A 13-week subchronic intravaginal toxicity study of pokeweed antiviral protein in mice.

Summary

Pokeweed antiviral protein (PAP), a 29-k Da plant-derived protein isolated from Phytolacca americana, is a broad-spectrum antiviral agent. PAP shows unique clinical potential to become the active ingredient of a non-spermicidal microbicide because of its potent in vivo anti-HIV activity, non-interference with in vivo sperm functions, and lack of cytotoxicity to genital tract epithelial cells. Over 13 weeks the subchronic and reproductive toxicity potential of an intravaginally administered gel formulation of PAP was studied in mice to support its further development as a vaginal microbicide. Female [B.sub.6][C.sub.3][F.sub.1] and CD-1 mice in subgroups of 20, were exposed intravaginally to a gel formulation containing 0, 0.025, 0.05, or 0.1% PAP, 5 days/week for 13 consecutive weeks. On a molar basis, these concentrations are 500--to 2000-times higher than the in vitro anti-HIV [IC.sub.50] value. After 13 weeks of intravaginal treatment, [B.sub.6][C.sub.3][F.sub.1] mice were evaluated for survival, body weight gain, and absolute and relative organ weights. Blood was analyzed for hematology and clinical chemistry profiles. Microscopic examination was performed on hematoxylin and eosin-stained tissue sections from each study animal. Placebo-control and PAP-dosed female CD-1 mice were mated with untreated males in order to evaluate if PAP has any deleterious effects on reproductive performance. There were no treatment-related mortalities. Mean body weight gain was not reduced by PAP treatment during the dosing period. The hemogram and blood chemistry profiles revealed lack of systemic toxicity following daily intravaginal instillation of PAP for 13 weeks. No clinically significant changes in absolute and relative organ weights were noted in the PAP dose groups. Extensive histopathological examination of tissues showed no increase in treatment-related microscopic lesions in any of the three PAP dose groups. Repeated intravaginal exposure of CD-1 mice to increasing concentrations of PAP for 13 weeks showed no adverse effect on their subsequent reproductive capability (100% fertile), neonatal survival (>90%) or pup development. Collectively, these findings demonstrate that repetitive intravaginal administration of PAP at concentrations as high as 2000 times its in vitro anti-HIV [IC.sub.50] value was not associated with local or systemic toxicity and did not adversely affect the reproductive performance of mice. PAP may be useful as an active ingredient of a safe vaginal microbicide for prevention of the sexual transmission of viruses, particularly of HIV-1.

Key words: 13-week intravaginal, antiviral proteins, HIV/AIDS, microbicide

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Introduction

The sexual transmission of human immunodeficiency virus, type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), continues to be the predominant mode of the pandemic spread of HIV/AIDS (Quinn, 1996). Worldwide, more than 80 percent of all adult HIV infections have resulted from heterosexual intercourse (CDC Report, 2001). Approximately 49%, or 19.2 million, of the 39.2 million adults living with HIV/AIDS worldwide are women (UN-AIDS report, 2001). Women are 4-16 times more likely to contract HIV from infected males than vice versa, and young women are especially vulnerable (Rivers and Aggleton, 2001). In the US, the proportion of AIDS cases among women has more than tripled, from 7% in 1985 to 25% in 2000 (Gayle, 2000). HIV/AIDS is currently the leading cause of death for African-American women between the ages of 25-44 and the sixth-leading cause of death for all American women in this age group (Hodge, 2001). In addition to HIV, an estimated 45 million Americans older than 14 suffer from genital herpes, and 20 million from HPV (ASHA report, 1998). More than one-half million each have sexually transmitted hepatitis B and HIV-1 (ASHA report, 1998).

The emergence of HIV/AIDS as a disease spread through sexual intercourse, combined with growing public awareness about the problems associated with other viral sexually transmitted diseases (STDs), has prompted the search for new, effective, and safe vaginal microbicides for curbing mucosal and perinatal viral transmission (Uckun and D'Cruz, 1998). Microbicides would provide protection by inactivating viruses or preventing viruses from replicating either in semen or the infected host cells that line the vaginal wall. Microbicides that are currently being investigated are directed mainly at prevention of pregnancy as well as protection against STDs (Uckun and D'Cruz, 1998; D'Cruz et al. 1998, 1999, D'Cruz and Uckun, 1999; D'Cruz et al. 2000). The availability of a non-spermicidal microbicide will be equally important for sexually active women to allow pregnancy while protecting both mother and fetus from HIV infection. The antiviral agent should have broad-spectrum activity, because HIV-1-infected men often have other sexually transmitted viruses in their semen, such as cytomegalovirus, herpes simplex virus, and hepatitis C virus (Belec et al. 1998). It must also be certain, however, that repeated short-term usage of vaginal microbicide poses no health risk for the female partner by producing local inflammation or systemic toxicity.

A number of plant-derived biologicals with antiviral properties are currently under investigation as experimental anti-HIV agents (Mori et al. 1998; Lee-Huang et al. 1995; Uckun et al. 1999). Of these, pokeweed antiviral protein (PAP), a 29-kDa naturally occurring protein isolated from the leaves of the pokeweed plant, Phytolacca americana, is particularly promising because it is well characterized and can be purified in relatively large amounts (Irvin, 1995; Irvin and Uckun, 1992; Tumer et al. 1999; Rajamohan et al. 1999a, b, c, 2000; Myers et al. 1991, 1997). Both the native and recombinant forms of PAP exhibit potent anti-HIV activity (Myers et al. 1997; Rajamohan et al. 1999a, b, c, 2000). PAP displays broad-spectrum antiviral activity against several pathogenic viruses including HIV-1, herpes simplex virus, cytomegalovirus, influenza virus, and polio virus (Irvin, 1995; Irvin and Uckun, 1992; Tumer et al. 1999). PAP inhibits HIV-1 replication with nanomolar [IC.sub.50] values. The anti-HIV activity of PAP is attributed to its unique ability to inhibit viral protein synthesis and depurinate HIV-1 RNA (Rajamohan et al. 1999b, c). The potent anti-HIV activity of PAP has led to the clinical use of PAP as an anti-HIV agent (Myers, 1991; Uckun et al. 1999). PAP conjugates of monoclonal antibodies recognizing CD4 or CD7 antigens on the cell surface effectively inhibit HIV-1 replication in normal T-cells at noncytotoxic concentrations (Zarling et al. 1990; Erice et al. 1993; Uckun et al. 1998). The activity of PAP conjugates has been tested against both zidovudine (ZDV/AZT)-sensitive and ZDV-resistant 22 clinical HIV-1 strains. Both ZDV-sensitive and ZDV-resistant clinical HIV-1 isolates were >4 log more sensitive to PAP than to ZDV (Zarling et al. 1990).

We recently reported that in vitro treatment of human sperm with PAP, even at a concentration 2000-times higher than its in vitro anti-HIV-1 activity showed no adverse effect on sperm motility, cervical mucus penetrability, sperm-egg binding and penetration (D'Cruz and Uckun, 2001a). PAP was nontoxic to normal female genital tract epithelial cells. Furthermore, PAP treatment of semen even at a concentration 2000 times higher than its in vitro anti-HIV-1 [IC.sub.50] value, prior to artificial insemination, had no significant adverse impact on maternal fertility and newborn parameters in the rabbit model (D'Cruz and Uckun, 2001b). These studies indicated unequivocally the clinical potential of PAP as a non-spermicidal microbicide. An important step in further developing PAP as a vaginal microbicide is a detailed preclinical study of its local and systemic toxicity potential following repeated intravaginal exposure. In this report, we describe the results of a 13-week subchronic study that examined the local, systemic, and reproductive toxicity potential of intravaginally administered PAP in [B.sub.6][C.sub.3][F.sub.1] and CD-1 mice, respectively.

Materials and Methods

Purification of PAP

PAP was purified from spring leaves of the pokeweed plant, P. americana, in four steps (Myers et al. 1991, 1997). Briefly, spring leaves were homogenized in a neutral pH buffer and centrifuged to sediment remaining cellular fragments. The supernatant was fractionated between 60%-95% saturation of ammonium sulfate and the precipitate dialyzed against a low-ionic-strength pH 7.5 buffer. The solution was passed through a DEAE cellulose column and the flowthrough fraction containing PAP was then applied to a cation exchange resin S-Sepharose column. The adsorbed PAP was eluted in a linear KCl gradient. The protein peak which eluted at 0.12 M KCl was taken as PAP. This fraction was dialyzed extensively against water and lyophilized for storage at -20[degrees]C. The procedure resulted in homogeneous PAP, with a purity of >99% as measured by both SDS-12% PAGE and analytical cation-exchange high-performance chromatography. Purified PAP induced concentration-dependent inhibition of HIV-1 replication in normal human peripheral blood mononuclear cells infected with the HIV-1 strain [HTLV.sub.IIIB] with an [IC.sub.50]p24 value of 14 [+ or -] 2 nM.

Gel formulation of PAP

A gel formulation of PAP for intravaginal delivery was prepared by mixing a stock solution of PAP (100 mg/ml) in saline with a mixture of seaspan carrageenan and Xantral polymer solutions, with sodium benzoate as a preservative. The mixing was performed at room temperature with a Caframo BDC 3030 mixer at 600 rpm until a translucent homogenous suspension was obtained, with desirable viscosity (~ 1000 centipoise), containing 0.025-0.10% PAP. The gel formulation did not cause drug precipitation. PAP-containing gel formulation was found to be very stable as judged by SDS-PAGE analysis of formulated protein over time.

Animals

Eighty female [B.sub.6][C.sub.3][F.sub.1] and eighty female CD-1 mice of 6 weeks of age and 80, proven fertile, 3-month old, male CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). They were assigned to groups of five per cage, in microisolator cages, and were provided with corncob or wood chip as bedding (Harlan Teklad, Madison WI). All assigned mice were uniquely identified with metal ear tags and ear notches. All cages, bedding, tap water, and laboratory diet (Teklad LM-485; Harlan Teklad) were sterilized. Water and food were available ad libitum. Throughout the duration of the study, the mice were maintained in a HEPA filtered room (alongside sentinel animals) kept at 22 [+ or -] 2[degrees]C with a relative humidity of 50 [+ or -] 10% and a 12-h light/dark cycle. Mice were quarantined and acclimated for 35 days prior to initiation of the studies. Animal studies were approved by the Parker Hughes Institute Animal Care and Use Committee and all animal husbandry operations were in compliance with the "Guide for the Care and Use of Laboratory Animals" (Institute of Laboratory Animal Resources, 1996).

Experimental design

Animal studies were conducted in accordance with Good Laboratory Practice (GLP) conditions. PAP was dissolved in a gel formulation at concentrations of 0.025, 0.05, and 0.10%. Eighty female [B.sub.6][C.sub.3][F.sub.1] as well as CD-1 mice were allocated to 4 subgroups of 20. They were given 50 [micro]l of intravaginal gel containing 0, 0.025, 0.05, or 0.10% PAP, respectively. The treatment period was 5 days per week for 13 consecutive weeks. Intravaginal instillation was performed inside a laminar flow hood using sterile procedures. All animals were observed individually and daily for signs of toxic effects. Body weights were obtained before exposure (day 0), weekly during exposure, and preceding sacrifice. At the end of the 13-week case study, blood samples were collected from [B.sub.6][C.sub.3][F.sub.1] mice for systemic toxicity studies (hematology and clinical chemistry). Blood collected from all [B.sub.6][C.sub.3][F.sub.1] mice exposed to gel with and without PAP was used for determination of each of the 13-hematology parameters and 18 clinical chemistry parameters. A complete necropsy was performed on all treated and control [B.sub.6][C.sub.3][F.sub.1] mice.

Hematology parameters

Hematology parameters were analyzed using an Abbot CELL-DYN 3200 multiparameter, automated hematology analyzer (Abbot Laboratories, Abbot Park, IL), which was standardized for mouse blood (D'Cruz and Uckun, 2001c). This instrument uses flow-cytometric techniques to generate the following hematologic measurements in heparin anticoagulated whole blood: red blood cells (RBC; [10.sup.4]/[micro]l), total and differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils as [10.sup.3]/[micro]l or %), hemoglobin concentration (HGB; g/dl), hematocrit (HCT; %), mean corpuscular volume (MCV; fl), mean cell hemoglobin (MCH; pg), mean cell hemoglobin concentration (MCHC; g/dl), red cell distribution width (RDW; %), platelets (PLT; [10.sup.4]/[micro]l), and mean platelet volume (MPV; fl).

Clinical chemistry profiles

Biochemical analyses were performed using a Boehringer Mannheim Hitachi 911 analyzer (Indianapolis, IN). Blood collected at sacrifice, which was obtained by the cardiac route following 13 weeks of intravaginal application from placebo and PAP-treated mice, was used for the determination of plasma levels of total protein (TP), albumin (ALB), globulin (G), albumin/globulin (ALB/G) ratio, blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (CHO), triglycerides (TG), aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), alkaline phosphatase (ALP), amylase (AMY), total bilirubin (TBIL), glucose (GLU), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and phosphorus (P), using reagents and methods provided by the manufacturer.

Necropsy and histopathology 

All [B.sub.6][C.sub.3][F.sub.1] mice from the control and PAP-treatment groups were sacrificed after 13 weeks of intravaginal exposure for complete necropsy and histopathological evaluation of tissues. At necropsy, the brain, thymus, heart, lungs, intestine, liver, pancreas, spleen, kidneys, and genital tract (cervix, vagina, uterus, and ovaries) as well as the bone (sternum, femur, and vertebra), bone marrow, large and small intestine, skeletal muscle, skin, spinal cord, spinal nerves, stomach, and urinary bladder were fixed in 10% buffered formalin solution, trimmed, embedded in paraffin, sectioned at 4-6 [micro]m, and stained with hematoxylin and eosin. Tissue sections were evaluated microscopically for all control and PAP-dosed groups.

Organ weights

The brain, thymus, heart, lungs, liver, pancreas, spleen, kidneys, and genital tract (ovaries, uteri, and cervicovagina) from each of 20 animals per dose were weighed at necropsy. Organ weights were recorded as absolute weights and as a percentage of body weight.

Fertility of female CD-1 Mice

Immediately after cessation of the 13-week intravaginal administration, CD-1 mice from control and PAP-treatment groups were caged (1:1) with untreated males of the same strain. The females were allowed to complete a pregnancy (21 [+ or -] 2 days). Litter size, neonatal weight, and condition of each offspring were determined. The pups were allowed to remain with the dam until day 5. The number of pups per litter and the mean weight of the pups surviving on day 5 were used to measure perinatal effects.

Statistical analysis

Group means and standard deviations were calculated from body weights, organ weights, hematology and clinical chemistry parameters and reproductive end-points. Statistical significance of the treated group mean with that of control group was analyzed by a one-way analysis of variance, followed by Dunnett's multiple comparison test using GraphPad Instat version 2.03 software (San Diego, CA). The incidence of histopathological changes and the number of offspring were evaluated by Fisher's exact probability test. Differences were considered statistically significant if p < 0.05.

Results

Animal observations and measurements

Mortality did not occur in any mouse group and there were no clinical signs attributed to intravaginal exposure of PAP-containing gel throughout these studies. All animals were clinically healthy and all groups gained weight during the 13-week treatment period. Mean body weight gain and final mean body weight of [B.sub.6][C.sub.3][F.sub.1] mouse groups exposed to increasing doses of PAP for 13 weeks were similar to those of the gel controls (Fig. 1).

Laboratory parameters

Complete blood counts of mice revealed no biologically significant differences between PAP-dosed and vehicle control mice. The values of hematologic parameters studied including red cell counts, leukocyte/lymphocyte counts, platelet counts, and hemoglobin were within normal limits (Table 1). Analysis of blood chemistry parameters for female mice revealed no clinically significant treatment-related differences between PAP-dosed and vehicle control groups (Table 2). A statistically significant increase in the mean AST values was observed only in the lowest (0.025%) dose group. In particular, even at the highest dose level, intravaginal PAP treatment did not cause alterations in kidney function (BUN and CRE), liver function (TBIL, AST, ALT, ALP, CHO, and TG), pancreas function (AMY, GLU), immunological function (G), nutritional status (TP), calcium (Ca), phosphorous (P), or serum electrolytes (Na, K, and Cl).

[FIGURE 1 OMITTED]

Necropsy/organ weights

Table 3 summarizes the terminal body weight, terminal absolute and relative organ weights of vehicle control and PAP-dosed [B.sub.6][C.sub.3][F.sub.1] mouse groups, respectively, at the conclusion of the 13-week study. There were no statistically significant treatment-related differences in absolute and relative organ weights of the brain, heart, intestines, kidneys, liver, lung, pancreas, reproductive organs, spleen, thymus, or urinary bladder between vehicle control and 0.025, 0.05 and 0.1% PAP-treatment groups, which were considered to be related to intravaginal PAP exposure. No treatment-related microscopic lesions were found at necropsy (see below).

Histopathology

Microscopic examination of tissue specimens taken from the placebo control and PAP-dosed [B.sub.6][C.sub.3][F.sub.1] mice did not reveal any treatment-related organ lesions (Table 4). The incidental lesions noted in kidney (15-30%) and uterus (20-35%) of PAP-treated mice were relatively low in comparison to the background incidence of such lesions in placebo-treated control mice (50% and 45% respectively; Table 4). These histologic lesions noted included mild to moderate hydronephrosis (5%), subacute interstitial nephritis (5-30%), pyelitits (10-20%), mild suppurative or nonsuppurative pyelonephritis (5%) of the kidney and mild cystic endometrial hyperplasia (15-45%), endometrial fibrosis (5%), and endometritis (5%) of uterus that could be expected to be seen normally in laboratory mice, and were distributed randomly throughout the study groups (Table 5). The interstitial nephritis of the kidney and endometrial hyperplasia of the uterus lesions was more pronounced in the vehicle control group. Other incidental lesions diagnosed were lipoma (5%) of the brain, myocardial degeneration (5%), and satellitosis of the heart (5%), ovarian cyst (5%), and urinary bladder cystitis (11%), found in a few animals only, were distributed randomly in the control and PAP-dosed groups. Microscopic examination did not reveal adverse effects of the test substance on the genital tract (cervico-vagina, uteri, and ovaries), which suggests lack of toxicity to repeated intravaginal exposure to PAP via gel formulation. No inflammatory changes were noted in the ovaries, uteri, or cervico-vaginas of mice following repeated intravaginal instillation of gel formulation, with and without increasing amounts of PAP. Notably, the epithelial lining and glandular architecture appeared normal.

Reproductive Performance

Table 6 summarizes the fertility parameters for CD-1 mice given intravaginally increasing concentrations of PAP for 13 weeks before mating. One CD-1 mouse in the 0.025% dose group succumbed to the muricidal behavior of the male. All other mice in all groups survived and were clinically healthy at the end of the fertility study. When gel control and PAP-treated female CD-1 mice were evaluated for their fertility immediately after cessation of the 13-week intravaginal administration, we observed no statistically significant adverse effect on fertility parameters in PAP-treated mice. Thirteen-week treatment of PAP at concentrations higher than 500 to 2000-times its in vitro anti-HIV I[C.sub.50] value had no statistically significant adverse effect on subsequent gestation length (22 [+ or -] 1 days), fertility (100% fertile), median litter size (n = 10-11), neonatal survival (>90%), pup morphology, or development. The mean neonatal and pup weights from mice exposed to 0.025%, 0.05% and 0.1% PAP were not lower than those of placebo-control group at birth and on lactation day 5.

Discussion

In the present study, intravaginal administration of increasing concentrations of PAP, a highly stable, potent and broad-spectrum antiviral protein, given to female [B.sub.6][C.sub.3][F.sub.1] mice via a gel formulation for 13 weeks, caused no treatment-related adverse effects on survival, mean body weight gain, mean body weight, absolute or relative organ weights or histopathology. PAP did not have an adverse effect on the function of the hematopoietic system, kidneys, liver, and pancreas or the nutritional status. Furthermore, repeated intravaginal exposure of female CD-1 mice to increasing concentrations of PAP for 13 weeks had no adverse effect on their subsequent reproductive capability, perinatal outcome, growth, or development of the offspring. Collectively, these findings demonstrate that repetitive intravaginal administration of PAP to yield effective antiviral concentrations in the vagina was not associated with local or systemic toxicity and did not adversely affect reproductive performance in mice.

PAP was discovered due to its ability to inhibit the transmission of tobacco mosaic virus in plants and it was subsequently demonstrated that the purified protein was equally effective against seven different viruses, each representing a different plant virus group (Irvin, 1995; Irvin and Uckun, 1992; Tumer et al. 1999). A comparison of the relative antiviral properties of a number of ribosome-inactivating proteins (RIPs), including PAP, indicated that of all the RIPs tested, none was as effective as PAP. Studies using virus infection of cultured mammalian cells demonstrated that PAP was an effective inhibitor of influenza virus, polio virus, herpes simplex virus, and HIV-1 (Irvin, 1995; Irvin and Uckun, 1992; Tumer et al. 1999; D'Cruz and Uckun, 2001a).

The anti-HIV-1 activity of PAP is attributed to its unique ability to preferentially inhibit viral protein synthesis and depurinate HIV-RNA. PAP is a site-specific RNA N-glycosidase which catalytically removes a single adenine base from a highly conserved "[alpha]-sarcin/ricin" (SR) loop of the large ribosomal RNA species in eukaryotic (28S rRNA) and prokaryotic (23S rRNA) ribosomes (Endo et al. 1988). This depurination of the SR loop results in irreversible inhibition of protein synthesis at the translation step by impairing both the elongation factor (EF) 1-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF-2 to the affected ribosome (Gessner and Irvin, 1980). Recent evidence indicates that L3, a highly conserved ribosomal protein at the peptidy ltransferase center, may provide a binding site for PAP, allowing depurination of the target adenine in its RNA substrate (Hudak et al. 1999). Aside from its ability to inhibit viral protein synthesis, PAP is also capable of directly depurinating viral RNA and DNA (Rajamohan et al. 1999b, c). PAP can inhibit translation by a mechanism other than ribosome depurination, by recognizing the cap structure and specifically depurinating the capped RNAs (Hudak et al. 2000). Furthermore, PAP also displays other viral RNA-specific effects in vivo and has been shown to inhibit ribosomal frameshifting and retrotransposition, a molecular mechanism used by many RNA viruses to produce Gag-Pol fusion proteins (Tumer et al. 1998).

In conclusion, sexual transmission of HIV is thought to be transmitted primarily in the form of infected cells in semen. The potent antiviral activity of PAP against HIV-1, herpes simplex virus, and cytomegalovirus may be useful for the reduction or elimination of these viruses in semen. The demonstrated lack of adverse effects of PAP sperm function and normal female genital tract epithelial cells (D'Cruz and Uckun, 2001a, b), as well as the low potential of intravaginally administered PAP to produce local, systemic or reproductive toxicity as showed in this study, indicates that PAP displays the very properties required of an ideal nonspermicidal microbicide and warrants further preclinical research and development. Recently, we have cloned the cDNA for PAP and expressed the biologically active and homogeneously pure recombinant PAP [rPAP] in bacteria and yeast (Rajamohan et al. 1999a, 2000). Using a molecular model of PAP-HIV-1 RNA interactions for the rational design of rPAP, we have engineered, produced, and identified two mutant rPAP proteins with superior anti-HIV activity. These proteins depurinate viral RNA preferentially and are more potent anti-HIV agents than native PAP or recombinant wild type PAP both in vitro and in vivo. The availability of nontoxic rPAP mutants with superior anti-HIV activity provides the basis for large-scale production of clinical-grade PAP for its development as a promising nonspermicidal, broad-spectrum antiviral microbicide to inactivate both cell-free and cell-associated viruses in genital tract secretions.

Table 1. Hematological findings for [B.sub.6][C.sub.3][F.sub.1] mice
given PAP intravaginally for 13 weeks.

Parameter                    PAP concentration (%)
                             0 (a)                0.025

RBC (X[10.sup.4]/[micro]l)   831 [+ or -] 43      819 [+ or -] 44
WBC (X[10.sup.4]/[micro]l)     2.2 [+ or -] 0.9     2.5 [+ or -] 1.4
LYM (%)                       73.6 [+ or -] 15.7   73.5 [+ or -] 16.4
NEU (%)                        3.1 [+ or -] 1.8     3.9 [+ or -] 2.5
Others (%) (b)                23.3 [+ or -] 15.0   23.0 [+ or -] 14.8
HGB (g/dl)                    12.4 [+ or -] 0.7    12.2 [+ or -] 0.7
HCT (%)                       44.0 [+ or -] 2.4    43.4 [+ or -] 2.4
MCV (fl)                      53.0 [+ or -] 0.4    53.0 [+ or -] 0.5
MCH (pg)                      14.9 [+ or -] 0.3    14.7 [+ or -] 0.6
MCHC (g/dl)                   28.2 [+ or -] 0.6    27.8 [+ or -] 1.2
RDW (%)                       17.7 [+ or -] 0.7    17.6 [+ or -] 0.8
PLT (X[10.sup.4]/[micro]l)   720 [+ or -] 152     726 [+ or -] 210
MPV (fl)                       5.1 [+ or -] 0.7     5.2 [+ or -] 0.8

Parameter                    PAP concentration (%)
                             0.05                 0.10

RBC (X[10.sup.4]/[micro]l)   823 [+ or -] 85      836 [+ or -] 95
WBC (X[10.sup.4]/[micro]l)     2.3 [+ or -] 0.8     2.4 [+ or -] 0.7
LYM (%)                       72.0 [+ or -] 11.7   77.9 [+ or -] 11.6
NEU (%)                        4.0 [+ or -] 2.2     2.6 [+ or -] 1.7
Others (%) (b)                24.0 [+ or -] 10.1   19.5 [+ or -] 10.8
HGB (g/dl)                    12.1 [+ or -] 1.1    12.2 [+ or -] 1.2
HCT (%)                       43.9 [+ or -] 4.5    44.4 [+ or -] 5.0
MCV (fl)                      53.3 [+ or -] 0.5    53.2 [+ or -] 0.3
MCH (pg)                      14.8 [+ or -] 0.4    14.7 [+ or -] 0.5
MCHC (g/dl)                   27.8 [+ or -] 0.6    27.6 [+ or -] 0.9
RDW (%)                       17.7 [+ or -] 0.6    17.6 [+ or -] 0.5
PLT (X[10.sup.4]/[micro]l)   740 [+ or -] 184     800 [+ or -] 168
MPV (fl)                       4.9 [+ or -] 0.4     4.8 [+ or -] 0.4

(a) Mean [+ or -] SD, n = 20.
(b) Others -- MONO, EOS, and BASO, RBC -- red blood cells; WBC -- white
blood cells; LYM -- lymphocytes; NEU -- neutrophils; MONO -- monocytes;
EOS -- eosinophils; BASO -- basophils; HGB -- hemoglobin concentration;
HCT -- hematocrit; MCV -- mean corpuscular volume; MCH -- mean cell
hemoglobin; MCHC -- mean cell hemoglobin concentration; PLT --
platelets; MPV -- mean platelet volume.

Table 2. Blood chemistry profiles for [B.sub.6][C.sub.3][F.sub.1] mice
given PAP intravaginally for 13 weeks.

Parameter                PAP concentration (%)
                     0 (a)                  0.025

TP     g/dl      4.7 [+ or -] 0.2       4.7 [+ or -] 0.3
ALB    g/dl      3.0 [+ or -] 0.1       2.9 [+ or -] 0.3
G      g/dl      1.6 [+ or -] 0.1       1.7 [+ or -] 0.1
ALB/G  g/dl      1.8 [+ or -] 0.2       1.7 [+ or -] 0.3
BUN    mg/dl    21 [+ or -] 3          22 [+ or -] 2
CRE    mg/dl     0.24 [+ or -] 0.06     0.25 [+ or -] 0.05
CHO    mg/dl   103 [+ or -] 8          99 [+ or -] 12
TG     mg/dl   125 [+ or -] 35        102 [+ or -] 36
AST    IU/l     86 [+ or -] 60        188 [+ or -] 220*
ALT    IU/l     46 [+ or -] 25         89 [+ or -] 124
ALP    IU/l     82 [+ or -] 10         75 [+ or -] 13
AMY    IU/l   1765 [+ or -] 156      1617 [+ or -] 181
TBIL   mg/dl     0.21 [+ or -] 0.1      0.25 [+ or -] 0.2
GLU    mg/dl   271 [+ or -] 38        257 [+ or -] 50
Ca     mg/dl     8.0 [+ or -] 0.2       8.1 [+ or -] 0.4
Na     mg/dl   150 [+ or -] 1         152 [+ or -] 2
K      mg/dl     3.6 [+ or -] 0.3       3.9 [+ or -] 0.5
Cl     mg/dl   108 [+ or -] 2         109 [+ or -] 2
P      mg/dl     4.6 [+ or -] 0.6       4.5 [+ or -] 0.8

Parameter               PAP concentration (%)
                     0.05                   0.10

TP     g/dl      4.7 [+ or -] 0.3       4.7 [+ or -] 0.2
ALB    g/dl      3.0 [+ or -] 0.2       3.1 [+ or -] 0.2
G      g/dl      1.7 [+ or -] 0.1       1.6 [+ or -] 0.1
ALB/G  g/dl      1.8 [+ or -] 0.2       1.9 [+ or -] 0.1
BUN    mg/dl    21 [+ or -] 2          22 [+ or -] 2
CRE    mg/dl     0.24 [+ or -] 0.05     0.24 [+ or -] 0.05
CHO    mg/dl    99 [+ or -] 6         108 [+ or -] 9
TG     mg/dl   110 [+ or -] 39        132 [+ or -] 32
AST    IU/l     73 [+ or -] 29         90 [+ or -] 80
ALT    IU/l     37 [+ or -] 16         39 [+ or -] 12
ALP    IU/l     86 [+ or -] 11         86 [+ or -] 7
AMY    IU/l   1624 [+ or -] 249      1798 [+ or -] 185
TBIL   mg/dl     0.16 [+ or -] 0.1      0.22 [+ or -] 0.2
GLU    mg/dl   262 [+ or -] 44        268 [+ or -] 48
Ca     mg/dl     8.2 [+ or -] 0.4       8.3 [+ or -] 0.3
Na     mg/dl   150 [+ or -] 3         150 [+ or -] 2
K      mg/dl     3.8 [+ or -] 0.7       3.6 [+ or -] 0.4
Cl     mg/dl   107 [+ or -] 2         105 [+ or -] 2
P      mg/dl     4.5 [+ or -] 0.8       4.6 [+ or -] 0.6

Mean [+ or -] SD for groups of 20 mice; * Significantly different from
control group (p < 0.05).

Table 3. Absolute and relative organ weights of
[B.sub.6][C.sub.3][F.sub.1] mice given PAP intravaginally for 13 weeks.

Organ       PAP concentration (%)
            0
            Absolute (g)             Relative (%)

Terminal
Body        30.4 [+ or -] 2.2
weight
Brain        0.57 [+ or -] 0.02      1.89 [+ or -] 0.08
Heart        0.15 [+ or -] 0.01      0.50 [+ or -] 0.04
Intestines   1.98 [+ or -] 0.32      6.53 [+ or -] 1.05
Kidney       0.20 [+ or -] 0.02      0.66 [+ or -] 0.06
Liver        1.82 [+ or -] 0.19      6.01 [+ or -] 0.64
Lung         0.31 [+ or -] 0.04      1.03 [+ or -] 0.15
Pancreas     0.22 [+ or -] 0.03      0.74 [+ or -] 0.12
Rep Org      0.17 [+ or -] 0.03      0.56 [+ or -] 0.12
Spleen       0.12 [+ or -] 0.01      0.40 [+ or -] 0.05
Thymus       0.05 [+ or -] 0.07 (a)  0.18 [+ or -] 0.26
Bladder      0.02 [+ or -] 0.00      0.08 [+ or -] 0.02

Organ       PAP concentration (%)
            0.025
            Absolute (g)         Relative (%)

Terminal
Body        29.7 [+ or -] 1.9
weight
Brain        0.57 [+ or -] 0.03  1.91 [+ or -] 0.10
Heart        0.14 [+ or -] 0.01  0.49 [+ or -] 0.03
Intestines   1.87 [+ or -] 0.21  6.31 [+ or -] 0.71
Kidney       0.19 [+ or -] 0.02  0.66 [+ or -] 0.07
Liver        1.73 [+ or -] 0.19  5.84 [+ or -] 0.64
Lung         0.31 [+ or -] 0.04  1.06 [+ or -] 0.14
Pancreas     0.21 [+ or -] 0.03  0.71 [+ or -] 0.09
Rep Org      0.16 [+ or -] 0.04  0.56 [+ or -] 0.15
Spleen       0.12 [+ or -] 0.02  0.43 [+ or -] 0.08
Thymus       0.03 [+ or -] 0.00  0.12 [+ or -] 0.02
Bladder      0.02 [+ or -] 0.00  0.08 [+ or -] 0.02

Organ       PAP concentration (%)
            0.05
            Absolute (g)         Relative (%)

Terminal
Body        30.0 [+ or -] 1.7
weight
Brain        0.58 [+ or -] 0.02  1.93 [+ or -] 0.07
Heart        0.15 [+ or -] 0.01  0.51 [+ or -] 0.04
Intestines   1.92 [+ or -] 0.18  6.41 [+ or -] 0.61
Kidney       0.21 [+ or -] 0.01  0.71 [+ or -] 0.06
Liver        1.69 [+ or -] 0.38  5.63 [+ or -] 1.27
Lung         0.32 [+ or -] 0.03  1.07 [+ or -] 0.11
Pancreas     0.22 [+ or -] 0.02  0.74 [+ or -] 0.08
Rep Org      0.18 [+ or -] 0.03  0.60 [+ or -] 0.09
Spleen       0.13 [+ or -] 0.01  0.44 [+ or -] 0.05
Thymus       0.04 [+ or -] 0.00  0.14 [+ or -] 0.02
Bladder      0.02 [+ or -] 0.00  0.09 [+ or -] 0.02

Organ       PAP concentration (%)
            0.10
            Absolute (g)             Relative (%)

Terminal
Body        30.4 [+ or -] 2.0
weight
Brain        0.59 [+ or -] 0.03  1.95 [+ or -] 0.0
Heart        0.15 [+ or -] 0.01  0.49 [+ or -] 0.03
Intestines   1.94 [+ or -] 0.1   6.38 [+ or -] 0.62
Kidney       0.20 [+ or -] 0.02  0.68 [+ or -] 0.06
Liver        1.82 [+ or -] 0.15  6.01 [+ or -] 0.51
Lung         0.32 [+ or -] 0.03  1.06 [+ or -] 0.10
Pancreas     0.23 [+ or -] 0.04  0.77 [+ or -] 0.13
Rep Org      0.19 [+ or -] 0.03  0.62 [+ or -] 0.10
Spleen       0.12 [+ or -] 0.01  0.42 [+ or -] 0.05
Thymus       0.04 [+ or -] 0.01  0.14 [+ or -] 0.0
Bladder      0.02 [+ or -] 0.00  0.08 [+ or -] 0.02

(a) Mean [+ or -] SD for groups of 18-20 mice.

Table 4. Incidence of microscopic lesions in [B.sub.6][C.sub.3][F.sub.1]
mice given PAP intravaginally for 13 weeks.

Tissue                 PAP concentration (%)
                       0      0.025  0.05  0.10

Bone and Bone marrow:
 Sternum                0/20  0/20   0/20  0/20
 Femur                  0/20  0/20   0/20  0/20
Brain                   0/20  1/20   0/20  0/20
Gut:
 Large Intestine        0/19  0/20   0/20  0/20
 Small Intestine        0/20  0/20   0/20  0/20
Heart                   1/20  0/20   0/20  1/20
Kidney                 10/20  6/20   3/20  6/20
Liver                   0/20  0/20   0/20  2/20
Lung                    2/20  0/20   0/20  0/20
Ovaries                 0/14  0/15   0/18  1/18
Pancreas                0/19  0/19   0/19  0/20
Skeletal Muscle         0/20  0/20   0/20  0/20
Skin                    0/17  0/19   0/20  0/20
Spinal Cord             0/19  0/19   0/20  0/19
Spinal Nerves           0/20  0/19   0/20  0/20
Spleen                  0/20  0/19   0/20  0/20
Thymus                  0/15  0/17   0/18  0/20
Urinary bladder         0/12  0/14   2/17  2/19
Uterus                  9/20  4/20   7/20  5/20
Cervix/Vagina           1/7   0/12   0/15  0/10

Table 5. Nature and incidence of microscopic lesions in [B.sub.6]
[C.sub.3][F.sub.1] mice given PAP intravaginally for 13 weeks.

Tissue                                          PAP concentration (%)
                                                  0    0.025  0.05  0.10

Brain                                            (20)  (20)   (20)  (20)
 Lipoma                                            0     1      0     0
Heart                                            (20)  (20)   (20)  (20)
 Mycardial degeneration, slight, left              0     0      0     1
   ventricle
   Satellitosis, focal, slight                     1     0      0     0
Kidneys                                          (20)  (20)   (20)  (20)
 Hydronephrosis, mild, chronic                     0     0      0     1
 Hydronephrosis, moderate, chronic                 1     0      0     0
 Interstitial nephritis, lymphocytic, slight,      6     1      1     3
   focal or multifocal, subacute
 Pyelitis, lymphocytic, slight, subacute           3     4      2     2
 Pyelonephritis, nonsuppurative, mild, chronic     0     1      0     1
 Pyelonephritis, suppurative, mild, chronic        1     0      0     0
Liver                                            (20)  (20)   (20)  (20)
 Hepatitis, necrosuppurative, slight, focal,       0     0      0     1
 acute-subacute
 Hepattis, nonsuppurative, slight, focal,          0     0      0     1
   subacute
Lung                                             (20)  (20)   (20)  (20)
 Pneumonia, nonsuppurative, slight, subacute       2     0      0     0
Ovary                                            (14)  (15)   (18)  (18)
 Ovarian cyst                                      0     0      0     1
Stomach                                          (20)  (20)   (20)  (20)
 Gastritis, suppurative, mild, subacute            1     1      1     0
Urinary bladder                                  (12)  (14)   (17)  (19)
 Cystitis, nonsuppurative, mild, subacute          0     0      1     2
 Cystitis, suppurative, moderate, subacute         0     0      1     0
Uterus                                           (20)  (20)   (20)  (20)
 Cystic endometrial hyperplasis, slight or mild    9     3      7     4
 Endometrial fibrosis, focal                       0     0      0     1
 Endometriosis, suppurative, slight or mild        1     1      0     0
Cervix/Vagina                                    (7)   (12)   (15)  (10)
 Cervicitis, suppurative, mild                     1     0      0     0

Numbers in parentheses are numbers of animals examined.

Table 6. Fertility Parameters for CD-1 mice given 0.025 to 0.1%
intravaginal administration of PAP for 13 weeks.

Parameter                 PAP concentration (%)
                          0                       0.025

No. of mice/group       20                       19
Pregnancy rate (%)      20/20 (100)              19/19 (100)
Mean litter size
  (median)              11.5 [+ or -] 3.0 (11)   10.3 [+ or -] 3.0 (10)
Mean neonatal
  weight (g)             1.69 [+ or -] 0.15       1.69 [+ or -] 0.15
Total litter size      231                      201
Viable litter size     208                      196
Viable litter size
  on lactation day 5   205                      181
Neonatal survival on
  lactation day 5 (%)   98.5                     92.3
Mean neonatal weight
  on lactation day       2.85 [+ or -] 0.38       2.80 [+ or -] 0.50
  5 (g)

Parameter                 PAP concentration (%)
                          0.05                    0.10

No. of mice/group        20                     20
Pregnancy rate (%)       20/20 (100)            20/20 (100)
Mean litter size
  (median)               11.3 [+ or -] 2.9      11.5 [+ or -] 3.0 (11)
                         (11)
Mean neonatal
  weight (g)             1.72 [+ or -] 0.18      1.71 [+ or -] 0.18
Total litter size      226                     230
Viable litter size     216                     223
Viable litter size
  on lactation day 5   200                     201
Neonatal survival on
  lactation day 5 (%)   92.6                    90.1
Mean neonatal weight
  on lactation day       3.01 [+ or -] 0.52      2.95 [+ or -] 0.58
  5 (g)

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O. J. D'Cruz (1,2,5), B. Waurzyniak (1,3), and F. M. Uckun (1,4)

(1) Drug Discovery Program,

(2) Departments of Reproductive Biology,

(3) Experimental Pathology

(4) Virology Parker Hughes Institute, St. Paul, Minnesota, U.S.A.

(5) Paradigm Pharmaceuticals, LLC, St. Paul, Minnesota, U.S.A.

Address

Osmond J. D'Cruz, Ph.D, Parker Hughes Institute, 2657 Patton Road, St. Paul, MN 55113, USA

Tel.: [phone]; Fax: [phone]; e-mail: [e-mail]