Preclinical Toxicological evaluation of the HIV entry inhibitor peptide 2P23 as a candidate rectovaginal microbicide

Zheng-qin Gao; Rui Fu

doi:10.1038/s41598-026-55638-y

. 2026 May 31;16:24313. doi: 10.1038/s41598-026-55638-y

Preclinical Toxicological evaluation of the HIV entry inhibitor peptide 2P23 as a candidate rectovaginal microbicide

Zheng-qin Gao ^1,^✉, Rui Fu ^1,^✉

PMCID: PMC13448178 PMID: 42225864

Abstract

Pre-exposure prophylaxis of HIV infection with a microbicide is one of the most effective ways to block HIV/AIDS transmission. In this study, a potent and long-acting HIV entry inhibitor peptide 2P23 was developed as a novel HIV microbicide, and its toxicity and safety were evaluated through animal model in vivo for the first time, which provided a new effective means for the prevention of HIV/AIDS. In order to evaluate the potential effect to cause human toxicity and possible health hazards likely to arise from repeated rectovaginal application of 2P23, we conducted an acute toxicity in which a dose of 300 mg/kg of 2P23 was administrated by rectovaginal three times within 24-hour in male and female Sprague Dawley rats. Furthermore, a 90-day repeated rectovaginal treatment tests in rats were used to evaluate the chronic and reproductive toxicity of 50, 100, 200 mg/kg of 2P23. The effects of 2P23 administration on morbidity, mortality, clinical signs, changes in body weight, food consumption, clinical pathology, organ weight, hematological and physiological biochemical parameter indicators, histopathology, changes in rectovaginal microbiota, fertility, and early embryonic development were assessed. There were no mortality or abnormal clinical signs, no significant changes in body weight gain or hematological and physiological biochemical parameter indicators, no changes in necropsy and histopathology findings of vital organs, and no significant changes in rectum and vagina microbiota associated with the administration of 2P23. There were no effects on fertility and early embryonic development in male and female rats. The results indicate that 2P23 does not cause significant adverse effects in the 90-day and suggest that the no observed adverse effect level is 300 mg. This study has important application value and good innovation for the development of anti-HIV agents for rectovaginal administration. The novel HIV microbicide is safe and effective for the prevention and control of HIV/AIDS sexually transmitted infections, which enriches people’s understanding of the novel HIV microbicide gel.

Keywords: Peptide, Rectovaginal administration, HIV entry inhibitor, Microbicide

Subject terms: Diseases, Drug discovery, Medical research, Microbiology

Introduction

In the early 1980s, a new and deadly disease emerged — Acquired immune deficiency Syndrome (also known as AIDS), which was caused by a retrovirus called Human immunodeficiency virus (HIV)¹. At present, approximately 40 million people worldwide are infected with HIV². However, this also indicates that there is still a significant gap in AIDS prevention efforts. The number of new infections in 2024 reached 1.3 million - almost no change compared to the previous year³. HIV is mainly divided into two types: HIV-1 and HIV-2. Among them, HIV-1 has a higher transmissibility and infection rate, and is the main cause of most HIV infections^4,5. Antiretroviral therapy (ART), condom, male circumcision, HIV microbicide, vaginal ring, AIDS counseling and other strategies have helped transform HIV infection from a fatal disease into a manageable chronic infectious disease^6–11. Currently, for People living with HIV (PLWH), the recommended treatment regimen is to adopt Highly active antiretroviral therapy (HAART)¹². When using HAART, an important issue is the possible adverse reactions caused by the drugs¹³. Tenofovir disoproxil fumarate (TDF) is an important drug, drawing much attention due to its serious adverse reaction - kidney function impairment^14,15. Sookaromdee et al. reported that in Thailand, there were cases where some AIDS patients receiving HAART developed acute kidney failure and ultimately died due to the use of TDF¹⁶. Quisada et al. reported that in patients with AIDS who received TDF treatment in Ghana, cases of mild to moderate kidney problems were more common¹⁷. Sarfo et al. reported that kidney problems were more common among AIDS patients receiving TDF treatment. Subsequently, he pointed out that this problem was related to the mortality risk of AIDS patients¹⁸. Brennan et al. reported that within a few years after starting to use TDF, the occurrence of kidney problems was more common¹⁹.

The widely available antiretroviral therapy has enabled many children who contacted HIV during the perinatal period to reach adolescence and adulthood. They began engaging in sexual activity and started making decisions regarding their reproductive health. Most men and women infected with HIV also desire to have children. Therefore, it is of great significance to study the long-term effects of the drugs on the reproductive systems and fertility of young patients. 2P23 is a novel HIV Entry inhibitor (EI) peptide, which can effectively inhibit the replication of HIV²⁰. Our previous studies evaluated anti-HIV activity and safety of 2P23 ⁹. 2P23 is an excellent candidate as a microbicide for Pre-exposure prophylaxis (PrEP) of HIV. HIV PrEP is a method for preventing HIV infection. It can effectively reduce the risk of HIV infection among high-risk groups. Its use has a relatively high degree of autonomy. Nonetheless, as of this writing there is a dearth of published information, including toxicological data, relating to 2P23. Thus, a study of 2P23’s potential toxic effects and determination of a safe dose are urgently needed. Rats are often used as animal models for experiments to assess the potential effects of drugs on the reproductive system and to detect the direct toxic effects of drugs on tissues^21–23. Globally, the majority of people are exposed to HIV via vaginal-penile sex (Heterosexual transmission) or rectum-penile sex (Homosexual transmission). Therefore, this study adopted the transmission route of rectal-vaginal contact. Thus, the present study was conducted using the rectovaginal exposure route^24–27. In order to assess the potential toxicity of 2P23 to Human toxicity (HT) and possible health hazards resulting from repeated rectovaginal application, we conducted this study to evaluate the single-dose acute toxicity of 2P23 in male and female Sprague-Dawley (SD) rats. Additionally, a 90-day repeated rectovaginal application was conducted on male and female SD rats to examine the chronic and reproductive toxicity of 2P23. This study was carried out in male and female SD rats, with males reaching sexual maturity at 6 weeks, as they develop rapidly during infancy. The growth activities of male SD rats are most active in the first 8 months after birth, so the experiment was conducted from 6 weeks to 18 weeks.

Materials and methods

HIV microbicide and animals

2P23 is a new type of short peptide, featuring an M-T bond-hook structure, HIV-2 sequence, and salt bridge-forming residues. This is a highly stable helical peptide that can strongly bind to surrogate targets derived from HIV-1, HIV-2 and Simian immunodeficiency virus (SIV). 2P23 can effectively inhibit HIV-1 and HIV-2 ^9,20. At SciLight Peptide Biological Technology Co., Ltd. (SciLight Peptide; Beijing, China), the peptide was synthesized on the rink amide 4-methylbenzhydrylamine (amide 4-methylbenzhydrylamine, MBHA) resin using standard solid-phase 9-fluororenyl methoxycarbonyl (FMOC) chemistry. It was analyzed by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine that the purity of the 2P23 peptide was greater than 95%. The concentration of 2P23 peptide was determined by ultraviolet absorption method, and theoretical calculations were conducted based on the molar extinction coefficients of tryptophan and tyrosine residues. The powder form of 2P23 peptide is provided by Scilight Peptide (Beijing, China). The phosphate-buffered saline (PBS) solution is used for preparing the 2P23 peptide solution. The 2P23 gel is prepared by mixing different concentrations of 2P23 peptide solutions with a mixture containing gel (hydroxyethyl cellulose, HEC), glycerol and preservatives (methyl 4-hydroxybenzoate), and then filtering through a 0.45-micron microporous membrane. The formulation of the vehicle control gel is the same, but it does not contain the active ingredient (2P23 peptide).

To avoid the influence of the environment on the experiment, the experiments are conducted in specific pathogen-free (SPF) level laboratories without specific pathogens. Male and female SD SPF rats (age 6 weeks) used in our study were supplied by the National Center for Rodent Laboratory Animal Resources (Beijing, China). The animals were adapted to laboratory conditions for 6 days after they arrived at the laboratory animal facility of the Institute for Laboratory Animal Resources (ILAR) of National Institutes for Food and Drug Control (NIFDC, Beijing, China). The animals were housed in individually ventilated cage (IVC) rack systems with a maximum capacity of one animal per cage at a temperature of 22 ± 3℃ and relative humidity (RH) of 50 ± 15% and fed standard food and water ad libitum. The animal rooms were maintained under a 12 h light-dark cycle and 10–20 air exchanges per hour²⁸. When the experiments on live animals are completed (i.e. at the end of the experiment), these animals will be euthanized in accordance with the 2020 version of the AVMA Guidelines for Euthanasia of Animals)²⁹. For rodents weighing 200 g to 1000 g, the exposure time to CO₂ was 5 min. The method involved inhaling carbon dioxide from a high-pressure oxygen cylinder. The INTTLLIGENT ANIMAL ENTHANASIASYSTEM for laboratory animals from Beijing Mingkang Purification Technology Co., Ltd. was used. The optimal rate of carbon dioxide filling was 30% to 70% of the volume of the euthanasia container per minute.

Acute toxicity test

In the acute toxicity test, 20 SPF male and female SD rats were randomly divided into two groups: vehicle control gel group and 300 mg/kg 2P23 gel group (Table 1). The experimental group received three rectovaginal treatments with a 300 milligram per kilogram body weight dose of 2P23 gel over a 24-hour period, while the vehicle control group received treatment with gel. Rectal lavage (RL) and cervical-vaginal lavage (CVL) were collected before treatment (baseline state) and 24 h after treatment by irrigating the rectal walls and cervix and the lateral vaginal walls with PBS. The animals were continuously observed for 14 days. At least two observations were conducted on the day of administration, and then one observation was carried out every day thereafter. Body weight and food intake were recorded. At the end of the experiment, the animals were euthanized and their organs were extracted for weighing. If any abnormal changes are observed in the organs with the naked eye, further pathological histological examination should be conducted^30–37.

Table 1.

Animal characteristics in the 2P23 gel acute toxicity, chronic toxicity and reproductive toxicity studies.

Groups	ID	Gender	Initial body weight	ID	Gender	Initial body weight
Acute toxicity study
Gel	1 M	Male	108.7 g	1 F	Female	98.0 g
Gel	2 M	Male	113.7 g	2 F	Female	92.5 g
Gel	3 M	Male	102.7 g	3 F	Female	105.8 g
Gel	4 M	Male	108.2 g	4 F	Female	108.4 g
Gel	5 M	Male	104.4 g	5 F	Female	104.9 g
300 mg/kg 2P23 gel	6 M	Male	110.0 g	6 F	Female	99.0 g
300 mg/kg 2P23 gel	7 M	Male	108.5 g	7 F	Female	108.4 g
300 mg/kg 2P23 gel	8 M	Male	102.3 g	8 F	Female	104.2 g
300 mg/kg 2P23 gel	9 M	Male	112.4 g	9 F	Female	102.6 g
300 mg/kg 2P23 gel	10 M	Male	104.6 g	10 F	Female	110.7 g
Chronic toxicity study
Gel	11 M	Male	107.7 g	11 F	Female	98.4 g
Gel	12 M	Male	100.6 g	12 F	Female	97.8 g
Gel	13 M	Male	116.7 g	13 F	Female	101.6 g
Gel	14 M	Male	108.3 g	14 F	Female	103.1 g
Gel	15 M	Male	111.4 g	15 F	Female	98.9 g
Gel	16 M	Male	105.8 g	16 F	Female	99.6 g
Gel	17 M	Male	102.5 g	17 F	Female	109.7 g
Gel	18 M	Male	100.0 g	18 F	Female	111.6 g
50 mg/kg 2P23 gel	19 M	Male	106.2 g	19 F	Female	112.0 g
50 mg/kg 2P23 gel	20 M	Male	110.3 g	20 F	Female	96.4 g
50 mg/kg 2P23 gel	21 M	Male	106.6 g	21 F	Female	103.2 g
50 mg/kg 2P23 gel	22 M	Male	98.7 g	22 F	Female	99.2 g
50 mg/kg 2P23 gel	23 M	Male	99.1 g	23 F	Female	100.9 g
50 mg/kg 2P23 gel	24 M	Male	109.3 g	24 F	Female	102.1 g
50 mg/kg 2P23 gel	25 M	Male	103.0 g	25 F	Female	100.7 g
50 mg/kg 2P23 gel	26 M	Male	104.7 g	26 F	Female	108.6 g
100 mg/kg 2P23 gel	27 M	Male	105.4 g	27 F	Female	101.7 g
100 mg/kg 2P23 gel	28 M	Male	119.0 g	28 F	Female	104.2 g
100 mg/kg 2P23 gel	29 M	Male	106.8 g	29 F	Female	101.6 g
100 mg/kg 2P23 gel	30 M	Male	111.4 g	30 F	Female	107.3 g
100 mg/kg 2P23 gel	31 M	Male	94.8 g	31 F	Female	102.7 g
100 mg/kg 2P23 gel	32 M	Male	112.0 g	32 F	Female	105.3 g
100 mg/kg 2P23 gel	33 M	Male	106.8 g	33 F	Female	102.1 g
100 mg/kg 2P23 gel	34 M	Male	102.5 g	34 F	Female	104.7 g
200 mg/kg 2P23 gel	35 M	Male	111.3 g	35 F	Female	113.1 g
200 mg/kg 2P23 gel	36 M	Male	97.0 g	36 F	Female	110.4 g
200 mg/kg 2P23 gel	37 M	Male	105.0 g	37 F	Female	102.3 g
200 mg/kg 2P23 gel	38 M	Male	110.2 g	38 F	Female	102.6 g
200 mg/kg 2P23 gel	39 M	Male	100.0 g	39 F	Female	106.3 g
200 mg/kg 2P23 gel	40 M	Male	111.0 g	40 F	Female	111.8 g
200 mg/kg 2P23 gel	41 M	Male	109.9 g	41 F	Female	98.2 g
200 mg/kg 2P23 gel	42 M	Male	107.6 g	42 F	Female	106.2 g
Reproductive toxicity study
Gel	43 M	Male	112.8 g	43 F	Female	111.7 g
Gel	44 M	Male	98.2 g	44 F	Female	107.7 g
50 mg/kg 2P23 gel	45 M	Male	106.1 g	45 F	Female	105.5 g
50 mg/kg 2P23 gel	46 M	Male	100.4 g	46 F	Female	103.6 g
100 mg/kg 2P23 gel	47 M	Male	100.4 g	47 F	Female	104.1 g
100 mg/kg 2P23 gel	48 M	Male	107.0 g	48 F	Female	97.0 g
200 mg/kg 2P23 gel	49 M	Male	109.7 g	49 F	Female	108.8 g
200 mg/kg 2P23 gel	50 M	Male	108.1 g	50 F	Female	101.5 g

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Chronic toxicity test

In the chronic toxicity test, 64 SPF male and female SD rats were randomly divided into four groups: vehicle control gel group, 50 mg/kg 2P23 gel group, 100 mg/kg 2P23 gel group, 200 mg/kg 2P23 gel group (Table 1). 2P23 gel (50, 100, 200 mg/kg body weight) or a vehicle control gel were rectovaginal administrated once daily at 8:00 to 9:00 every morning for 90 days. Before the start of the treatment (baseline) and 24 h after the treatment, samples of RL and CVL were collected continuously for 90 days. The observed indicators include general indicators (such as the appearance, behavior, response to stimuli, secretions, excretions, etc. of the animals), the condition of animal deaths (time of death, reactions before death, etc.), changes in animal body weight (weighing the animals once before treatment and once before the end of the experiment when the animals were sacrificed, and weighing the animals multiple times during the observation period), food intake, etc. Record all the death situations, symptoms, onset time, severity, duration, etc. At the end of the experiment, euthanize the animals and extract their organs for weighing. Changes in the volume, color, texture, etc. of any tissue or organ were all recorded. The vital organs were fixed, dehydrated, embedded, sectioned, dewaxed, stained with hematoxylin and eosin (H&E), and observed under an optical microscope^38–44.

Reproductive toxicity testtive toxicity test

In the reproductive toxicity test, 16 SPF male and female SD rats were randomly divided into four groups: vehicle control gel group, 50 mg/kg 2P23 gel group, 100 mg/kg 2P23 gel group, and 200 mg/kg 2P23 gel group (Table 1). 2P23 gel (50, 100, 200 mg/kg body weight) or a vehicle control gel were rectovaginal administrated once daily at 8:00 to 9:00 every morning for 77 days. After 77 consecutive days of rectovaginal treatment, a female rat that was confirmed to be fertile and in estrus was paired with another male rat from the same group. The 0th day of gestation (GD0) was determined by examination the vaginal plug (a white plug occupying most of the cervical junction) and the presence of sperm in the vaginal smear the female is observed with a light microscope. The timing of pregnancy is as follows: Even if the mating occurred the previous night, the date when vaginal smear shows positive sperm and/or the cervical plug is present is regarded as the 0th day of pregnancy. The analysis of semen quality and embryo counting were conducted using standard methods^45–54. The vital signs and death situations were monitored twice a day. RL and CVL samples were collected. Body weight and food intake were recorded. Anticoagulated blood and serum of the animals were collected, and hematology and physiological biochemical indicators were determined using an automatic blood and chemical analyzer. At the end of the experiment, the animals were euthanized and their organs were weighed. Organs that show abnormal manifestations under the naked eye need to undergo further histological examination^55–66.

Rectal and vaginal microbiota analyses

In the acute toxicity test, RL and CVL samples were randomly chosen as follows: (1) AT0 (baseline): AT0M, AT0F; (2) AT1 (after 24 h administration): vehicle control gel group: AT1MG, AT1FG; 2P23 gel group: AT1MP, AT1FP; (3) AT2 (after 48 h administration): vehicle control gel group: AT2MG, AT2FG; 2P23 gel group: AT2MP, AT2FP; (4) AT3 (after 72 h administration): vehicle control gel group: AT3MG, AT3FG; 2P23 gel group: AT3MP, AT3FP; (5) AT4 (after 96 h administration): vehicle control gel group: AT3MG, AT3FG; 2P23 gel group: AT3MP, AT3FP. In the chronic toxicity test, the RL and CVL samples were randomly chosen as follows: (1) CT0 (baseline): CT0M, CT0F; (2) CT1 (after 30 days administration): vehicle gel group: CT1MG, CT1FG; 200 mg/kg 2P23 gel group: CT1MPH, CT1FPH; 100 mg/kg 2P23 gel group: CT1MPM, CT1FPM; 50 mg/kg 2P23 gel group: CT1MPL, CT1FPL; (3) CT2 (after 60 days administration): vehicle gel group: CT2MG, CT2FG; 200 mg/kg 2P23 gel group: CT2MPH, CT2FPH; 100 mg/kg 2P23 gel group: CT2MPM, CT2FPM; 50 mg/kg 2P23 gel group: CT2MPL, CT2FPL; (4) CT3 (after 90 days administration): vehicle gel group: CT3MG, CT3FG; 200 mg/kg 2P23 gel group: CT3MPH, CT3FPH; 100 mg/kg 2P23 gel group: CT3MPM, CT3FPM; 50 mg/kg 2P23 gel group: CT3MPL, CT3FPL. In the reproductive toxicity test, RL and CVL samples were randomly chosen as follows: ST (after 90 days administration): 200 mg/kg 2P23 gel group: STMPH, STFPH; 100 mg/kg 2P23 gel group: STMPM, STFPM.

The total microbiota community genomic DNAs of RL and CVL were extracted using the E.Z. N. A Soil DNA Kit (Omega, USA). The concentrations of DNA were determined using Qubit 2.0 (Life, USA). V3-V4 regions of microbiota 16 S rRNA gene were amplified using Kapa Hifi Hot Start Ready Mix (2×) (Takara Bio Inc., Japan) by PCR (forward primer 341 F: 5’-CCTACGGGNGGCWGCAC-3’; reverse primer 805R: 5’-GACTACHVGGGTATCTAATCC-3’)⁶⁷. The free primers and primer dimer species in the amplified products were purified using Am Pure XP beads. Samples were sent to Shanghai Sangon Biotechnology Co., Ltd., China, for library construction using Universal Illumina adapters and indexes. Depending on coverage requirements, all the libraries can be pooled for one run. The amplified products in each reaction mixture were aggregated in equal molar ratios according to their concentration. Sequencing was performed using the Illumina MiSeq system (Illumina, San Diego, CA, USA). After sequencing, data were collected as follows: (1) PEAR (V0.9.6) software was used to assemble 2 short Illumina readings based on the overlap and process FASTQ files to generate separate FASTA and QUAL files for analysis using standard methods; (2) Sequences with ambiguous bases and lengths greater than 480 base pairs (bp) were removed, and the maximum allowable homopolymer length was 6 bp⁶⁸. Sequences shorter than 200 bp were removed; (3) All the same sequences were combined into one; (4) Sequences were aligned according to the customized reference database; (5) Integrity of indexes and adapters were checked, and all indexes and adapter sequences were deleted; (6) Noise was removed using PRE Cluster tools. Chimera UCHIME was used to detect Chimera. All software was in the mothur package. We resubmitted the valid sequences of each sample to the RDP classifier to identify archaea and bacterial sequences. Species richness and diversity statistics, including coverage, Chao1, Ace, Simpson and Shannon-Ever, were calculated using mothur. The modified pipeline is described on the mothur website. Finally, all effective microbiota sequences without primers were submitted for data analysis⁶⁹. Operational taxonomic units (OTUs) were established de novo using UCLUST, and 97% sequence homology was truncated⁷⁰. OTUs for regions V3–V4 were specified by the Ribosomal Database Project (RDP) Naive Bayes classifier^71,72. Sequence was compared to the Greengenes core set using the Python nearest alignment space termination (PyNAST) aligner⁷³. Phylogenetic trees were generated using Fast Tree, and dilution curves were drawn to calculate alpha and beta diversity of the samples performed by QIIME⁷⁴. Similarities between microbial communities were identified using principal coordinate analysis (PCoA), which relies on unweighted and weighted Uni Frac. UCHIM software was used to detect and remove chimeric sequences based on the “RDP GOLD” database. OTU cluster analysis was performed according to the Galaxy online platform process. The BIOM file obtained by the QIIME software was uploaded to the Galaxy website for predictive analysis of the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICURSt) functional genes. The information could be obtained by referring to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology class 1 and class 2 functional gene classes to obtain the functional composition of the predicted genome⁷⁵.

Sperm characteristics analysis

The histopathological examination of reproductive organs is the most sensitive method for evaluating the effects on sperm production. When histopathological examination is difficult to carry out, and when further evidence and in-depth study of the functional characteristics are needed, sperm analysis can provide similar information to the above-mentioned histopathological examination. Sperm analysis can be regarded as a valuable experimental method. The mating experiment is a sensitive method for examining the effects of the test substance on sperm maturity, sperm vitality, and behavioral changes (i.e., sexual desire). During sperm analysis experiment (including sperm count, sperm motility, and sperm morphology), the samples for testing were taken from the epididymis. The results of the sperm analysis serve as an evaluation of fertility. After dissection, the sperm was collected immediately and was suspended in PBS. For the sperm count, motility and morphology analysis, a total of 10 µL of sperm suspension was placed on a slide. The sperm count was expressed as 1 million sperm cells per milliliter of suspension. The sperm motility was expressed in percentage of forward motile sperm while sperm count was expressed as million sperm cells per mL of suspension. Meanwhile, for sperm viability assessment, a thick smear was done using 10 µL of sperm suspension and adding 10 µL of Eosin stain on the slides. The dead sperm will take up the eosin stain and appear pinkish while normal live sperm will not take up the eosin stain and appear white in color. Count at least 200 sperm with an optical microscope and assess sperm viability by using the total number of sperm detected alive. In order to assist the sperm morphology observation, a thin smear of sperm suspension was done for Giemsa staining and the percentage of abnormal sperm morphology was calculated. The morphological abnormalities of 200 sperms were examined per slide. The data are obtainable as a percentage of abnormal sperm morphology. The sperm characteristics analysis was performed in triplicate per rat in accordance with the guidelines by World Health Organization (WHO) while guidelines by Industrial Reproductive Toxicology Discussion Group were used to analyze the rat sperm abnormal morphology^76–80.

Fertility and early embryonic development study

Pregnancy termination in pregnant female rat at 13–15 days of gestation was sufficient to evaluate the impact on fertility or reproductive function. Luteal number and implantation number of pregnant uterus were recorded, and the survival and death of embryos were observed^81–86.

Statistical analyses

All values were presented as the mean ± SEM (standard error of mean). The data were analyzed using one-way analysis of variance (ANOVA). Statistical analyses were performed using GraphPad Prism software version 7 (GraphPad Software Inc., San Diego, USA). Analysis method of the Advantage Species Heatmap graph: A heatmap can use color variations to reflect the data information in a two-dimensional matrix or table. It can visually represent the magnitude of data values by using the defined shades of color. Data can be clustered according to needs based on species, function or sample similarities. The clustered data can then be represented on a heatmap. Species or functions with high abundance and low abundance can be grouped together. Through color gradients and similarity levels, the similarities and differences in community composition or functions among multiple samples at various classification levels can be reflected. Software name: R. Version number: 3.6.0. URL link: https://www.r-projet.org.

The analysis method of relative abundance plots: By using statistical analysis methods, the community structure of the samples at different classification levels can be observed. When comparing the community structure analyses of multiple samples together, the changes can also be observed. Depending on whether the research subjects are a single sample or multiple samples, the results may be presented in different ways. Typically, more intuitive forms such as pie charts or bar charts are used for presentation. Unless otherwise specified, by default, species with an abundance proportion less than a certain threshold (1%) in all samples will be classified as “others”, while the remaining species will be analyzed as dominant species. Software name: R (The R Project for Statistical Computing). Version number: 3.6.0. URL link: https://www.r-projet.org.

Analyzing the statistical data of the research is a means to clarify its results. The main content is to determine the relationship between each variable and its distribution (descriptive statistics), as this determines how to conduct inter-group comparisons. Reproductive toxicity observation indicators usually show a non-normal distribution, and the diversity of their distribution can manifest as various situations ranging from approximately continuous to extreme. The “significance” test (a deductive statistical method) is only used to assist in interpreting the results. The interpretation of the results must be based on biological rationality. The conclusion that the difference from the control group results has no biological significance merely because it lacks “statistical significance” is rash. To some extent, it is also unwise to assert that a “statistically significant” difference must have a biological significance. Especially for those events with skewed distributions and low incidence rates (such as embryo death), the weight of statistics in the research is very low.

Results

Acute toxicity test

After a continuous 14-day observation, all the animals showed normal conditions, no acute toxicity symptoms were observed, and no deaths occurred. The appearance and behavior were normal. Compared with the vehicle control gel group, the 2P23 gel group showed no significant changes in body weight (Fig. 1) and food intake (Fig. 2). The absolute organ weights (Fig. 3) and relative organ weights (Fig. 4) of the vehicle control gel group showed no significant differences compared with the 2P23 gel group. Figure 5 shows that the microbial communities of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria remained stable in the rectum and vagina of these male and female rats at AT0 (baseline), AT1 (after 24 h administration), AT2 (after 48 h administration), AT3 (after 72 h administration) and AT4 (after 96 h administration) during the acute toxicity test. According to the toxicological standards and the experimental results, the 2P23 gel at a concentration of 300 mg/kg can reasonably be regarded as non-toxic.

Fig. 1 — Body weight changes in the acute toxicity test.

Fig. 2 — Food consumption changes in the acute toxicity test.

Fig. 3 — Absolute organ weight changes in the acute toxicity test.

Fig. 4 — Relative organ weight changes in the acute toxicity test.

Fig. 5 — Microbiota changes in the acute toxicity test. **(a)** Relative Abundance Bar Chart. The horizontal axis represents the sample numbers, while the vertical axis shows the relative abundance ratio of the species. The colors correspond to the names of each species at this taxonomic level. The widths of different color blocks represent the relative abundance ratios of different species. **(b)** Abundance Heatmap. Each row represents a species, each column represents a sample, and each small square’s color represents the relative abundance of the species in the sample. The darker the color (red), the higher the abundance of the species; the bluer the color, the lower the abundance of the species. Above the graph, there are color blocks, and the samples from the same group have the same color. Software name: R. Version number: 3.6.0. URL link: https://www.r-projet.org.

Chronic toxicity test

During the 90-day trial period, neither the 2P23 gel group nor the vehicle control gel group of animals exhibited any abnormal behaviors. Tables 2 and 3 present the hematological and physiological biochemical parameter indicators of the four groups of animals after 90 days of treatment. Obviously, there were no significant differences in the hematological and physiological biochemical parameters among these groups. Figures 6 and 7 show that, in the chronic toxicity test, compared with the vehicle control gel group, there were no significant changes in the body weight and food intake of these animals. The organ weight can serve as a meaningful indicator of treatment-related changes in repeated toxicity studies (regardless of whether corresponding histopathological examinations were conducted)^87,88. Significant changes in organ weight are common indicators of organ alterations caused by chemical factors^89,90. Figure 8 shows that there was no significant difference in organ weight between the 2P23 gel groups and the vehicle control gel groups. We monitored the changes in organ weight and histopathological changes of the animals treated with 2P23 gel. During the experiment, no treatment-related deaths, dead animals, or clinical symptoms were observed in the group treated with 2P23 gel. At the end of the study, histopathological examinations were conducted on all organs. Microscopic observation showed that no significant pathological changes were observed in the organs under study of the female and male rats treated with 2P23 gel after the chronical toxicity test. As shown in Fig. 9, no abnormal pathological changes were observed in the brains, hearts, lungs, livers, spleens, kidneys, intestines, testicles and ovaries of all the animals. Figure 10 showed that the microbial communities of Bacteroidetes, Proteobacteria, Bacteroidia, Gemmatiales, and Actinobacteria remained stable in the rectum and vagina of male and female rats at CT0 (baseline), CT1 (30 days after administration), CT2 (60 days after administration), and CT3 (90 days after administration) during the chronic toxicity test. These results indicates that the 2P23 gel will not disrupt the ecological balance of the vaginal and rectal microbiota. 2P23 gel is safe and non-toxic. It is suitable for HIV PrEP.

Table 2.

Hematological parameter indicators in the chronic toxicity test.

Item	Group	Gel	50 mg/kg 2P23 gel	100 mg/kg 2P23 gel	200 mg/kg 2P23 gel
WBC (×10⁹/L)	Male	9.8 ± 1.0	10.4 ± 1.1	9.9 ± 2.0	8.0 ± 1.1
WBC (×10⁹/L)	Female	4.9 ± 1.1	5.7 ± 1.1	5.1 ± 1.7	6.0 ± 2.1
RBC (×10¹²/L)	Male	5.5 ± 0.3	5.5 ± 0.4	5.8 ± 0.5	5.4 ± 0.4
RBC (×10¹²/L)	Female	5.0 ± 0.6	5.1 ± 0.4	4.8 ± 0.3	4.9 ± 0.3
HGB (×g/L)	Male	112 ± 4	110 ± 10	115 ± 6	112 ± 10
HGB (×g/L)	Female	104 ± 16	109 ± 10	105 ± 10	106 ± 6
PLT (×10⁹/L)	Male	411 ± 67	469 ± 63	458 ± 59	479 ± 17
PLT (×10⁹/L)	Female	451 ± 100	422 ± 45	403 ± 66	467 ± 57
NEUT%	Male	12.3 ± 6.9	7.2 ± 1.6	5.9 ± 3.0	6.5 ± 2.5
NEUT%	Female	9.2 ± 5.3	7.1 ± 2.9	5.5 ± 2.7	5.4 ± 0.7
LYMPH%	Male	83.3 ± 7.3	87.9 ± 2.6	89.7 ± 3.6	88.9 ± 3.4
LYMPH%	Female	86.6 ± 4	88.6 ± 2.1	90.7 ± 3	89.1 ± 0.3
MONO%	Male	0.8 ± 0.3	1.2 ± 0.4	0.6 ± 0.3	0.54 ± 0.3
MONO%	Female	0.5 ± 0.1	0.4 ± 0.2	0.5 ± 0.4	0.7 ± 0.6
EOS%	Male	3.4 ± 0.7	3.5 ± 0.9	3.6 ± 1.2	3.9 ± 1.0
EOS%	Female	3.4 ± 1.8	3.6 ± 0.8	3.1 ± 0.8	4.6 ± 1.6
BASO%	Male	0.08 ± 0.08	0.04 ± 0.05	0.02 ± 0.04	0.08 ± 0.04
BASO%	Female	0.08 ± 0.04	0.08 ± 0.08	0.08 ± 0.1	0.1 ± 0.07
HCT (%)	Male	30.9 ± 1.6	30.7 ± 2.9	32.3 ± 2	30.3 ± 3
HCT (%)	Female	28.9 ± 3.6	30.0 ± 2.9	28.8 ± 2.1	29.3 ± 1.9
MCV (fL)	Male	55.4 ± 1.6	55.2 ± 1.7	55.4 ± 1.4	55.1 ± 1.4
MCV (fL)	Female	57.8 ± 1.1	58.8 ± 1.1	59.0 ± 1.8	59.0 ± 2.0
MCH (pg)	Male	20.1 ± 0.7	19.8 ± 0.4	19.7 ± 0.8	20.4 ± 0.8
MCH (pg)	Female	20.7 ± 0.8	21.4 ± 0.3	21.6 ± 0.8	21.4 ± 1.1
MCHC (g/L)	Male	363 ± 8	360 ± 4	355 ± 10	370 ± 10
MCHC (g/L)	Female	358 ± 14	365 ± 7	366 ± 10	362 ± 10
RDW (%)	Male	13.1 ± 0.4	13.5 ± 0.8	13.2 ± 0.4	13.3 ± 0.2
RDW (%)	Female	12.8 ± 0.6	12.7 ± 0.2	13.3 ± 0.5	13.3 ± 0.2
PCT (%)	Male	0.1 ± 0.02	0.1 ± 0.02	0.1 ± 0.02	0.2 ± 0.02
PCT (%)	Female	0.1 ± 0.04	0.1 ± 0.01	0.1 ± 0.02	0.2 ± 0.03
MPV (fL)	Male	3.7 ± 0.1	3.8 ± 0.07	4.0 ± 0.1	4.3 ± 0.4
MPV (fL)	Female	3.7 ± 0.1	3.7 ± 0.08	4.3 ± 0.1	4.4 ± 0.2
PDW (%)	Male	14.9 ± 0.2	15.0 ± 0.3	15.6 ± 0.3	16.2 ± 0.8
PDW (%)	Female	15.0 ± 0.3	15.2 ± 0.4	16.6 ± 0.5	16.4 ± 0.4

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RBC: total erythrocyte count (red blood cells). RDW: red cell distribution width. HGB: hemoglobin concentration. HCT: hematocrit. MCV: mean corpuscular volume. MCH: mean corpuscular hemoglobin. MCHC: mean corpuscular hemoglobin concentration. PLT: platele count. MPV: mean platelet volume. PCT: plateletocrit. PDW: platelet distribution width. WBC: total leucocyte count (white blood cells). NEU: neutrophils absolute value. LYMPH: lymphocyte absolute value. MONO: monocyte absolute value. EOS: eosinophil absolute value. BASO: basophil absolute value. ALT: alanine aminotransferase.

Table 3.

Physiological biochemical parameter indicators in the chronic toxicity test.

Item	Group	Gel	50 mg/kg 2P23 gel	100 mg/kg 2P23 gel	200 mg/kg 2P23 gel
ALT (U/L)	Male	50 ± 8	42 ± 6	41 ± 4	43 ± 7
ALT (U/L)	Female	44 ± 16	43 ± 1	44 ± 3	51 ± 12
AST (U/L)	Male	161 ± 17	93 ± 24	135 ± 32	108 ± 27
AST (U/L)	Female	172 ± 91	121 ± 33	127 ± 15	125 ± 36
TP (kat/L)	Male	65.9 ± 4.7	66.6 ± 2.2	67.6 ± 2	67.3 ± 3.1
TP (kat/L)	Female	75.8 ± 7.3	72.6 ± 4.1	71.1 ± 3.9	76.7 ± 3.0
ALB (kat/L)	Male	27.5 ± 1.5	27.1 ± 0.8	27.5 ± 0.6	27.7 ± 0.8
ALB (kat/L)	Female	35.7 ± 3.2	32.9 ± 1.8	34 ± 2.0	35.4 ± 2.1
ALP (U/L)	Male	187 ± 32	130 ± 15	148 ± 21	179 ± 29
ALP (U/L)	Female	68 ± 15	83 ± 20	93 ± 15	115 ± 62
BUN (mmol/L)	Male	6.2 ± 0.8	5.9 ± 0.3	6.7 ± 0.5	7.2 ± 1.2
BUN (mmol/L)	Female	6.8 ± 1.3	7.9 ± 1.7	7.9 ± 1.3	6.9 ± 0.8
SCr (µmoI/L)	Male	26.9 ± 4.4	26.8 ± 3.2	26.3 ± 1.9	34.7 ± 13.7
SCr (µmoI/L)	Female	24.2 ± 3.5	36.3 ± 22.5	34.8 ± 7.1	26.9 ± 3.9
GLU (mmol/L)	Male	6.1 ± 0.5	7.3 ± 0.8	6.2 ± 0.7	7.7 ± 0.7
GLU (mmol/L)	Female	6.4 ± 0.5	6.9 ± 1.1	6.2 ± 0.3	7.1 ± 0.6
CA (mmol/L)	Male	2.5 ± 0.08	2.5 ± 0.07	2.5 ± 0.05	2.5 ± 0.1
CA (mmol/L)	Female	2.6 ± 0.1	2.6 ± 0.09	2.5 ± 0.1	2.6 ± 0.1
P (mmol/L)	Male	2.5 ± 0.1	2.5 ± 0.2	2.5 ± 0.1	2.3 ± 0.1
P (mmol/L)	Female	2.4 ± 0.5	2.1 ± 0.1	2.1 ± 0.3	2.0 ± 0.2
CHO (mmol/L)	Male	3.1 ± 0.7	3.1 ± 0.6	3.3 ± 0.2	3.4 ± 0.5
CHO (mmol/L)	Female	2.9 ± 0.6	3.2 ± 0.9	3.4 ± 0.4	3.3 ± 0.6

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AST: aspartate aminotransferase. TP: total protein. ALB: albumin. ALP: alkaline phosphatase. BUN: blood urea nitrogen. SCr: serum creatinine. GLU: blood glucose. CA: calcium. P: phosphorus. CHO: total cholesterol. TG: triglyceride.

Fig. 6 — Body weight changes in the chronic toxicity test.

Fig. 7 — Food consumption changes in the chronic toxicity test.

Fig. 8 — Absolute organ weight changes in the chronic toxicity test.

Fig. 9 — Histological examination of vital organs: **(a)** Brain, **(b)** Heart, **(c)** Lung, **(d)** Liver, **(e)** Spleen, **(f)** Kidney, **(g)** Intestine, **(h)** Testis and **(i)** Ovary.

Fig. 10 — Microbiota changes in the chronic toxicity test. **(a)** Relative Abundance Bar Chart. The horizontal axis represents the sample numbers, while the vertical axis shows the relative abundance ratio of the species. The colors correspond to the names of each species at this taxonomic level. The widths of different color blocks represent the relative abundance ratios of different species. **(b)** Abundance Heatmap. Each row represents a species, each column represents a sample, and each small square’s color represents the relative abundance of the species in the sample. The darker the color (red), the higher the abundance of the species; the bluer the color, the lower the abundance of the species. Above the graph, there are color blocks, and the samples from the same group have the same color. Software name: R. Version number: 3.6.0. URL link: https://www.r-projet.org.

Reproductive toxicity test

In the reproductive toxicity test, the number of SPF male and female SD rats in each group were sufficient for conducting meaningful data analysis. In present study, three dose groups (50 mg/kg 2P3 gel, 100 mg/kg 2P3 gel, 200 mg/kg 2P3 gel) and the vehicle control groups (gel) were set up. With this design, it was able to determine the “dose level without adverse effects” in the reproductive toxicity. The results of the present 2P23 gel repeated rectovaginal dose toxicity study demonstrated that administering 2P23 gel did not adversely affect most of the toxicological factors in male and female rats. Tables 4 and 5 show that there were no significant differences in hematological and physiological biochemical parameters between the gel group and the 2P23 gel group. Figures 11 and 12 show that there were no significant differences in body weight and organ weight between the gel group and the 2P23 gel group. At the scheduled autopsy, no obvious signs were observed in the rats. As shown in Fig. 13, histopathological analyses of the tissues of the brain, cerebellum, brainstem, spinal cord, pituitary gland, thyroid gland, salivary gland, pancreas, adrenal gland, larynx, trachea, lungs, heart, thymus, spleen, lymph nodes, esophagus, stomach, intestines, rectum, liver, kidneys, ureters, bladder, ovaries, fallopian tubes, uterus, vagina, testicles, epididymis, epidermis, dermis, cartilage, bones, and muscles showed that the structures of all groups were normal. The entire process of rat spermatogenesis (including sperm maturation) takes 63 days. Repeated dosing toxicity tests can provide information on the potential effects of the test substance on sperm production. The treatment with 2P23 did not significantly reduce the sperm count, motility and survival rate in the epididymis (Table 6; Fig. 14). On the 14th day of pregnancy, the rats were euthanized and underwent cesarean section. The ovaries from the bilateral uterine horns were removed, and the number of corpora lutea was counted. The pregnant uterus was carefully dissected, weighed, and examined. According to the previously described method, the total number of implantation sites in each pregnant uterus, as well as the number of live, resorbed, or dead fetuses, were recorded^91–98. Table 7 summarized the relevant exposure results of the 2P23 in the prenatal developmental toxicity study. During the entire experimental process, the number of offspring and the abortion situation of any four groups of mother rats were not recorded. Uteri obtained from pregnant dams via cesarean section operations were weighed and carefully examined for abnormalities. Those obtained from the four groups revealed a normal distribution of implanted fetuses between the two horns (Fig. 15). The uterus showed normal distribution of fetuses in the two horns in the gel group and 2P23 group. No obvious reduction in the recorded uterine weights of dams from the 2P23 groups, compared with the control group, was observed. No obvious changes in the total number of implantation sites were revealed in 2P23 groups. There was no obvious increase in the percentage post-implantation loss index in 2P23 group when compared to the control group. The sequencing results of 16 S rRNA genes indicated that in the rectum and vagina, the composition of the bacterial community (at the phylum level) of Firmicutes and Proteobacteria remained stable after 90 days administration 2P23 gel high dose group: STFPH and 2P23 gel middle dose group: STFPM (as shown in Fig. 16). The results demonstrate that 2P23 gel is safe and non-toxic. Therefore, 2P23 gel is suitable as a candidate microbicide for HIV PrEP.

Table 4.

Hematological parameter indicators in the reproductive toxicity test.

Item	Group	Gel	50 mg/kg 2P23 gel	100 mg/kg 2P23 gel	200 mg/kg 2P23 gel
WBC (×10⁹/L)	Male	13.4 ± 1.6	11.8 ± 1.3	11.4 ± 0.7	11 ± 0.2
WBC (×10⁹/L)	Female	9.5 ± 0.7	9.4 ± 0.8	7.9 ± 0.5	9.8 ± 1.4
RBC (×10¹²/L)	Male	7.1 ± 0.2	7.1 ± 0.07	7.0 ± 0.1	7.7 ± 0.3
RBC (×10¹²/L)	Female	6.0 ± 0.1	5.8 ± 0.06	6.3 ± 0.02	6.3 ± 0.2
HGB (×g/L)	Male	152.5 ± 4.9	151.5 ± 4.9	147.5 ± 3.5	155.5 ± 10.6
HGB (×g/L)	Female	143.5 ± 0.7	143 ± 3.5	143 ± 1.4	147 ± 2.8
PLT (×10⁹/L)	Male	496 ± 226	571 ± 67	593 ± 64	567 ± 5
PLT (×10⁹/L)	Female	524 ± 38	430 ± 31	462 ± 94	532 ± 65
NEUT%	Male	30.3 ± 12.4	28 ± 0.4	29 ± 0.2	30.6 ± 3.0
NEUT%	Female	28.8 ± 2.8	49.4 ± 2.5	16.6 ± 2.1	22.1 ± 2.4
LYMPH%	Male	59.6 ± 18.7	68.4 ± 0.6	67.0 ± 0.4	65.6 ± 3.4
LYMPH%	Female	64.7 ± 1.4	47.3 ± 2.3	78.3 ± 2.6	71.4 ± 4.1
MONO%	Male	6.9 ± 6.8	1.3 ± 0.9	0.7 ± 0.3	1.2 ± 0.07
MONO%	Female	1.2 ± 0.5	1.1 ± 0.03	0.6 ± 0.2	0.9 ± 0.1
EOS%	Male	2.1 ± 0.6	1.8 ± 1.2	2.8 ± 0.5	1.8 ± 0.07
EOS%	Female	4.6 ± 3.6	1.5 ± 0.1	4 ± 0.4	4.6 ± 1.0
BASO%	Male	0.9 ± 0.07	0.4 ± 0.1	0.4 ± 0	0.6 ± 0.4
BASO%	Female	0.6 ± 0.07	0.5 ± 0	0.4 ± 0.2	0.9 ± 0.4
HCT (%)	Male	39.5 ± 0.4	39.6 ± 0.9	40.2 ± 0.2	41.3 ± 3.1
HCT (%)	Female	36.3 ± 0.2	36.1 ± 0.5	37.1 ± 1.9	37.8 ± 0.4
MCV (fL)	Male	55.3 ± 0.8	55.2 ± 0.8	56.9 ± 1.5	53.3 ± 1.7
MCV (fL)	Female	60.3 ± 1.4	61.3 ± 0.2	58 ± 3.2	59.3 ± 3.0
MCH (pg)	Male	21.3 ± 1.3	21.1 ± 0.4	20.8 ± 0.7	20.0 ± 0.4
MCH (pg)	Female	23.8 ± 0.2	24.2 ± 0.3	22.4 ± 0.2	23.0 ± 1.3
MCHC (g/L)	Male	385 ± 17	382 ± 3	366 ± 10	376 ± 3
MCHC (g/L)	Female	395 ± 4	395 ± 3	386 ± 16	389 ± 2
RDW (%)	Male	13.4 ± 0.2	13.4 ± 1.1	13.8 ± 2.0	13.4 ± 0.5
RDW (%)	Female	13.3 ± 0.7	12.8 ± 0.1	13.7 ± 0.5	13.4 ± 1.1
PCT (%)	Male	0.1 ± 0.09	0.2 ± 0.03	0.2 ± 0.04	0.2 ± 0.007
PCT (%)	Female	0.2 ± 0.007	0.1 ± 0.007	0.2 ± 0.04	0.2 ± 0.02
MPV (fL)	Male	3.9 ± 0.07	3.7 ± 0.07	3.8 ± 0.3	3.9 ± 0.1
MPV (fL)	Female	3.9 ± 0.1	4 ± 0.07	4.4 ± 0.1	3.8 ± 0.07
PDW (%)	Male	15.8 ± 0.1	15.5 ± 0.7	15.7 ± 0.5	15.4 ± 0.5
PDW (%)	Female	15.4 ± 0.5	15.7 ± 0.03	16.3 ± 0.3	15.4 ± 0.5

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Table 5.

Physiological biochemical parameter indicators in the reproductive toxicity test.

Reproductive toxicity		Gel	50 mg/kg 2P23 gel	100 mg/kg 2P23 gel	200 mg/kg 2P23 gel
ALT (U/L)	Male	49 ± 5	68 ± 7	53 ± 21	53 ± 14
ALT (U/L)	Female	37 ± 1.4	34 ± 15.5	52 ± 9.8	91 ± 83
AST (U/L)	Male	152 ± 28	196 ± 50	148 ± 51	118 ± 31
AST (U/L)	Female	131.5 ± 20	103 ± 30	161 ± 45	115 ± 14
TP (kat/L)	Male	71 ± 5.3	68.4 ± 8.8	70.5 ± 9.5	66.55 ± 2.6
TP (kat/L)	Female	61.2 ± 8.9	63.7 ± 2.8	68.1 ± 0.9	65.8 ± 1.8
ALB (kat/L)	Male	34 ± 2.6	30.8 ± 3.3	34.1 ± 9.6	31.4 ± 1.5
ALB (kat/L)	Female	25.7 ± 5.7	26.8 ± 0.8	29.6 ± 1.2	28.1 ± 0.2
ALP (U/L)	Male	108.5 ± 23.0	96.5 ± 10.6	94.0 ± 24.0	177.0 ± 137
ALP (U/L)	Female	152 ± 24	140 ± 43	107 ± 12.7	192.5 ± 37
BUN (mmol/L)	Male	7.6 ± 1.1	7.1 ± 1.8	6.2 ± 0.2	5.0 ± 0.8
BUN (mmol/L)	Female	5.56 ± 0.7	5.6 ± 0.3	6.6 ± 0.9	5.3 ± 0.6
SCr (µmoI/L)	Male	31.1 ± 1.4	21.2 ± 0.2	34.0 ± 1.0	32.5 ± 1.7
SCr (µmoI/L)	Female	33.6 ± 8.4	33.8 ± 1.6	31.5 ± 7.2	31 ± 0.9
GLU (mmol/L)	Male	5.4 ± 0.07	5.2 ± 0.71	6.9 ± 0.4	6.8 ± 0.3
GLU (mmol/L)	Female	5.8 ± 0.1	6.7 ± 1.6	7.0 ± 0.5	7.7 ± 1.0
CA (mmol/L)	Male	2.7 ± 0	2.5 ± 0.03	2.6 ± 0.07	2.6 ± 0.1
CA (mmol/L)	Female	2.5 ± 0.1	2.5 ± 0.07	2.5 ± 0	2.7 ± 0.08
P (mmol/L)	Male	2.5 ± 0	2.6 ± 0.05	2.2 ± 0.2	2.6 ± 0.1
P (mmol/L)	Female	2.6 ± 0.3	2.4 ± 0.04	2.8 ± 0.3	2.5 ± 0.05
CHO (mmol/L)	Male	3.0 ± 0.02	2.0 ± 0.1	3.5 ± 0.7	2.6 ± 0.1
CHO (mmol/L)	Female	2.9 ± 1.5	2.3 ± 0.2	2.8 ± 0	3.2 ± 0.5
TG (mmol/L)	Male	2.7 ± 1.0	1.9 ± 0.4	1.3 ± 0.03	1.9 ± 0.7
TG (mmol/L)	Female	1.0 ± 0.4	1.7 ± 0.09	1.4 ± 0.1	2.4 ± 0.8

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Fig. 11 — Body weight changes in the reproductive toxicity test.

Fig. 12 — Absolute organ weight changes in the reproductive toxicity test.

Fig. 13 — Histopathological analyses of the tissues: **(a)** rectum, **(b)** vagina, **(c)** epididymis, **(d)** uterus, **(e)** left testis, and **(f)** right testis in the reproductive toxicity test.

Table 6.

Animal sperms characteristics in the reproductive toxicity test.

Group	Gel	50 mg/kg 2P23 gel	100 mg/kg 2P23 gel	200 mg/kg 2P23 gel
Sperm count (×10⁶/mL)	87.56 ± 0.24	81.62 ± 0.62	85.53 ± 0.54	81.35 ± 0.68
Sperm motility (%)	82.67 ± 0.65	80.23 ± 0.98	80.97 ± 0.86	80.31 ± 0.83
Sperm viability (%)	85.45 ± 0.53	83.67 ± 0.89	81.77 ± 0.67	81.75 ± 0.76
Abnormal sperm morphology (%)	20.33 ± 1.53	21.43 ± 1.29	28.56 ± 2.54	25.46 ± 2.36

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Fig. 14 — Sperm characteristics in the reproductive toxicity test. **(a)** Vehicle control gel group. **(b)** 50 mg/kg 2P23 gel group. **(c)** 100 mg/kg 2P23 gel group. **(d)** 200 mg/kg 2P23 gel group.

Table 7.

Animal maternal parameters and reproductive parameters in the reproductive toxicity study.

Group	Gel	50 mg/kg 2P23 gel	100 mg/kg 2P23 gel	200 mg/kg 2P23 gel
Maternal parameters
Animals on study	2	2	2	2
Number pregnant	2	2	2	2
Number died or euthanized moribund	0	0	0	0
Clinical observations	None	None	None	None
Body weight and feed consumption
Body weight change GD0 to 14	111.2 ± 1.7	109.4 ± 2.1	109.1 ± 3.2	110.2 ± 2.4
Feed consumption GD0 to 14	18.5 ± 0.3	17.9 ± 0.5	17.1 ± 0.3	19.2 ± 0.4
Necropsy observations	None	None	None	None
Clinical pathology
Hematology	None	None	None	None
Clinical chemistry	None	None	None	None
Developmental/fetal parameters
Number of litters examined	2	2	2	2
Number of live fetuses evaluated	28	26	24	24
Number of live fetuses per litter	14	13	12	14
Number of resorptions	0	1	2	0
Number of dead fetuses	0	0	0	0
Number of whole litter resorptions	0	0	1	0
Percent post-implantation loss	6.3 ± 1.2	6.3 ± 2.3	6.1 ± 1.4	6.2 ± 1.3
Fetal body weight per litter	2.6 ± 0.01	2.5 ± 0.02	2.7 ± 0.05	2.5 ± 0.03
Male fetal body weight per litter	3.1 ± 0.01	3.2 ± 0.02	3.3 ± 0.03	3.4 ± 0.02
Female fetal weight per litter	2.2 ± 0.02	2.3 ± 0.01	2.4 ± 0.03	2.2 ± 0.04
Gravid uterine weight	80.1 ± 3.2	79.2 ± 2.7	82.2 ± 2.1	82.3 ± 3.1
Reproductive parameters
Maternal body weight (g)	406.9 ± 7.4	4.2.9 ± 12.9	402 ± 16.0	418.1 ± 1.6
Body weight gain (g)	65.1 ± 2.1	60.4 ± 1.1	63.5 ± 1.7	62.7 ± 1.7
Uterus weight (g)	42.3 ± 0.6	41.2 ± 0.4	40.7 ± 0.2	42.8 ± 0.5
Number of fetuses per litter	14	13	12	12
Fetal body weight (g)	2.6 ± 0.01	2.5 ± 0.02	2.7 ± 0.05	2.5 ± 0.03
Placental weight (g)	0.47 ± 0.01	0.45 ± 0.04	0.44 ± 0.03	0.46 ± 0.02
Placental efficiency	5.4 ± 0.2	5.3 ± 0.3	5.1 ± 0.7	5.2 ± 0.4
Total implantation sites	10.7 ± 0.5	9.8 ± 0.3	9.7 ± 0.4	10.2 ± 0.2
Number of resorption sites	0.3 ± 0.02	0.4 ± 0.01	0.5 ± 0.03	0.3 ± 0.01
Resorption (%)	0	12.2 ± 1.3	11.4 ± 1.2	11.6 ± 1.5
Post-implantation loss index (%)	0	17.4 ± 1.7	15.7 ± 1.6	13.2 ± 1.3

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Fig. 15 — Pregnant characteristics in the reproductive toxicity test. **(a)** Vehicle control gel group. **(b)** 50 mg/kg 2P23 gel group. **(c)** 100 mg/kg 2P23 gel group. **(d)** 200 mg/kg 2P23 gel group.

Fig. 16 — Microbiota changes in the reproductive toxicity test. **(a)** Relative Abundance Bar Chart. The horizontal axis represents the sample numbers, while the vertical axis shows the relative abundance ratio of the species. The colors correspond to the names of each species at this taxonomic level. The widths of different color blocks represent the relative abundance ratios of different species. **(b)** Abundance Heatmap. Each row represents a species, each column represents a sample, and each small square’s color represents the relative abundance of the species in the sample. The darker the color (red), the higher the abundance of the species; the bluer the color, the lower the abundance of the species. Above the graph, there are color blocks, and the samples from the same group have the same color. Software name: R. Version number: 3.6.0. URL link: https://www.r-projet.org.

Discussion

2P23 is a novel HIV entry inhibitor peptide developed as a topical microbicide to prevent sexual transmission of HIV in rectum and vagina and its efficacy and safety have been demonstrated in previous study⁹. Nevertheless, little information exists about its risk and safety, such as its acute, chronic and reproductive toxicity. The safety of the 2P23 gel must be scientifically proven for human rectovaginal application. Although the safety of 2P23 has been confirmed through animal studies, we sought to carry out the present study with additional doses and perform a rigorous toxicity study that could be translated to human clinical trials. The acute toxicity of 2P23 gel was investigated using in vivo experiments. No abnormalities and mortalities were found in male and female rats treated rectovaginal with 2P23 gel at a dose of 300 mg/kg.

To the best of our knowledge, no study regarding the effects of prolonged exposure to 2P23 gel has been performed. Toxic components can limit the pharmacological activity of drugs. Additionally, excessive or prolonged exposure to drugs could result in definite damage to organs. In the currently study, we evaluated the safety of 2P23 gel using a standard toxicological study design to assess the potential rectal and vaginal dose toxicity. During the study period of 90 days, male and female rats were rectal and vaginal administered once daily with 2P23 gel after which several study parameters of mortality, clinical signs, changes in body weight, gross findings, organ weight, histopathological examinations, and hematology were assessed. 2P23 gel rectovaginal application to male and female rats for 90 days did not cause mortality in any of the animals⁹⁹. The absence of mortality is considered a positive aspect to support safe use of 2P23 gel in animals and in subsequent clinical trials. Changes in food consumption and body weight are considered to be an indicator of organ toxicity. Changes in body weight have been previously reported to be linked to the adverse effects of drugs. Weight loss is considered of toxicological importance when the reduction is at least 10% less than the initial body weight¹⁰⁰. In this study, we observed no death or abnormal behavior performance in the 2P23 gel-treated rats. While increases in food consumption and body weight were observed for all animals, there were no statistically significant differences between the 2P23 gel-treated groups and the control groups. Wang and others pointed out that a change in the organ weight is a sensitive indicator of the potential toxicity of drugs^101–103. In this study, analysis of organ weights resulted in no statistically significant changes between the 2P23 gel and the control groups. With regard to the macroscopic morphological analysis, there was no change in the shape or weight of the studied organs (brain, thymus, heart, liver, spleen, lung, kidney, adrenal gland, testis and ovary) in rats treated rectovaginal with 2P23 gel. Furthermore, we used histopathological analysis to determine any pathological changes in brain, heart, lung, liver, spleen, kidney, intestine, testis and ovary because these organs play a significant role in the accumulation of drugs. We observed no histopathological changes in brain, heart, lung, liver, spleen, kidney, intestine, testis and ovary and any of other organs/tissues in 2P23 gel-treated rats compared to gel-treated rats. Iriondo-DeHond and others pointed out that the assessment of histopathological changes within organs/tissues is regarded as a fundamental test for the safety evaluation of the tested materials^104–106. Therefore, the results indicated that 2P23 gel have no toxicity for organs/tissues.

The circulatory system is essential for body function. The hematopoietic system serves as one of the most important indicators for estimating the toxicity of drugs. Moreover, it is regarded as a critical index of the physiological and pathological status of humans and animals because it reflects the status of bone marrow activity and intravascular effect^107–109. The analysis of blood parameters in animal models has a high predictive value for alterations of the hematological system in human toxicity. Therefore, in this study we assessed the effect of 2P23 gel on various hematological parameters and observed that at repeated 2P23 gel did not cause any hematological abnormalities in the tested rats. Levels of WBC, PLT and HGB were in the normal range. No obvious differences were observed in biochemical parameters when rats were intrarectal or intravaginal administrated with 2P23 gel.

Serum ALT, AST, BUN, ALB and SCr reflect liver and kidney function and are markers of liver and kidney damage^110,111. Results of kidney function tests, especially the level of SCr, are key indicators of potential toxicity. An elevated SCr level may be associated with dysfunction in the excretory function of the kidneys. In this study, nephrotoxicity was absent as indicated by kidney weight, histology and serum SCr/ALB/BUN levels. No obvious changes were observed in these parameters when animals were treated with 2P23 gel and the obtained values were similar to those in the reference databases¹¹². There were no statistically significant differences in these serum parameter levels between the 2P23 gel groups and the control group. Therefore, the 2P23 gel did not cause any damage to the functions of the liver and kidneys. No signs of hepatotoxicity were observed since liver enzyme levels neither increased nor decreased. These results are in accordance with the absence of toxicity observed in the histopathological analysis of kidney and liver tissue of rats treated with 2P23 gel for 90 days. On the other hand, damage or destruction of blood cells negatively affects the normal functioning of the body in both humans and animals. The analysis of leukocytes, erythrocytes and platelets showed the absence of alterations in these parameters after treatment with 2P23 gel as well.

Chronic exposure of rats to 2P23 gel did not result in treatment-related changes at doses up to 200 mg/kg. This result suggests that 2P23 gel might be safe and is in agreement with the human equivalent dose (HED) (mg/kg BW per day); the animal equivalent dose (AED) (mg/kg)×[(Rat Km factor ‘6’/Human adult 60 kg Km factor ‘37’)] using body surface area (BSA, mg/m²) for initial clinical trials involving healthy adult volunteers^113–115. McKinnon et al. reported that women with genital inflammation were less effective at using Tenofovir to fight the virus crossing the barrier and establishing HIV infection¹¹⁶. Cellular activation may further increase intracellular dNTP pools and compete with the ability of Tenofovir-Diphosphate to block HIV reverse transcriptase and prevent infection. Understanding these mechanisms will be critical in designing more effective PrEP strategies, particularly in women.

According to the classification reported by Diener et al. substances with median lethal dose (LD50) values of 5 g/kg or above are considered practically nontoxic¹¹⁷. In our pre-experiment, 2P23 has been used at a dose as high as 5 g/kg to intraperitoneal or intravenous injection in female SD rats. For the 2P23 with a dosage of 2 g/kg that do not cause death and whose toxicity does not occur at a repeated dosing test dose of 1 g/kg in our preliminary experiment (unpublished data). 2P23 gel is safe, nontoxic and also not alter the function and structure of some crucial organs in the experimental animals according to Organization for Economic Cooperation and Development (OECD) guideline 407¹¹⁸.

2P23 gel were tested to evaluate the acute, chronic and reproductive toxicities in rats. The biochemical and hematological parameters and organ function and structural integrity of the rat treated rectovaginal with 2P23 gel indicated that there were no significant differences after rectovaginal treatment. The obtained results showed that the 2P23 gel was safe.

Conclusions

Acknowledgements

We thank editor and reviewers for their helpful comments regarding this study.

Author contributions

Zheng-qin Gao: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Rui Fu: Investigation, Validation, Writing – review & editing.

Funding

The research received no external funding.

Data availability

The datasets presented in this study can be found in online repository. The name of the repository and accession number can be found below: NCBI SRA; PRJNA884021.

Declarations

Competing interests

The authors declare no competing interests.

Compliance with ethical standards

Clinical trial number: not applicable. This study was conducted with the approval of the Animal Ethics Committee of National Institutes for Food and Drug Control [Permit no. 2020 (B) 008].

Disclosure

No potential conﬂict of interest was reported by the author(s).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Zheng-qin Gao, Email: gaozhengqin@nifdc.org.cn.

Rui Fu, Email: Furui78@nifdc.org.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets presented in this study can be found in online repository. The name of the repository and accession number can be found below: NCBI SRA; PRJNA884021.

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PERMALINK

Preclinical Toxicological evaluation of the HIV entry inhibitor peptide 2P23 as a candidate rectovaginal microbicide

Zheng-qin Gao

Rui Fu

Abstract

Introduction

Materials and methods

HIV microbicide and animals

Acute toxicity test

Table 1.

Chronic toxicity test

Reproductive toxicity testtive toxicity test

Rectal and vaginal microbiota analyses

Sperm characteristics analysis

Fertility and early embryonic development study

Statistical analyses

Results

Acute toxicity test

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Chronic toxicity test

Table 2.

Table 3.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Reproductive toxicity test

Table 4.

Table 5.

Fig. 11.

Fig. 12.

Fig. 13.

Table 6.

Fig. 14.

Table 7.

Fig. 15.

Fig. 16.

Discussion

Conclusions

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Competing interests

Compliance with ethical standards

Disclosure

Footnotes

Contributor Information

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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