Target: Bacterial RNA polymerase (inferred from rifamycin class mechanism; no specific IC50/Ki provided in this study) [1]
MIC for intracellular M. tuberculosis: 0.015-0.06 μg/mL (depending on strain); MBC: 0.06-0.5 μg/mL [1]
MIC for extracellular M. tuberculosis: 0.06 μg/mL for H37Rv and Erdman, 0.06 for Atencio; MBC: 0.25-0.5 μg/mL [1]
The primary molecular target of Rifapentine is bacterial DNA-dependent RNA polymerase (RNAP). Rifapentine, like other rifamycins, binds to the β-subunit of bacterial RNA polymerase, specifically to a pocket in the RNAP active site that is distinct from the catalytic site. By binding to RNAP, Rifapentine inhibits the initiation of RNA synthesis, blocking the transcription of bacterial genes. This prevents the synthesis of essential proteins and leads to bacterial cell death. The compound's high activity against M. tuberculosis is attributed to its potent inhibition of the mycobacterial RNAP. Rifapentine's activity against intracellular bacteria is particularly important for the treatment of tuberculosis, as M. tuberculosis can survive and replicate inside macrophages. The compound's metabolism to 25-O-desacetylrifapentine, which retains about half of the parent compound's activity, contributes to its prolonged antibacterial effect.

The activities of rifampin and rifapentine against Mycobacterium tuberculosis residing in human monocytederived macrophages were determined. The MIC and MBC of rifapentine for intracellular bacteria were two- to four-fold lower than those of rifampin. For extracellular bacteria, this difference was less noticeable.
Cell Assay: Rifapentine inhibits the function of DNA-dependent RNA polymerase in strains of M. tuberculosis, while inducing no effect on mammalian cells. Both Rifapentine and its active metabolite, 25-desacetylrifapentine, localize within monocyte-derived macrophages, thus allowing for intracellular inhibition of M. tuberculosis at a greater kill rate as compared with that of the parent or metabolite alone. Rifapentine is deacetylated in the liver and induces cytochrome P450 much less than rifampin. Rifapentine has shown higher bacteriostatic and bactericidal activities (MICs and MBCs) than RMP, especially against intracellular bacteria growing in human monocyte-derived macrophages.

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In Vitro: Rifapentine (DL 473) MICs against extracellular M. tuberculosis were 0.06 μg/mL for H37Rv and Erdman strains, and 0.06 μg/mL for Atencio strain; MBCs were 0.25-0.5 μg/mL. For intracellular bacteria in human macrophages, MICs were 0.015-0.06 μg/mL and MBCs were 0.06-0.5 μg/mL, which were two- to eight-fold lower than those of rifampin. [1]
The metabolite 25-desacetyl-rifapentine had MICs of 0.06-0.12 μg/mL (extracellular) and 0.03-0.12 μg/mL (intracellular); MBCs of 0.5-1.0 μg/mL (extracellular) and 0.25-1.0 μg/mL (intracellular). Combination of rifapentine and its metabolite showed additive effects with fractional inhibitory concentration coefficients of 0.75-1 and fractional bactericidal concentration coefficients of 1-1.5. [1]
Intracellular accumulation: In human monocytes, the intracellular/extracellular concentration ratio for rifapentine was 24.0 ± 3.9 (mean), for its metabolite 7.2 ± 2.4, and for rifampin 4.4 ± 1.9. This ratio was independent of extracellular concentration (10, 20, 40 μg/mL). [1]
After a single 2-hour pulsed exposure of infected macrophages to 3.0 μg/mL of rifapentine, the number of viable M. tuberculosis (H37Rv) decreased from 6.6×10³ CFU per 2×10⁵ macrophages on day 0 to 1.0×10² on day 4, 4.3×10¹ on day 7, and 3.0×10¹ on day 14. For Erdman strain: from 1.0×10⁴ to 2.1×10², 4.5×10², 3.6×10² respectively. For Atencio strain: from 9.7×10³ to 1.2×10², 2.6×10¹, 7.9×10². This effect was greater than that of rifampin at the same concentration for two of three strains. [1]
When infected macrophages were exposed for 72 hours to changing concentrations mimicking human pharmacokinetics after one 600 mg dose of rifapentine (max 17.25 μg/mL, area under curve 474 μg·h/mL, half-life 14.54 h), the number of viable bacteria decreased by 2-3 log₁₀ units during the first 7 days, with some rebound on days 14 and 21. This effect was superior to one or three daily doses of rifampin mimicking human pharmacokinetics. [1]
When weekly 72-hour exposures to rifapentine (changing concentrations mimicking blood levels) were repeated for 4 weeks, the initial decline was maintained without rebound, and only a few colonies were isolated by day 28. [1]
In vitro antibacterial activity of Rifapentine is characterized by its potency against M. tuberculosis and other mycobacteria. Against M. tuberculosis H37Rv, Rifapentine has MIC values of 0.015-0.06 μg/mL, which are 5-10 times lower than those of rifampicin. Against other mycobacteria, including M. avium complex and M. kansasii, Rifapentine also shows potent activity. The compound is active against both extracellular and intracellular bacteria, with activity against intracellular bacteria being particularly important for the treatment of tuberculosis. Rifapentine also shows activity against Gram-positive bacteria, including Staphylococcus aureus and Streptococcus pneumoniae, and against Neisseria gonorrhoeae. The compound's activity is time-dependent and is affected by the pH and the presence of serum proteins. Rifapentine shows synergistic activity with other antituberculosis drugs, including isoniazid, pyrazinamide, and ethambutol. Resistance to Rifapentine is mediated by mutations in the rpoB gene, which encodes the β-subunit of RNA polymerase.

Rifapentine inhibits bacterial RNA synthesis by binding to the β-subunit of DNA-dependent RNA polymerase in susceptible species. Rifapentine is generally more active than rifampicin against sensitive strains of M. tuberculosis. Rifapentine significantly increases the rate of antipyrine and pentobarbital metabolism in vivo. Rifapentine also increases liver weight, the content of liver microsomal protein and cytochrome P-450, the activity of NADPH-cytochrome C reductase and NADPH oxidase. Rifapentine combined with isoniazid (INH) and pyrazinamide (PZA) administered daily results in an apparent clearance of M.tuberculosis organisms in the lungs and spleens of infected mice after 10 weeks of treatment
In vivo activity of Rifapentine has been demonstrated in animal models and in clinical use for the treatment of tuberculosis. In mouse models of tuberculosis, Rifapentine administered orally at doses of 10-20 mg/kg shows superior efficacy compared to rifampicin, with more rapid bacterial clearance and shorter treatment duration required. In clinical studies, Rifapentine is used for the treatment of tuberculosis, including both drug-susceptible and drug-resistant forms. The compound is typically administered once weekly during the continuation phase of tuberculosis treatment, in combination with isoniazid, due to its long half-life. Rifapentine is also used for the treatment of latent tuberculosis infection, with a shorter and more convenient dosing regimen compared to other agents. The compound's high activity against intracellular bacteria makes it particularly effective for the treatment of tuberculosis. The compound's in vivo efficacy depends on achieving adequate plasma concentrations and penetration into tissues, including the lungs and macrophages.

For in vitro antibacterial susceptibility testing with Rifapentine, the following protocol is used following CLSI guidelines: M. tuberculosis isolates are cultured in Middlebrook 7H9 broth supplemented with 10% OADC and 0.05% Tween 80 at 37°C. For MIC determination, Rifapentine is serially diluted two-fold in 96-well plates in 7H9 broth to achieve final concentrations ranging from 0.001 to 1 μg/mL. The bacterial suspension is added to each well to achieve a final inoculum of approximately 5 × 10⁵ CFU/mL. The plates are incubated at 37°C for 7-14 days. The MIC is determined as the lowest concentration of the compound that completely inhibits visible bacterial growth. For time-kill assays, bacteria are treated with Rifapentine at concentrations of 1-4 × MIC, and aliquots are plated on 7H10 agar at various time points for CFU enumeration. For assessment of intracellular activity, human monocyte-derived macrophages are infected with M. tuberculosis, treated with Rifapentine, and bacterial survival is assessed by CFU enumeration after macrophage lysis.

Cell Assay: Human monocyte-derived macrophages were obtained by Ficoll-Hypaque gradient centrifugation from peripheral blood. Monocytes were adhered to plastic petri plates and cultivated for 7 days in RPMI 1640 with 5% unheated human serum to mature into macrophages. Macrophage monolayers were infected with M. tuberculosis suspension at ~10⁶ CFU/mL to achieve ~5 bacteria per macrophage. After 1-hour incubation, extracellular bacteria were washed off. Infected macrophages were incubated in RPMI 1640 with 1% human serum at 37°C in 7% CO₂. For MIC/MBC determinations, infected macrophages were exposed to various drug concentrations for 8 days. Viable counts were determined on days 0, 4, and 8 by plating serial dilutions of macrophage lysates on 7H11 agar. [1]
For pulsed exposure experiments, infected macrophages were exposed to 3.0 μg/mL of rifapentine for 2 hours, then medium was replaced with drug-free medium and incubated for 2 weeks. Samples were taken on days 0, 4, 7, and 14 for CFU determination. [1]
For pharmacokinetic simulation experiments, infected macrophages were exposed to changing concentrations of rifapentine and its metabolite for 72 hours (first 72 hours of each week). The schedule for rifapentine: 12.0 μg/mL for 18 h, then 7.0 μg/mL for 24 h, then 3.0 μg/mL for 30 h. For the metabolite: 5.0 μg/mL for 42 h then 3.0 μg/mL for 30 h. After exposure, cells were kept in drug-free medium. CFU were determined on days 0, 3, 7, 14, 21, and 28. [1]
For determining intracellular concentrations, monocytes were purified by adherence, detached, and adjusted to 2×10⁷ cells/mL. Drugs at 10, 20, or 40 μg/mL were added and incubated for 2 h at 37°C. Cells were separated from extracellular fluid by velocity gradient centrifugation using oil (DC550 and paraffin oil). Cell pellets were resuspended, lysed by sonication, and drug concentrations measured by bioassay using Micrococcus luteus on antibiotic agar. Intracellular concentration was calculated based on cell volume (421 μm³ per monocyte). [1]
For in vitro cell-based assays with Rifapentine, the following typical protocol is used: For cytotoxicity assessment, mammalian cells (e.g., HepG2 liver cells, THP-1 macrophages) are cultured in appropriate media at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 5,000-10,000 cells per well and allowed to adhere overnight. Rifapentine is dissolved in DMSO and diluted in culture medium to final concentrations ranging from 0.01 to 100 μg/mL. Cells are treated for 24-72 hours. Cell viability is assessed using the MTT or CellTiter-Glo assay to determine the CC50. For assessment of immune modulation, macrophages are treated with Rifapentine and stimulated with LPS, and cytokine production (TNF-α, IL-6, IL-10) is measured by ELISA. For assessment of intracellular activity, macrophages are infected with M. tuberculosis and treated with Rifapentine, and bacterial survival is assessed by CFU enumeration. For assessment of hepatic metabolism, hepatocytes or liver microsomes are incubated with Rifapentine, and the formation of 25-O-desacetylrifapentine is measured by HPLC or LC-MS/MS.

For in vivo animal studies with Rifapentine, the following general protocol is used: For tuberculosis efficacy studies, female BALB/c mice (6-8 weeks old, 18-22 g) are infected intranasally or by aerosol with M. tuberculosis H37Rv (10⁵-10⁶ CFU). After 3-4 weeks of infection, mice are randomized into treatment groups (n=8-10 per group). Rifapentine is formulated in a suitable vehicle (e.g., 0.5% methylcellulose) and administered orally at doses of 5, 10, 20, and 40 mg/kg, once daily or once weekly, for 4-8 weeks. Control groups receive vehicle only or standard tuberculosis drugs (rifampicin, isoniazid). At the end of the treatment, mice are euthanized, and the lungs and spleen are collected for bacterial CFU enumeration. Lung tissue is also processed for histopathological examination (H&E and acid-fast staining). For pharmacokinetic studies, blood samples are collected at various time points after dosing, and plasma concentrations of Rifapentine and its metabolite 25-O-desacetylrifapentine are analyzed by LC-MS/MS.

Absorption, Distribution and Excretion
Rapid and adequate absorption from the gastrointestinal tract. Following a single oral dose of 600 mg of radiolabeled rifapentine in healthy volunteers (n=4), 87% of the 14C-labeled rifapentine was recovered from urine (17%) and feces (70%). 70.2 ± 9.1 L Apparent oral clearance = 2.51 ± 0.14 L/h [male tuberculosis patients treated with 600 mg rifapentine in combination with isoniazid, pyrazinamide, and ethambutol] Apparent oral clearance = 1.69 ± 0.41 L/h [female tuberculosis patients treated with 600 mg rifapentine in combination with isoniazid, pyrazinamide, and ethambutol] [ethambutol] Metabolisms/Metabolites Liver

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ADME/Pharmacokinetics: The pharmacokinetic parameters used to simulate human blood concentrations after one 600 mg oral dose of rifapentine were: maximum concentration 17.25 μg/mL for rifapentine and 5.73 μg/mL for its metabolite 25-desacetyl-rifapentine; area under the curve 474 μg·h/mL for rifapentine and 257 μg·h/mL for the metabolite; elimination half-life 14.54 h for rifapentine and 13.23 h for the metabolite. [1]
The intracellular/extracellular concentration ratio for rifapentine in human monocytes was 24.0 ± 3.9, indicating high intracellular accumulation. [1]
The pharmacokinetic properties of Rifapentine are characterized by its long half-life, which allows for once-weekly dosing. After oral administration, Rifapentine is absorbed with a bioavailability of approximately 70-80% in humans. Peak plasma concentrations are reached within 5-6 hours (Tmax). The compound has a large volume of distribution (approximately 2-5 L/kg), indicating extensive tissue distribution. Plasma protein binding is very high (>97%). The elimination half-life is approximately 14-18 hours in humans, which is significantly longer than that of rifampicin (3-4 hours). Rifapentine is metabolized primarily in the liver by deacetylation to 25-O-desacetylrifapentine, which retains about half of the parent compound's activity. The compound and its metabolite are excreted primarily in the feces (approximately 50-60%) and urine (approximately 20-30%). The compound's pharmacokinetics are dose-dependent, with nonlinearity observed at higher doses due to saturation of clearance pathways. Rifapentine is a potent inducer of cytochrome P450 enzymes (CYP3A4), which can cause drug-drug interactions with other medications.

Hepatotoxicity
Due to the limited use of rifapentine, its effects on the liver are not as well-defined as those of rifampin, but may be similar. Therefore, long-term use of rifapentine can lead to mild, transient increases in serum transaminase levels in 2% to 7% of patients; these abnormalities usually do not require dose adjustment or discontinuation. There are currently no reports of clinically significant liver injury caused by rifapentine, but its potential to cause acute liver injury may be similar to that of rifampin. Because rifapentine is often used in combination with isoniazid and/or pyrazinamide (two other known hepatotoxic drugs), the cause of acute liver injury in patients treated with rifapentine may be difficult to determine as being caused by a single drug; some evidence suggests that these combination therapies are more likely to cause liver injury than either drug alone. Typically, rifamycin-induced liver injury develops within 1 to 6 weeks, with an initial serum enzyme profile that is usually hepatocellular, but unlike isoniazid and pyrazinamide, cholestatic and mixed patterns may also occur. Extrahepatic manifestations of rifapentine hepatotoxicity, such as fever, rash, arthralgia, edema, eosinophilia, and autoantibody formation, are uncommon. Rifapentine has not been proven to be hepatotoxic. Probability score: E (Unproven, but suspected cause of clinically significant liver injury). Pregnancy and Lactation Use ◉ Overview of Lactation Use
The amount of rifapentine and its metabolites in breast milk is insufficient to treat tuberculosis in breastfed infants. The US Centers for Disease Control and Prevention and other professional agencies state that women taking rifapentine should not be discouraged from breastfeeding. Infants should be monitored for signs of hepatotoxicity. Breast milk may appear reddish-orange. ◉ Effects on Breastfed Infants
No published information found as of the revision date. ◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein binding rate
97.7% (bound to plasma proteins)

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The toxicity profile of Rifapentine is well-established from clinical use. Common side effects include gastrointestinal disturbances (nausea, vomiting, diarrhea), headache, and rash. Hepatotoxicity (elevated liver enzymes) is the most significant adverse effect and is more common with Rifapentine than with rifampicin. The risk of hepatotoxicity is increased in patients with pre-existing liver disease, alcoholism, and concomitant use of other hepatotoxic drugs. Rifapentine can also cause flu-like syndrome, hemolytic anemia, thrombocytopenia, and renal impairment. The compound is a potent inducer of CYP3A4 and can reduce the efficacy of other drugs metabolized by this enzyme, including oral contraceptives, anticoagulants, and antiretrovirals. Rifapentine is contraindicated in patients with known hypersensitivity to rifamycins. The compound should be used with caution in patients with hepatic impairment and in pregnant women. Monitoring of liver function is recommended during therapy.

Ann Pharmacother.1999 Nov;33(11):1203-10;Antimicrob Agents Chemother.1995Sep;39(9):2073-7;Drugs.1998 Oct;56(4):607-16; discussion 617.

Rifapentine is an N-alkylpiperazine, N-iminopiperazine compound belonging to the rifamycin class. It is an anti-tuberculosis drug and a treatment for leprosy. Rifapentine is a prescription antibacterial drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of active pulmonary tuberculosis (also known as tuberculosis). Rifapentine is also FDA-approved for the treatment of latent tuberculosis infection to prevent the development of active tuberculosis. Tuberculosis can be an opportunistic infection of HIV. Rifapentine is an antibiotic used to treat tuberculosis. It works by inhibiting the activity of DNA-dependent RNA polymerase in susceptible cells. Specifically, it interacts with bacterial RNA polymerase but does not inhibit mammalian enzymes. Rifapentine is a rifamycin-based antimycobacterial drug. Rifapentine is a rifamycin antibiotic with a structure and activity similar to rifampin and rifabutin, and is often used in combination with other drugs to treat tuberculosis, especially with once-weekly or twice-weekly dosing regimens. Rifapentine is associated with transient and asymptomatic elevations in serum transaminases, which may be one of the causes of clinically apparent acute liver injury. Rifapentine is a long-acting cyclopentyl-substituted derivative of rifamycin, used to treat mycobacterial infections. See also: Rifapentine hydrochloride (its active ingredient). Indications: For the treatment of pulmonary tuberculosis. Mechanism of Action: Rifapentine has high antibacterial and bactericidal activity, particularly effective against bacteria growing within macrophages derived from human monocytes. Rifapentine inhibits DNA-dependent RNA polymerase in susceptible strains of Mycobacterium tuberculosis. Rifapentine works by inhibiting DNA-dependent RNA polymerase, leading to RNA synthesis inhibition and cell death. Pharmacodynamics: Rifapentine is an antibiotic that inhibits the activity of DNA-dependent RNA polymerase in susceptible cells. Specifically, it interacts with bacterial RNA polymerase but does not inhibit mammalian RNA polymerase. It has bactericidal activity and broad-spectrum antibacterial activity against most Gram-positive and Gram-negative bacteria (including Pseudomonas aeruginosa) and Mycobacterium tuberculosis. Due to the rapid emergence of resistant bacteria, its use is limited to the treatment of mycobacterial infections and a few other indications. Rifampin is well absorbed after oral administration and widely distributed in tissues and fluids throughout the body, including cerebrospinal fluid. It is metabolized in the liver and excreted primarily via bile, with a small amount excreted in the urine; however, no dose adjustment is necessary for patients with renal insufficiency.

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Additional Info: Rifapentine (DL 473) is a cyclopentyl rifamycin derivative with a longer elimination half-life (about five times longer than rifampin) and higher intracellular accumulation in macrophages. The study demonstrated that one dose of rifapentine had a greater long-lasting effect against intracellular M. tuberculosis than one or three daily doses of rifampin in a human macrophage model mimicking pharmacokinetic curves. Weekly exposure to rifapentine for 4 weeks maintained bacterial decline without rebound, supporting its potential for intermittent treatment regimens (e.g., once-weekly administration) and possibly ultrashort therapy for tuberculosis. The metabolite 25-desacetyl-rifapentine also had activity, but did not enhance rifapentine’s effect in combination. [1]
Rifapentine (DL 473) (CAS# 61379-65-5) is a semisynthetic rifamycin antibiotic for tuberculosis and leprosy. It has a molecular formula of C47H64N4O12 and a molecular weight of 877.03 g/mol. It is 10 times more active than rifampicin against M. tuberculosis. Future research could focus on optimizing dosing regimens to reduce toxicity and improve adherence, developing new rifamycin derivatives with improved activity and safety profiles, and investigating the use of Rifapentine in combination with other agents for the treatment of drug-resistant tuberculosis.

C47H64N4O12

877.03

876.452

C, 64.37; H, 7.36; N, 6.39; O, 21.89

61379-65-5

Rifapentine-d9

135403821

Solid powder

1.4±0.1 g/cm3

969.3±65.0 °C at 760 mmHg

179-180ºC

540.0±34.3 °C

0.0±0.3 mmHg at 25°C

1.625

2.58

6

15

6

63

1730

9

OC(C1=C(C2=O)C(O[C@@]2(O/C=C/[C@@H]([C@H]([C@@]([C@@H]3C)([H])OC(C)=O)C)OC)C)=C(C)C(O)=C1C(O)=C4NC(/C(C)=C\C=C\[C@@H]([C@@H]([C@@H](C)[C@H]3O)O)C)=O)=C4/C=N/N5CCN(C6CCCC6)CC5

WDZCUPBHRAEYDL-GZAUEHORSA-N

InChI=1S/C47H64N4O12/c1-24-13-12-14-25(2)46(59)49-37-32(23-48-51-20-18-50(19-21-51)31-15-10-11-16-31)41(56)34-35(42(37)57)40(55)29(6)44-36(34)45(58)47(8,63-44)61-22-17-33(60-9)26(3)43(62-30(7)52)28(5)39(54)27(4)38(24)53/h12-14,17,22-24,26-28,31,33,38-39,43,53-57H,10-11,15-16,18-21H2,1-9H3,(H,49,59)/b13-12+,22-17+,25-14-,48-23+/t24-,26+,27+,28+,33-,38-,39+,43+,47-/m0/s1

3-(N-(4-Cyclopentyl-1-piperazinyl)formimidoyl)rifamycin

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 50 ~100 mg/mL ( 57.01 ~114.02 mM )
H2O : ~0.67 mg/mL (~0.76 mM)
Ethanol : ~10 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (2.85 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)