Rifapentine is a semi-synthetic, broad-spectrum bactericidal agent, with the molecular formula C47H64N4O12 and a molecular weight of 877.04, please refer to Figure 1. Its chemical designation is 3-[4-cyclopentyl-1-piperazinyl-iminomethyl]-rifamycin SV (Temple and Nahata, 1999). Rifapentine has been in clinical use in China since it was included in the 1996 edition of the Chinese National Essential Medicine List (Zheng et al., 2017). The U.S. Food and Drug Administration (FDA) (2001) approved rifapentine for the treatment of tuberculosis in 1998 (Roehr, 1998). As a derivative of rifamycin B, rifapentine belongs to the rifamycin family of antibiotics. It appears as a brick-red or dark red crystalline powder that is both odorless and tasteless. The compound dissolves easily in methanol and chloroform, has limited solubility in ethanol and acetone, and is almost completely insoluble in water and ether (Jenkin, 2017).

Figure 1. The chemical formula, structural formula, and spatial structure diagram of Rifapentine.

In vitro studies reveal that rifapentine possesses significant antimicrobial efficacy against M. tuberculosis, with a minimum inhibitory concentration (MIC) ranging from 0.03 to 0.25 mg/L. This makes it 2 to 10 times more potent than Rifampin (Rastogi et al., 2000; Bemer-Melchior et al., 2000; Mor et al., 1995; Arioli et al., 1981). Clinically, a bi-weekly treatment regimen with rifapentine achieves results comparable to daily Rifampin therapy, but with fewer side effects (Van den Boogaard et al., 2009). Follow-up studies 3 years post-treatment have shown a bacteriological relapse rate of 2.6% with rifapentine (Jarvis and Lamb, 1998), demonstrating its substantial long-term effectiveness in treating pulmonary tuberculosis with a relatively low recurrence rate. Moreover, rifapentine exhibits strong antibacterial activity against most Gram-positive bacteria, although its effectiveness against Gram-negative bacteria is weaker (Wu et al., 2015; Albano et al., 2021). When combined with isoniazid, rifapentine’s suppressive effects on Mycobacterium tuberculosis significantly exceed those observed with the sole use of Rifampin and isoniazid (Sterling et al., 2011). Additionally, rifapentine has been shown to prevent tuberculosis in HIV-positive individuals (Swindells et al., 2019).

Tuberculosis is an infectious disease caused by the M. tuberculosis complex, primarily affecting various organs throughout the body, with the lungs being the most common site (Daniel, 2006). This disease is one of the leading infectious diseases worldwide, with extremely high incidence and mortality rates, claiming over one million lives each year (Glaziou et al., 2018). According to the World Health Organization’s (WHO) 2022 Global Tuberculosis Report, there were 10.6 million new cases of tuberculosis in 2021, with an estimated 1.6 million deaths (Daley, 2019). Tuberculosis remains one of the significant epidemics among the top ten causes of death globally. Thus, the pharmacokinetics of drugs related to the treatment of tuberculosis will continue to be a focus of pharmacological research for an extended period.

Antituberculosis medications are broadly divided into first-line and second-line drugs. First-line agents include isoniazid, Rifampin, pyrazinamide, ethambutol, rifabutin, and rifapentine, each possessing a distinct mechanism of action. Notably, rifapentine’s pharmacological action is characterized by its binding to the subunit of DNA-dependent RNA polymerase, which inhibits bacterial RNA synthesis, halts the RNA transcription process, and ceases the synthesis of DNA and proteins, while having no impact on RNA polymerase in human and animal cells (Brodolin, 2014). Animal studies have demonstrated that this drug exhibits certain hepatotoxic effects and may have teratogenic impacts on fetuses (Jenkin, 2017). Recently, rifapentine has garnered considerable attention in clinical settings due to its pronounced antimicrobial properties and relatively low resistance rates (Rosenthal et al., 2007; Daniel et al., 2000; Conte et al., 2000).

Research indicates substantial interindividual variability in the bioavailability of rifapentine during administration (Francis et al., 2019; Savic et al., 2014; Weiner et al., 2004). The absorption of the drug in the gastrointestinal tract is slow and incomplete, influenced significantly by the presence of food. For example, bioavailability increases by 55% when a 600 mg tablet is taken with food, compared to fasting, with corresponding increases of 44% and 43% in peak concentration (Cmax) and area under the curve (AUC0-∞), respectively (Keung et al., 1995). Volunteer studies have also recorded the time to reach peak concentration (Tmax) as ranging between 4.8 and 6.6 h (Jarvis and Lamb, 1998; Temple and Nahata, 1999). The drug’s protein binding rate exceeds 98% (Keung et al., 1998), and its oral half-life ranges from 14 to 16 h (Egelund et al., 2014). In vivo, rifapentine is predominantly distributed in the liver, with secondary distribution in the kidneys and high concentrations in other tissues, though it poorly penetrates the blood-brain barrier (Zurlinden et al., 2016). In the liver, rifapentine undergoes deacetylation by esterase to form 25-desacetyl rifapentine. This metabolite deacetylates more slowly than Rifampin, significantly reducing protein binding and resulting in the formation of inactive 3-formylrifamycin upon hydrolysis (Langdon et al., 2005). The drug and its metabolites primarily undergo hepatic-intestinal recycling; some are excreted into the intestine through bile, where they can be reabsorbed and then expelled with the feces, with only a minor portion eliminated via urine (Reith et al., 1998). Moreover, as rifapentine is a hepatic enzyme inducer, it accelerates the metabolism of itself and several other medications, requiring careful monitoring of drug interactions during clinical combination therapies and home medication management (Ling et al., 2023; Zheng et al., 2017).

In conclusion, rifapentine is a crucial antituberculosis drug, making the study of its pharmacokinetics in the human body especially important. Given the limited public data on its bioequivalence studies, this research conducted a single-dose cross-over oral trial with rifapentine capsules in healthy adult males, using both the test and reference formulations. By estimating the pharmacokinetic parameters and assessing bioavailability, this study evaluates the bioequivalence of the test drug to the reference, aiming to provide cost-effective, high-quality options for patient treatment. Additionally, this project has developed a rapid method to detect rifamycin-class drugs in human plasma, offering a benchmark for the rapid testing of similar medications.

Participants were instructed to fast overnight for more than 10 h. The following morning at 7:00 a.m., they administered the prescribed medication on an empty stomach, accompanied by 250 mL of warm water. Each capsule contained 0.15 g of the formulation. The dosage was established at 0.6 g per dose (4 capsules), based on the package insert of the reference formulation and a thorough review of clinical study data for the test formulation (Jarvis and Lamb, 1998; Dooley et al., 2008; Weiner et al., 2004; Bock et al., 2002).

The study was designed as a single-center, open-label, randomized trial. Participants were divided into two groups, A and B, with 10 individuals in each group. A two-period crossover design was used, with specific dosing protocols detailed in Supplementary Table S1. Participants were instructed not to consume alcohol, caffeinated beverages, or juice the day before and during the trial. After fasting for 10 h, participants took their assigned medication on an empty stomach the following morning. Group A took four capsules of the test formulation of rifapentine, and Group B took four capsules of the reference formulation of rifapentine, each with 250 mL of warm water. Participants were not allowed to drink water within the first 2 h after dosing and were provided a standardized meal (low-fat diet) 4 h post-dose. After a 1-week washout period, the groups switched medications. Each participant in the same group took their medication at 2-min intervals, and blood samples were also drawn at 2-min intervals. Blood samples were protected from light, placed in an ice bath, and centrifuged quickly for plasma separation, then stored at −70°C. Based on the results of a preliminary trial, 4 mL of blood was drawn from the antecubital vein into a heparinized tube, centrifuged at 4°C at 4,000 r/min for 10 min, and the plasma was then stored at −70°C for future analysis. The washout period lasted for 7 days, with the second cycle of medication administration and blood sampling mirroring the first cycle.

Accurately dispense 100 μL of mixed blank plasma from six different sources and process following the 'Plasma Sample Processing Method,’ beginning with 'vortex for 30 s,’ to produce Figure 2A. Introduce 10 μL of a 160 μg/mL rifapentine standard solution and 10 μL of the internal standard solution, both diluted with 100 μL acetonitrile, to generate Figure 2B. Add rifapentine standard solution to the blank plasma to achieve a 16 μg/mL drug-containing plasma concentration, and process as per the 'Plasma Sample Processing Method’ to obtain Figure 2C. Collect plasma samples from Subject No. 1, 4 hours post-drug administration, and follow the 'Plasma Sample Processing Method’ to produce Figure 2D. Rifapentine’s retention time is approximately 0.82 min, and the internal standard, rifampin, is around 0.62 min. The results confirm that endogenous substances in the plasma do not interfere with the quantification of rifapentine and the internal standard.

Figure 2. UPLC chromatogram of Rifapentine in plasma. (A) Chromatogram of the blank plasma sample. (B) Chromatogram of rifapentine standard (16 μg/mL) and I.S. rifampin standard (62.2 μg/mL). (C) Chromatogram after adding rifapentine (16 μg/mL) and rifampin standard (62.2 μg/mL) to blank plasma. (D) Chromatogram of rifapentine and rifampin in plasma from subject No. 1, 4 h after oral administration of rifapentine capsules.

Accurately dispense 10 μL of rifapentine standard solutions at various concentrations into seven blank centrifuge tubes, and add 100 μL of blank plasma to create plasma samples containing rifapentine at concentrations of 0.05, 0.25, 0.50, 1.00, 5.00, 10.00, and 20.00 mg per liter. Proceed according to the 'Plasma Sample Processing Method,’ starting from the step where 10 μL of the rifampin internal standard solution (622 μg/mL) is precisely added. Analyze the samples to establish a standard curve for rifapentine. The concentration of the test substance (X, μg/mL) is plotted on the x-axis, and the ratio of the peak area of the test substance (As) to that of the internal standard (Ai) (As/Ai) is plotted on the y-axis, represented as Y. Linear regression is performed using a weighting factor (weight coefficient: 1/X2) to obtain the linear equation for the standard curve. The average standard curve results, validated over 3 days, are presented in Supplementary Tables S2–S4, and Supplementary Figure S1, which includes three standard curves per day. The findings confirm that rifapentine maintains a robust linear relationship within the concentration range of 0.05–20.00 μg/mL, encompassing the plasma concentrations of rifapentine in humans, with a quantitation limit of 0.05 μg/mL (S/N > 3).

Take 100 μL of blank plasma and add 10 μL of a 0.5 μg/mL rifapentine standard solution to prepare a sample with a final rifapentine concentration of 0.05 μg/mL in the plasma. On the second day of method validation, analyze six samples and calculate the concentration of each sample using the day’s standard curve. The data are presented in Supplementary Table S5. The results demonstrate that the UPLC-UV method can quantify rifapentine in plasma with a lower limit of quantification (LLOQ) of 0.05 μg/mL.

Dispense 10 μL of rifapentine standard solution at varying concentrations into separate blank centrifuge tubes, then add 100 μL of blank plasma to prepare samples with drug concentrations of 0.1, 2.5, and 16 μg/mL, representing low, medium, and high concentration levels in the plasma. Follow the 'Plasma Sample Processing Method’ for preparation. Conduct this preparation and analysis across three analytical batches on different days, with each batch containing low, medium, and high concentration levels, analyzing six samples per concentration. Calculate the intra-day and inter-day relative standard deviation (RSD), which was found to be less than 10%. The results are documented in Supplementary Table S6.

Begin by adding 100 μL of blank plasma into each empty centrifuge tube, followed by the precise addition of 10 μL of rifapentine standard solution at various concentrations. Proceed according to the 'Standard Curve and Lower Limit Quantitation Plasma Sample Handling Method’ to prepare samples at low, medium, and high concentrations. Conduct three analyses per concentration, determining the respective drug peak area, As(H), and the internal standard peak area, Ai(H). Subsequently, mix 10 μL of rifapentine standard solution at varying concentrations with 10 μL of internal standard solution, dilute with acetonitrile, and perform an injection analysis to ascertain the corresponding drug peak area As(D) and internal standard peak area Ai(D). The extraction recovery rates are calculated as follows: for the drug, As(H)/As(D) × 100%; for the internal standard, Ai(H)/Ai(D) × 100%. These results are detailed in Supplementary Table S7, which shows that the extraction recovery rates for low, medium, and high concentrations range from 102.60% to 109.55%, and the internal standard recovery rates vary from 99.19% to 112.04%.

Start by adding 10 μL of rifapentine standard solution at different concentrations into blank centrifuge tubes, followed by 100 μL of blank plasma to prepare plasma samples with drug concentrations of 0.10 and 16.00 μg/mL at low and high levels, respectively. Prepare five sets of these concentrations, with each set comprising three samples. For one set, follow the 'Plasma Sample Processing Method’ and analyze the samples after leaving them at room temperature for 0, 24, and 36 h to evaluate the stability of plasma samples under room temperature conditions. Also assess the stability of plasma samples after thawing for 8 h. Another set is to be frozen at −70°C and subjected to three freeze-thaw cycles, then processed and analyzed according to the 'Plasma Sample Processing Method’ to evaluate the stability after repeated freeze-thaw cycles. Additionally, freeze two more sets at −70°C and analyze them after 15 and 30 days, respectively, following the 'Plasma Sample Processing Method’ to assess the long-term stability under freezing conditions, with each set analyzed in triplicate. Concurrently, evaluate the stability of the rifapentine standard solution after remaining at room temperature for 8 h and after being frozen at −20°C for 30 days. The results, as shown in Supplementary Tables S8, S9, indicate an RSD of less than 10%, demonstrating good stability of rifapentine under room temperature, repeated freeze-thaw, and long-term freezing conditions.

To reduce systematic errors, an accompanying standard curve is generated for each batch of sample analyses to calculate the concentrations of medication in the blood. This curve is constructed using a weighted regression method (W = 1/X2). Additionally, quality control samples at low, medium, and high concentrations—0.1, 2.5, and 16 μg/mL, respectively—are measured. The results can be found in Supplementary Tables S10, S11.

Nineteen participants orally administered 0.6 g of either the test or the reference formulation of rifapentine capsules. The rifapentine plasma concentration-time data were obtained using UPLC, as shown in Supplementary Tables S12, S13. Plasma concentration-time curves for the nineteen healthy participants who took the reference formulation can be found in Figure 3, while those for the test formulation are presented in Figure 4, and a comparison of the mean values is depicted in Figure 5.

Figure 3. Plasma concentration-time curve of the drug after 19 subjects orally administered the reference formulation.

Figure 4. Plasma concentration-time curve of the drug after 19 subjects orally administered the test formulation.

Figure 5. Average plasma concentration-time curves of the drug after 19 subjects orally administered either the test formulation or the reference formulation.

Cmax and Tmax are measured values. Other parameters are calculated using Equations 1, 2. The results are presented in Table 1 and 2. Relative bioavailability is calculated according to Equation 3. The ratio of AUC0-84, denoted as F, is used as the numeric value for relative bioavailability, and the ratio of AUC0-∞, denoted as F′, is used as a reference value. The results for F and F′ are shown in Table 3. The variance analysis results for ln AUC0-84, ln AUC0-∞, and ln Cmax between the two formulations are presented in Tables 4–6. These results indicate no statistically significant differences between the formulations for ln AUC0-84, ln AUC0-∞, and ln Cmax; no statistically significant period differences for ln Cmax; however, significant period differences exist for ln AUC0-84 and ln AUC0-∞ (P < 0.05); and highly significant inter-individual differences for ln AUC0-84, ln AUC0-∞, and ln Cmax (P < 0.001). The results of the two-sided one-sample t-tests and the [1-2α]% confidence interval analysis for ln AUC0-84, ln AUC0-∞, and ln Cmax are shown in Tables 7–9. The results in Tables 7, 8 indicate that the null hypothesis H0 for the pharmacokinetic parameters ln AUC0-84 and ln AUC0-∞ of the two formulations is rejected in favor of the alternative hypothesis H1, and the results demonstrate that the [1-2α]% confidence intervals for ln AUC0-84 and ln AUC0-∞ fall within the 80%–125% range, indicating bioequivalence in terms of absorption between the two formulations. Table 9 shows that the null hypothesis H0 for the pharmacokinetic parameter ln Cmax is rejected in favor of H1, and the results indicate that the [1-2α]% confidence interval for ln Cmax falls within the 70%–143% range, suggesting bioequivalence in peak concentration between the two formulations. The non-parametric test results for Tmax are presented in Table 10.

Table 1. Pharmacokinetic parameters of the 0.6 g reference formulation of oral rifapentine capsules in 19 subjects.

Table 2. Pharmacokinetic parameters of the 0.6 g test formulation of oral rifapentine capsules in 19 subjects.

Table 3. Relative bioavailability and Cmax percentages (CT/CR) for test formulation (T) and reference formulation (R).

Table 4. Analysis of Variance Results for lnAUC0-84 Between Two Formulations.

Table 5. Analysis of Variance Results for lnAUC0-∞ Across Two Formulations.

Table 6. Analysis of Variance Results for ln Cmax Between Two Formulations.

Table 7. Equivalence Analysis for ln AUC0-84 Using Two Sided One Sample t-Test.

Table 8. Equivalence Analysis for ln AUC0-∞ Using [1-2α]% Confidence Interval Method.

Table 9. Equivalence Analysis for ln Cmax Using Two Way One Sided t-Test.