 37357, HD 42884, HD 42889, and AI 54352 and American Foundation for AIDS Research grant 02667. Back

2 Correspondence: Osmond J. D'Cruz, Parker Hughes Institute, 2657 Patton Road, St. Paul, MN 55113. FAX: 651 628 9891; odcruz{at}ih.org Back

Received: 4 June 2003.

First decision: 24 June 2003.

Accepted: 28 July 2003.


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