Formulation, development and characterization of fosfomycin tromethamine pessaries for the treatment of vaginal infections

Mahboob Ul Haq; Attiqa Naz; Abdul Baseer; Sajjad Ahmad; Basmah F Alharbi; Muhammad Khurram

doi:10.1038/s41598-025-29721-9

. 2025 Dec 1;16:381. doi: 10.1038/s41598-025-29721-9

Formulation, development and characterization of fosfomycin tromethamine pessaries for the treatment of vaginal infections

Mahboob Ul Haq ^1,², Attiqa Naz ^1,^✉, Abdul Baseer ³, Sajjad Ahmad ⁴, Basmah F Alharbi ^5,^✉, Muhammad Khurram ²

PMCID: PMC12770539 PMID: 41326543

Abstract

Background The present study investigated the formulation, development, and characterization of Fosfomycin pessaries for the treatment of genitourinary tract infections. To achieve higher local concentration and to avoid the systemic exposure of the body towards Fosfomycin, vaginal pessaries have been developed in this research work. Methodology The Fosfomycin pessaries were prepared using the hand rolling method. After the formulation of the Fosfomycin pessaries, pre-formulation studies were conducted to evaluate the drug-excipient compatibility, followed by in vivo studies. Results The pre-formulation studies using FTIR (Fourier Transform Infra-Red) spectrophotometric analysis showed no significant interaction of Fosfomycin with the base and formation of Fosfomycin pessaries. Furthermore, DSC (differential scanning calorimetry) and TGA (thermogravimetric analysis) analysis confirm the formulation stability. The physicochemical analysis of Fosfomycin pessaries, including liquefaction time and breaking force, showed the formulation within the pharmacopeia standard range. Furthermore, the pharmacokinetic and in vitro release studies showed that Fosfomycin pessaries, in contrast to the Fosfomycin suspension, exhibited higher elimination time, higher absorption rate, and area under the curve. The antibacterial results of the Fosfomycin pessaries showed significant activity against E. coli using in vitro and in vivo studies. Conclusion The in vitro and in vivo results revealed that Fosfomycin pessaries have improved pharmacokinetics and increased antibacterial activity.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-29721-9.

Keywords: Fosfomycin, Pessaries, Pre-formulation, Pharmacokinetics, Blood, Drug development, Molecular docking simulation, Enzyme inhibitor

Subject terms: Drug discovery, Medical research, Microbiology

Introduction

Fosfomycin is an antibacterial agent and is considered to be the drug of choice for the treatment of urinary tract infections¹. The Fosfomycin is active against both gram-positive and gram-negative bacteria and interferes with the formation of the peptidoglycan precursor UDP N-acetylmuramic acid (UDPMurNAc) during peptidoglycan biosynthesis². The drug is available in two oral formulations: Fosfomycin calcium and Fosfomycin tromethamine³. Fosfomycin is active against Escherichia coli, Staphylococci, Enterococci, Haemophilus spp., and most enteric gram-negative bacteria⁴.

The pharmacokinetic properties of Fosfomycin tromethamine revealed that the drug has 34% to 41% bioavailability after oral administration⁵. Fosfomycin calcium has 2–2.5.5 times less absorption than Fosfomycin Tromethamine, i.e., only 10–30%⁴. The elimination half-life of Fosfomycin calcium is 5.7 h and the Fosfomycin Tromethamine half-life is 2 h. Moreover, it is excreted in urine in unchanged form⁶. The age of an individual does not influence Fosfomycin absorption rate; however, the food intake reduces it^5,7. After oral administration of a 30 mg/kg dose, the mean peak concentration (C_max) observed was 3.60 ± 0.96 µg/ml with a calculated T_max of 3.00 ± 0.00 h⁸. A single 3-gm dose of Fosfomycin Tromethamine can push the serum concentration (C_max) to 26.1 (± 9.1) µg/mL within 2 h⁹.

The dosage form of any drug significantly influences its pharmacokinetic and pharmacodynamic properties¹⁰. There are several types of dosage forms available, such as oral, IV, IM, SC, etc^11,12.. All these routes of administration have advantages and disadvantages. The most common limitation of these dosage forms is their generalized action, thus ultimately causing severe adverse effects¹³. Similarly, sometimes the infections are limited to the special tissues or organs of the body and direct targeting of such infections is very fruitful¹⁴. This direct application not only avoids the systemic toxicity but also provides an increased concentration of drug directly delivered to the diseased site¹⁵. The genitourinary tract infection is very common in females and multiple gram-negative and anaerobic bacteria are involved, which are very problematic, especially for females having either comorbidities or old age¹⁶. When the invading microbes overwhelm the normal flora, the symptoms of vaginitis develop. The Lactobacillus acidophilus bacteria are normally present in the vagina and establish the acidic environment of vagina by releasing bacteriocin and hydrogen peroxide¹⁶. When the level of the lactobacillus acidophilus is decreased, different aerobic and anaerobic bacteria colonize the vagina and cause infection¹⁷. The Fosfomycin oral powder is employed for the treatment of uncomplicated UTIs and achieves a reasonable concentration in the urinary system¹⁸. However, several adverse effects, such as nausea and vomiting, are common. Additionally, for local vaginal infections, Fosfomycin is not suitable⁵. In order to overcome these pharmacokinetic constraints of Fosfomycin, an alternative and effective drug delivery system of Fosfomycin tromethamine needs to be developed. In this connection, pessaries dosage is the rational option to maximize the site-specific bioavailability of Fosfomycin. After literature survey, it is inferred that no work has been reported so far on pessaries dosage form of Fosfomycin^19,20.

Bacterial vaginosis is often associated with a high recurrence rate and various complications have been reported, including pelvic inflammatory diseases and pre-term birth²¹. Currently, bacterial vaginosis is treated with clindamycin and metronidazole oral or intravaginal formulations²². However, the emergence of resistance, systemic adverse effects, and recurrence of vaginal infections (50–70% within a year) necessitate the development of newer treatment approaches^22,23. The Fosfomycin antibiotic serves as an important alternative for the treatment of bacterial vaginosis by irreversibly inhibiting the bacterial cell wall and its extensive use has been reported against the UTI²⁴. Nevertheless, its potential use against bacterial vaginosis using pessaries dosage form has not been explored yet. Previously, for the treatment of bacterial vaginosis, oral and cream formulations have been focused on, which leads to suboptimal local administration and poor retention of the drug²⁵. Despite its broad-spectrum antibacterial activity, Fosfomycin is associated with variable and poor oral bioavailability due to its instability in the gastrointestinal tract and poor absorption. By formulating the Fosfomycin vaginal Pessary, Fosfomycin will be available at a higher concentration at the site of infection, thereby avoiding the harsh gastrointestinal environment. The current study aimed at the formulation development of Fosfomycin tromethamine pessaries for the treatment of vaginal infection and it’s in vitro and in vivo evaluation. The idea is to have a prolonged drug release, deliver a higher concentration of drug at the intended site and reduce the recurrence rate. Furthermore, it was anticipated that this formulation would also help in avoiding the Fosfomycin-induced systemic toxicity in contrast to oral formulations.

Materials and methods

Chemicals and reagents

The cocoa butter base and Fosfomycin used in this research are provided by MEDLINE Technology, Pakistan. The equipment and other reagents used were: glass vials, glass containers, septum stoppers, dissolution apparatus, and distilled water as the dissolution medium. Similarly, the E. coli (O157:H7 strain) culture suspension, phosphate-buffered saline, anesthetics agent, centrifugation machine (Thermo Scientific, United States), analytical balance Mettler-Toledo (Germany), and compound microscope (Shandong) attached with the camera were also utilized in the present study. The chemicals and reagents used were of analytical grade. Overall, the study on the formulation development and characterization of Fosfomycin tromethamine pessaries is depicted in Fig. 1. The methods applied for the preparation of pessaries are similar to those for suppository preparation, which include the Hand rolling method²⁶. The hand-rolling method was used for the preparation of pessaries containing Fosfomycin tromethamine²⁷. It is the oldest method for pessaries preparation and can be used when only a few pessaries are required²⁸. In this method, a plastic-like mass was prepared by triturating the bases, i.e., cocoa butter and Fosfomycin, in a mortar, and then the mass was formed into a ball as reported previously²⁹. The pessaries formulation was optimized using varying amounts of drug and bases with the help of Design-expert software (Stat-Ease, Inc. Minneapolis, MN 55418)³⁰. The dose of Fosfomycin was kept at 30 mg/kg, and thickness of the pessaries was 2 mm in diameter and 6 mm in length³¹.

Fig. 1 — Sequential stepwise presentation of the research conducted herein.

Study design

The Fosfomycin tromethamine pessaries formulation was developed and characterized with necessary modifications. The following steps were used during the study design. (1) Pre-formulation phase, (2) Formulation development phase, (3) In vitro and in vivo evaluation phase. Each step of the methodology presented herein is conducted in accordance with the relevant guidelines and regulations.

Pre-formulation phase

This phase included the compatibility studies, which aimed to evaluate the interaction between the Fosfomycin and cocoa butter base and stability of the drug in the cocoa butter base using FTIR (Fourier-Transform Infrared Spectroscopy), DSC (Differential Scanning Calorimeter), and TGA (Thermogravimetric analysis) analysis³².

Drug-suppository base interaction

The FTIR analysis was conducted to investigate the interaction of the excipients, i.e., cocoa butter, with the Fosfomycin utilizing the FTIR spectrophotometer (thermonicolet). The FTIR analysis was conducted for the drug i.e., Fosfomycin alone, cocoa butter base, and the Fosfomycin pessaries dosage form containing both Fosfomycin and cocoa butter base FTIR spectra were recorded for the individual components of Fosfomycin pessaries and pessary formulation. The samples were scanned between 400 and 4000 cm^{− 1} in transmittance mode³³.

DSC analysis

The DSC was used to study the thermal stability of Fosfomycin pessaries and to investigate the Fosfomycin compatibility with the cocoa butter during the formulation³⁴. The DSC analysis was conducted for the Fosfomycin, cocoa butter and Fosfomycin-loaded cocoa butter pessaries using the DSC 60 instrument (Schimadzu, Japan). The three samples mentioned earlier were wrapped in the aluminum pans after weighing the individual components. Subsequently, the DSC thermograms under the nitrogenous atmosphere were taken as read out, having the temperature ranging from the ambient temperature up to 300 °C at a 10 °C/min heating rate. The heat flow vs. temperature graph was plotted in the DSC thermogram³⁵.

TGA analysis

The pessaries were subjected to the TGA analysis to assess the thermal stability of the Fosfomycin-loaded pessaries³⁶. The sample for the TGA analysis was prepared by weighing 300 mg of suppository, and it was ensured that the sample was in film form for uniform distribution of the heat. Before the TGA analysis, any residual moisture content was removed to ensure that the results were not affected. The Perkin Elmer (TGA 4000, USA) equipment was used for the TGA analysis. After the sample preparation, the environmental factors were controlled, such as nitrogen gas; the flow rate was maintained at 50 ml/min, and inert gas was used to maintain an inert environment³⁷. The heating of the sample started from 35 °C to a final temperature of 600 °C, and the heating rate was maintained at 10 °C. For the accurate measurement of the weight, the balance was calibrated to avoid any discrepancy³⁸. The sample was placed in the aluminum holder to ensure the resistance and stability of the temperature involved in the TGA analysis³⁹. During the analysis, the weight loss was monitored, and the TGA analyzer monitored changes in the mass at every 1 s with respect to temperature at regular intervals⁴⁰. In order to increase the reliability and reproducibility of the data, the TGA analysis was performed in triplicate⁴¹. The data was analyzed to assess the changes in the weight of the suppository with time. The thermal stability of the suppositories was analyzed from the extent of weight loss and decomposition features of the formulation⁴².

Formulation development phase

Entrapment efficiency

The entrapment efficiency was determined to quantify the amount of Fosfomycin present in the suppositories using the UV-Visible spectrophotometry⁴³. A fixed mass of the suppository formulation (30 mg Fosfomycin and 270 mg Cocoa butter) was dispersed in the desired volume, i.e., 100 ml PBS at pH 7.4 and temperature of 37 °C using continuous stirring. The standard curve was used to quantify the amount of Fosfomycin in different calibration standards. The calculation of the Entrapment Efficiency was performed using the following formula⁴⁴.

The initial mass of pessary was 30 mg, while the mass of the drug, which was determined using the UV-Visible spectroscopy, shows the actual amount of concentration measured in the drug content. During the % drug content measurement, the whole pessary was used⁴⁵.

Muco-adhesion

Muco-adhesion is an important parameter to evaluate the attachment of pessaries with vaginal mucosa using female rabbits⁴⁶. A modified physical balance with a glass vial with an upside-down position hung from one hand was used⁴⁷. A small segment of the vagina was retained on the glass container with the help of a septum stopper. Another glass container was made fixed with the help of glue and duct tape. A drop of Fosfomycin pessaries was placed on the vaginal mucosa, which was fixed on the glass container. Muco-adhesive force/per unit area was expressed in dyne/cm2⁴⁸

Physicochemical evaluation

The objective of these physicochemical parameters is to confirm whether a stable pessary formulation has been formed or not⁴⁷.

Disintegration test

For the disintegration time measurement, the disintegration apparatus (Curio Tech, Laboratory, Punjab, Pakistan) was used. The disintegration test was performed by simulating the vaginal environment and to evaluate the disintegration of the suppository in medium⁴⁹. The disintegration test was done to evaluate whether the pessary is completely dissolved, softened sufficiently, or completely dispersed⁵⁰.

Liquefaction test

The liquefaction test was employed to assess the melting or softening of the suppository at body temperature and subsequently, release the drug appropriately⁵¹. The time of the liquefaction varies according to the base of the formulation and the specification of the formulation⁵². The fat-based suppository should liquefy within 30 min, while the water-soluble suppository must liquefy within 60 min. The liquefaction behavior of the pessaries was observed by keeping the pessaries in the temperature-controlled water bath, i.e., 37 ± 0.5 °C, to simulate the temperature of the vagina. For liquation, each pessary was placed in the glass vial to observe the complete liquefaction of the pessary. The time required for the conversion of the solid pessary into liquid was recorded⁵³.

Breaking test

The breaking time test, also called the rupture test, is usually employed for the suppository dosage form to assess the mechanical strength of suppository, which can enable it to withstand the handling and still release the drug effectively upon administration⁵⁴. The base and the formulation composition significantly influence the breaking time⁵⁵. The exact values of the breaking time or officially recognized but suppository during handling and administration must maintain its integrity⁵⁶. The breaking strength of the pessary was evaluated using compression method⁵⁷. The flat platform was used where the pessary was positioned, and at a constant speed, the force was applied centrally until the pessary was fractured. The maximum applied force, which resulted in the fracture of the pessary, was recorded as breaking strength⁵⁸. To ensure the reliability and reproducibility, the mean values with standard deviation were presented⁵⁹.

Standard curve

The Fosfomycin was quantified in the in vitro media using an established analytical method i.e., a non-destructive quantitative tool reported by⁶⁰, which was used with necessary modifications⁴⁵. Briefly, the stock solution was prepared by dissolving 30 mg of Fosfomycin in phosphate buffer saline (PBS) at a pH of 7.4 and the final volume was made 100 ml by adding PBS. The concentration of the Fosfomycin stock solution was 0.3 mg/ml or 300 µg/ml. From the stock solution, 5 serial dilutions were prepared, such as 30 µg/ml, 25 µg/ml, 20 µg/ml, 15 µg/ml, 10 µg/ml, and 5 µg/ml solutions. The absorbance was recorded at 254 nm using the UV-VIS spectrophotometer. The standard curve was plotted by plotting the concentration on the x-axis, and absorbance on the y-axis⁴⁵.

In vitro release

The in vitro drug release from the developed Fosfomycin pessaries was conducted using the USP recommended dissolution apparatus, i.e., the paddle method⁶¹. The release kinetics were studied at body temperature (36.5 °C) at 80 rpm in 500 mL of water as the dissolution medium. The Fosfomycin pessaries, having a specific mass (30 mg Fosfomycin and 270 mg cocoa butter base), were taken in a semipermeable membrane tube which was closed with a clamp on both ends⁶². Subsequently, these semi-permeable membranes holding the pessaries formulation were placed in the dissolution medium, i.e., water. After predetermined time intervals, e.g., 0, 20, 40, 60, 80, 100, 120, 140, 160 and 180 min, a fixed volume of dissolution medium (2 ml) was taken out from the dissolution vessel, followed by the addition of the same amount of dissolution medium to maintain the sink conditions⁶³. The samples taken out were filtered, followed by drug analysis using the pre-established analytical method using UV-Visible spectrophotometric standard curve method^64,65. The Fosfomycin saturation solubility was experimentally determined in the simulated vaginal fluid (SVF), and the dissolution medium was chosen to be at least 3-fold higher than the amount of the solvent required for the total drug in the single pessary to completely solubilize, to fulfill the criteria of the sink condition. At the pre-determined time interval, a fixed volume of 2 ml was withdrawn and replaced with an equal amount of fresh and pre-warmed buffer to keep the volume constant and maintain the sink condition during the entire experiment⁶⁶.

In vivo evaluation

Animals

The female rabbits were procured from the animal facility (Veterinary Research Institute, Peshawar) and all activities were performed in the Basic Medical Sciences Lab, Abasyn University, Peshawar. The rabbits were acclimated to the lab environment and were provided with free access to water and food. The animals’ activities were performed according to the Animal Ethical Committee of Abasyn University, Peshawar. Ethical approval was granted (Approval number of the study is Ref. No. IERC-AUP 2024-011). During the whole experiment, it was made to avoid unnecessary harm to the animals and minimum number of animals were used. At the end of the study, the animals were euthanized humanely by the 50:50 mixture of Pentobarbital sodium (at the dose of 100 mg/kg via intraperitoneal (IP) route) and Lidocaine Hydrochloride (10 mg/ml intraperitoneal). The rabbits were monitored until the lack of heartbeat was noted for > 60 s before tissue harvest⁶⁷.

Induction of vaginal infection

Twenty (20 µl) of E. coli culture suspension having a dose of 2 × 10⁷ CFU was administered vaginally to the rabbits as a study group. As a control group, an equivalent amount of phosphate-buffered saline (PBS) was administered vaginally in the rabbits⁶⁸. The rabbits of all the groups were placed in a comfortable position for up to 3 min for the administration of pessaries⁶⁹. Afterwards, incubation time was recorded, which is 3 to 4 days⁷⁰. In short, 20 rabbits (each group consisting of 5 animals) were divided into four groups, i.e., normal control (received no drug and no E. coli), negative control received only E. coli, Fosfomycin pessaries (received E. coli and Fosfomycin pessaries) and Fosfomycin suspension group (received E. coli and Fosfomycin oral suspension). Both the Fosfomycin pessaries and Fosfomycin oral suspension group received equivalent dose, i.e., 30 mg/kg body weight³¹. The Rabbits were left for 12 h overnight fasting before the experimental procedure⁷¹. Both the treatment groups received the Fosfomycin pessaries and Fosfomycin oral suspension at the same time after the induction of the disease via E. coli⁷². After three hours of administration of drugs, the vaginal swabs were collected, and the bacteria was allowed to grow on the petri dishes for the growth of the bacteria. After the experiment, the E. coli concentration or the number of colonies in all groups was compared. Similarly, the in vitro antibacterial studies were conducted to evaluate the effect of the Fosfomycin pessaries and Fosfomycin suspension against the genitourinary tract infections. The purpose of the experiment was to study the comparative analysis of the two formulations against the E. coli, which is very commonly involved in the pathogenesis of UTIs⁷².

Blood sampling

The dose of Fosfomycin was optimized at different concentrations in the pessaries during in vivo studies⁷³. After dose optimization, the rabbits were divided into two groups (each group containing 6 rabbits)⁷⁴. The rabbits of both groups were fasted for 12 h before starting the experimental procedure. The rabbits were sedated with the anesthetic agent, i.e., Xylazine and Ketamine (16 mg + 60 mg, i.p, respectively) combination, placed on a surgical table in the supine condition, and fixed with holding clips. One group of rabbits was vaginally administered with Fosfomycin pessaries with an optimized dose, while the second group was administered orally with Fosfomycin powder (using the clinically available dos9e for comparison with the optimized dose). After a predetermined time, blood samples of 300 µl were collected from the veins of both rabbit groups. In each sampling time, a normal saline solution was administered to each rabbit to avoid blood thickening⁷⁵. The collected samples were centrifuged using a centrifugation speed at 8,000 g for 15 min to extract plasma⁷⁶. Plasma was preserved at −20 °C and the Plasma Fosfomycin concentration was determined using a developed analytical method⁷⁷.

Pharmacokinetic evaluation

The pharmacokinetic parameters (the drug concentration time curve (AUC) from zero to infinity, half-life (t_1/2) and the elimination constant (K_el)) were assessed using the non-compartmental analysis⁷⁸. The maximum plasma concentration of Fosfomycin (C_max) and the time required to obtain the maximum plasma concentration (T_max) were calculated from the time versus plasma concentration curve⁷⁹. The pharmacokinetics of suspension and pessaries delivery systems were compared⁸⁰.

Histopathological analysis of vagina

The H and E staining was performed to assess the effect of Fosfomycin pessaries on the vagina integrity and the results were compared with the normal control and Fosfomycin suspension. The H and E staining was performed and the various factors that were evaluated include inflammation and infiltration. A vaginal segment having a length of 4 cm was isolated from the treatment rabbits (being administered with Fosfomycin pessaries) after 12 h from administration of pessaries dosage form. Afterwards, it was washed with normal saline solution by adding the 10% neutral carbonate buffered formaldehyde. The vaginal segments were embedded in paraffin and separated into 3 ~ 4 μm segments⁷⁴. Then it was stained with hematoxylin and eosin for damage assessment. The untreated vagina tissues were used as a normal control (negative control). All the tissues were morphologically examined and visualized using a Chinese 25-600X Digital Camera USB LED Light Microscope. The vaginal segments were observed for inflammation, injuries, or any degenerative lesions. Furthermore, vaginal swabs were obtained from all the recruited animals, and the swabs were cultured to assess the bacterial growth. The bacteria were grown using Petri dish method, the number of bacterial colonies was assessed in both groups, and the results were compared⁸¹.

In-silico studies

Protein preparation and development of a compound library

The selected protein structure, MurF, was retrieved from the Protein Data Bank (PDB: 4QDI)⁸², followed by potential inhibitory compounds of cocoa butter major compounds were retrieved from literature⁸³. The WADDAICA29 server (https://heisenberg.ucam.edu:5000/), which employs a deep learning approach for scaffold hopping to aid in drug modification and novel drug design, was used in this process⁸⁴.

Characterization of MurF pockets and molecular docking analysis

The binding pocket of the MurF structure was identified using the CASTp 3.0 server (http://sts.bioe.uic.edu/castp)⁸⁵, while trj_cavity version 2.1 was used to detect druggable cavities in the targeted protein⁸⁵. Moreover, blind docking was conducted using AutoDock 4.2.40. Molecular docking was carried out using PyRx 0.8⁸⁶. The grid was tailored to encompass the active site, specifically^87,88. For each ligand, up to 100 binding poses were considered for grid refinement, but only the most favorable pose per compound was retained⁸⁹. Redundant structures were subsequently removed from the dataset. The final ranking of ligand-target interactions was based on calculated binding free energy (in kcal/mol)⁹⁰.

Molecular dynamics simulation

Molecular dynamics simulations were performed for MurF in complex with the top lead compounds using the Ff19SB force field⁹¹, as implemented in AMBER22⁹². The simulations were initiated from the best docked structure. Each system was placed in an OPC water model with a 0.4 Å buffer between the protein and the box boundary and neutralized with sodium ions⁹³. Electrostatic interactions were computed using the Particle Mesh Ewald (PME) method⁹⁴ with a real-space cutoff of 10 Å, a PME order of 4, and a relative tolerance of 10⁻⁵ between long- and short-range electrostatics. Short-range interactions, including Lennard–Jones and real-space electrostatic interactions, were evaluated with a 10 Å neighbor list and truncated at 9 Å⁹⁵. The system temperature was maintained at 300 K using the SHAKE thermostat⁹⁶, and hydrogen bonds were constrained using the Langevin algorithm. Energy minimization was conducted using the steepest descent method until the maximum force was reduced to below 10 kJ/mol⁹⁷. The production run was conducted for 100 ns. The simulation trajectories were analyzed using the CPPTRAJ module⁹⁸ and plotting was done via XMGRACE⁹⁹.

Binding free energy calculation

Binding free energy was estimated using the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) approaches^90,100. Analyses were conducted on the final 5 ns of each molecular dynamics trajectory. The MMGBSA tool, integrated with AMBER, was employed for these calculations¹⁰¹. Snapshots for each protein ligand complex were extracted at 100 ps intervals throughout the selected trajectory segment¹⁰².

Statistical analysis

The results of the study were presented as Mean ± S.D. The assays were performed in triplicate. The results of the study were analyzed using the unpaired t-test (for in vitro release study) and One-Way ANOVA (Analysis of Variance)¹⁰³ followed by the post hoc Tukey’s Multiple comparison (for the anti-bacterial activity)¹⁰⁴. The pharmacokinetic parameters were evaluated using the PK solver ADD-in in Excel and all the required parameters were related to the pharmacokinetics, assessed for both Fosfomycin pessaries and Suspension. The data was analyzed using GraphPad Prism version 5 software and a P-value less than 0.05 was chosen for statistical significance¹⁰⁵.

Results

Preparation of fosfomycin pessaries

The Fosfomycin pessaries were prepared using the hand-rolling method as discussed in the method Sects^106,107. The required amount of Fosfomycin was weighed, finely powdered and mixed with the base, which is already softened using the spatula to form a homogenous mixture. The details are listed in Table 1. The torpedo or cylindrical shape pessaries were made using hands (wearing gloves) and it was ensured for easy insertion by making one end tapered. The formed pessaries were kept for 15–30 min either in a refrigerator or on a cold marble slab for hardening. For further refinement of the shape, the pessaries can be rolled again. To inhibit the melting and sticking of the formed pessaries, wrap them in the appropriate covering material, i.e., foil or wax paper and store them in the refrigerator before use. As the concentration of the Fosfomycin increases in the suppositories, the gelation temperature also decreases. As drug concentration increases the gel strength also increases; however, the gel strength decreases with the increase in the water concentration. The bio-adhesive strength for all the formulations was determined to study the strength of the formulation, which binds to the mucosal surfaces. The higher the bio-adhesive strength, the higher will be the absorption and retention of the drug within the vagina, as shown in Table 1. The results showed that when the concentration of the cocoa butter base is increased and the Fosfomycin concentration is reduced, the bio-adhesion of the formulation also increased and vice versa.

Table 1.

The concentration of cocoa butter, fosfomycin, and bio-adhesive force of fosfomycin pessaries.

Composition	Cocoa butter (w)	Fosfomycin (w)	Bioadhesive force (×10²dyne/cm²)
F1	60	40	9.7 ± 0.3
F2	65	35	11.2 ± 0.5
F3	70	30	12.5 ± 0.2
F4	75	25	12.78 ± 0.4
F5	80	20	13.6 ± 0.3
F6	85	15	13.8 ± 0.5

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The prepared Fosfomycin-loaded cocoa butter Pessaries were subjected to the viscosity assessment viscometers (NDJ-8 S). In the sample, viscometer rotor was immersed and at a constant shear rate, the viscosity was determined¹⁰⁷. The reading of the viscometer was studied for 20 min at an interval of 2 min and the strength of the viscosity was recorded in milliPascal-seconds. The results of the viscosity showed that the viscosity is low from 0 to 2 min and exhibits a more fluid consistency of the formulation. However, with the passage of time, the viscosity increases, and the solidification of the formulation takes place gradually. The higher viscosity of the formulation improves the drug’s retention, while the controlled release of the Fosfomycin was confirmed by a gradual increase in the viscosity. Furthermore, the viscosity also enables the retention of the pessaries within the site of administration for a longer period of time and doesn’t allow the leakage of the formulation as shown in Fig. 2.

Fig. 2 — The rheology of the Fosfomycin-loaded cocoa butter pessaries using time vs. viscosity as parameters for comparison.

Pre-formulation studies using FTIR analysis

During the FTIR analysis all the samples were scanned between the spectral range of 500–4000 cm⁻¹. The spectra were taken in transmittance mode. The FTIR spectra indicate the molecular structure of the Fosfomycin only and show the Fosfomycin pure absorption bands. The significant peaks correspond to the region of 1000–1200 cm⁻¹, indicating the P-O-C stretching vibrations due to the presence of the phosphonic acid group. While the O-H stretching due to the hydroxyl group is shown around the 3000–3500 cm⁻¹. These peaks reveal the presence of a unique functional group associated with the Fosfomycin. The green line shows the FTIR spectra of the cocoa butter base, which shows characteristic typical absorption peaks related to the lipids. The C-H stretching vibration due to the aliphatic chain is represented by the presence of a significant peak around the 2800–3000 cm⁻¹ band, and the presence of the C = O stretching vibration due to the presence of the ester groups (showing the presence of the triglycerides) is represented by the peak around the 2800–3000 cm⁻¹. Furthermore, the presence of the additional bands around the 1000–1500 cm⁻¹ band is due to C-O and C-C vibrations and indicates the complex lipid nature of the base. The black line represents the Fosfomycin pessary formulation FTIR spectra and exhibits the features of both components in the formulation. The Fosfomycin pessary retains the key features of both components in the formulation and shows that the preservation of the molecular integrity of both components is maintained in the Formulation. The formulation FTIR spectra do not have new peaks, which is indicative of the minimal degradation and interaction between the two components in the formulation and hence, showed that both components are compatible in the formulation Fig. 3.

Fig. 3 — The FTIR analysis of the combined cocoa butter base, Fosfomycin only, and Fosfomycin Pessary.