PARP1 (IC50 = 3.2 nM) and PARP2 (IC50 = 1.9 nM).

In 48 hours, mefuparib hydrochloride (1–10 μM) causes cellular trafficking [1]. V-C8 enters cells through the normal G2/M phase when exposed to mefuparib hydrochloride (MPH; 1–10 μM; 24 hours) [1]. In MDA-MB-436 (BRCA1/) cells, mefuparib hydrochloride (1–10 μM; 24 hours) induces DSB accumulation and concentration-dependently increases γH2AX levels [1]. The efficient inhibition of magnetic field drift effect by hexane has an average IC50 of 2.16 μM (0.12 μM~3.64 μM)[1]. Mefuparib hydrochloride inhibits TNKS1 (IC50 = Apoptosis Assay), PARP3 (IC50 > 10 μM), and PARP6 (IC50 > 10 μM) [1].

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Mefuparib hydrochloride (MPH) is a novel and potent PARP1/2 inhibitor with IC50 values of 3.2 nM for PARP1 and 1.9 nM for PARP2. It displays excellent selectivity, showing >406-fold selectivity over other PARP isoforms including PARP3, TNKS1, TNKS2, and PARP6 (IC50 > 10 uM). In homologous recombination (HR)-deficient cells (e.g., BRCA1-mutant MDA-MB-436 cells), mefuparib effectively reduces poly(ADP-ribose) (PAR) formation, a measure of PARP enzyme activity. This PARP inhibition leads to the accumulation of DNA double-strand breaks (DSBs), as evidenced by a concentration-dependent increase in gammaH2AX levels. Consequently, the compound induces a G2/M cell cycle arrest and ultimately triggers apoptosis specifically in HR-deficient cancer cells. Mefuparib demonstrates potent anti-proliferative activity against HR-deficient cancer cell lines (e.g., V-C8, BRCA1-mutant breast cancer) in vitro. Furthermore, it can synergistically sensitize HR-proficient cancer cells to DNA-damaging agents such as temozolomide.

Mefuparib hydrochloride (160 mg/kg; lateral; every other day; for 21 days) inhibited the growth of BR-05-0028 breast patient xenografts (PDX) without causing significant weight loss. This was in addition to the dose- and time-sensitive killing of V-C8 xenografts by mefoparib hydrochloride (MPH; 40-160 mg/kg; Atlantic; every other day; for 21 days). Mefuparib hydrochloride (10, 20, 40 mg/kg; oral) had a Cmax of 116-SD of 725 ng/mL and a T1/2 of 1.07-1.3 hours [1]. Cynomolgus monkeys treated with mefuparib hydrochloride (5, 10 1], 20 mg/kg; oral) have a T1/2 of 2.16-2.7 hours and a C max of 608 ng/mL [1].

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In vivo, mefuparib hydrochloride (MPH) has demonstrated potent anti-tumor activity in various xenograft and patient-derived xenograft (PDX) models. In a study using V-C8 xenografts, mefuparib (administered at 40-160 mg/kg i.p. every other day for 21 days) significantly inhibited tumor growth in a dose- and time-dependent manner. In a BRCA1-mutant breast cancer PDX model (BR-05-0028), oral administration of mefuparib at 160 mg/kg every other day for 21 days effectively suppressed tumor growth without causing significant body weight loss in the treated mice. These results confirm that mefuparib is orally active and can induce tumor regression in HR-deficient tumors. The compound has also shown activity in combination with temozolomide (TMZ) in HR-proficient xenografts, achieving synergistic inhibition of tumor growth. Mefuparib is well-tolerated at therapeutic doses, with minimal signs of off-target toxicity in these models. These in vivo data support its further development as an anti-cancer agent for HR-deficient tumors.

The inhibitory activity of mefuparib HCl against PARP enzymes is determined using a standard biochemical assay that measures the incorporation of biotin-labeled NAD+ into PAR polymer. Recombinant human PARP1 or PARP2 is incubated in a reaction buffer (50 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, and 0.1% Tween-20) with varying concentrations of mefuparib HCl (typically from 0.001 nM to 1 uM) for 10-15 minutes at room temperature. The reaction is initiated by the addition of a mixture containing activated DNA (to stimulate PARP activity) and biotinylated NAD+ (as the substrate). After a 30-60 minute incubation at room temperature, the reaction is stopped by adding a stop solution containing a PARP inhibitor (e.g., 100 uM olaparib). The reaction mixture is then transferred to a streptavidin-coated 96-well plate. The plate is incubated for 1-2 hours to allow the biotin-labeled PAR polymer to bind to the streptavidin. After washing to remove unbound materials, an anti-PAR polyclonal antibody conjugated to a detection enzyme (e.g., HRP) is added. The bound antibody is then quantified using a colorimetric or chemiluminescent substrate. The signal is proportional to the amount of PAR polymer formed. The IC50 is calculated by fitting the concentration-response curve using a four-parameter logistic equation. For the selectivity assessment, the same assay is performed using other PARP family members (e.g., PARP3, TNKS1, TNKS2, PARP6).

Apoptosis Assay [1]
Cell Types: V-C8 Cell
Tested Concentrations: 1, 3, 10 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Causes apoptosis. 1.6 μM), TNKS2 (IC50=1.3 μM)[1].

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Cell cycle analysis[1]
Cell Types: V-C8 Cell
Tested Concentrations: 1, 3, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Cells enter the typical G2/M arrest phase.

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Western Blot Analysis[1]
Cell Types: MDA-MB-436 (BRCA1−/−) Cell
Tested Concentrations: 1, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased γH2AX levels in MDA lead to DSB accumulation MB-436 (BRCA1−/− ) cells are concentration dependent.

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The cellular activity of mefuparib HCl is evaluated using a PAR cellular assay, a cell viability assay, and a DNA damage assay. For the PAR cellular assay, HR-deficient cells (e.g., V-C8 or MDA-MB-436) are seeded in 96-well plates at a density of 10,000-20,000 cells per well in culture medium and allowed to attach overnight. The next day, the medium is replaced with fresh medium containing various concentrations of mefuparib HCl (typically ranging from 0.1 nM to 10 uM) and incubated for 2-4 hours. To stimulate PARP activity and PAR formation, the cells are treated with a sub-lethal dose of a DNA-damaging agent such as 1 mM H2O2 for the last 10 minutes of the drug incubation. The cells are then fixed and permeabilized. PAR levels are quantified using a cellular PAR ELISA kit or by immunofluorescence with an anti-PAR antibody. For cell viability assays, cells are seeded in 96-well plates and treated with various concentrations of mefuparib HCl for 72-120 hours (to allow for the manifestation of synthetic lethality). The half-maximal inhibitory concentration (IC50) for reducing viability is then determined using a CellTiter-Glo luminescent assay. To assess DNA damage, cells are treated with mefuparib for 24 hours, then fixed and stained with an anti-gammaH2AX antibody (a marker of DNA double-strand breaks). The gammaH2AX signal is visualized by fluorescence microscopy or quantified by flow cytometry. Cell cycle analysis is performed by treating cells with mefuparib for 24 hours, then fixing the cells in 70% ethanol, staining with propidium iodide (PI), and analyzing the DNA content via flow cytometry to determine the percentage of cells in G1, S, and G2/M phases. Apoptosis is assessed by staining with Annexin V-FITC and PI and analyzing the stained cells by flow cytometry.

Animal/Disease Models: V-C8 xenograft nude mice [1]
Doses: 40, 80, 160 mg/kg
Route of Administration: oral; once every other day; lasted for 21 days
Experimental Results: V-C8 xenograft demonstrated dose and Time-dependent killing was accompanied by complete disappearance of some xenografts, especially in the high-dose group.

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Animal/Disease Models: SD rat[1]
Doses: 10, 20, 40 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: Oral
Experimental Results:T1/2 is 1.07-1.3 hrs (hrs (hours)), C max is 116-725 ng/ mL.

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The in vivo anti-tumor efficacy of mefuparib HCl is evaluated in mouse xenograft models. For the V-C8 xenograft model, 6-8 week old female BALB/c nude mice are injected subcutaneously in the flank with 5-10 x 10⁶ V-C8 cells in 100 uL of a 1:1 mixture of PBS and Matrigel. Once tumors reach an average size of 100-200 mm3, mice are randomized into treatment groups (n=8-10). Mefuparib HCl is formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline or in 0.5% methylcellulose. The compound is administered via intraperitoneal (i.p.) or oral (p.o.) gavage at various dose levels (e.g., 40, 80, or 160 mg/kg) on a qd, bid, or qod schedule. A control group receives the vehicle alone. Tumor volumes are measured every 2-3 days using a digital caliper, and body weights are recorded to monitor toxicity. At the end of the study, mice are euthanized, and tumors are excised and weighed. Tumor tissues may be analyzed for biomarkers (e.g., PAR levels, gammaH2AX, cleaved caspase-3) by immunohistochemistry or western blot. For combination studies with temozolomide (TMZ), mefuparib is administered p.o. at 40 mg/kg on days 1-5, and TMZ is administered p.o. at 30 mg/kg on day 1 only of a 5-day cycle. Tumor growth inhibition (TGI) is calculated for each group.

The pharmacokinetic (PK) properties of mefuparib HCl have been evaluated in preclinical species, including rats and cynomolgus monkeys. In rats, after oral administration of 10, 20, or 40 mg/kg, mefuparib is rapidly absorbed, reaching a peak plasma concentration (Cmax) of 116-725 ng/mL within 0.5-1 hour (Tmax). The elimination half-life (t1/2) is relatively short, ranging from 1.07 to 1.3 hours. In cynomolgus monkeys, after oral administration of 5, 10, or 20 mg/kg, the compound exhibits a longer half-life of 2.16 to 2.7 hours, with a Cmax of 608 ng/mL at the 20 mg/kg dose. The compound has demonstrated good oral bioavailability in these species. The low clearance and moderate volume of distribution suggest that it can achieve and sustain levels sufficient for target inhibition. These favorable PK properties support its once- or twice-daily oral dosing schedule. The compound is primarily metabolized, and its metabolites have been identified.

Preclinical toxicity of mefuparib HCl has been assessed in standard in vitro and in vivo studies. In vitro, mefuparib shows low potential for genotoxicity in the Ames test and the in vitro micronucleus assay. In repeated-dose toxicity studies in rats and dogs, the primary toxicities were consistent with the on-target effects of PARP inhibition, including bone marrow suppression (thrombocytopenia and neutropenia) and gastrointestinal toxicity (vomiting and diarrhea). These effects were dose-dependent and reversible upon cessation of treatment. The no-observed-adverse-effect-level (NOAEL) was determined in these studies. The overall profile suggests a manageable safety margin when administered at therapeutically effective doses. Off-target effects are minimal due to the high selectivity of mefuparib for PARP1/2 over other PARP family members.

[1]. Novel PARP1/2 inhibitor mefuparib hydrochloride elicits potent in vitro and in vivo anticancer activity, characteristic of high tissue distribution. Oncotarget. 2017 Jan 17;8(3):4156-4168.

[2]. Cancer-Cell-Membrane-Coated Nanoparticles with a Yolk-Shell Structure Augment Cancer Chemotherapy. Nano Lett. 2020 Feb 12;20(2):936-946.

Mefuparib hydrochloride is the hydrochloride salt of Mefuparib. It is a potent poly(ADP-ribose) polymerase inhibitor with significant anticancer activity and can inhibit SARS-CoV-2 virus in cell culture. It can be used as an anticoronavirus drug, an antitumor drug, an EC 2.4.2.30 (NAD(+) ADP-ribosyltransferase) inhibitor, and an apoptosis inducer. It contains a Mefuparib (1+) domain.

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Mefuparib HCl (also known as CVL-218) is a next-generation PARP inhibitor that is being developed for the treatment of various cancers, including breast, ovarian, prostate, and pancreatic cancers, particularly those with BRCA mutations or other HR deficiency (HRD). It has advanced into clinical trials. A Phase 1 clinical study evaluated the safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of mefuparib in patients with advanced solid tumors. The results showed that it was well-tolerated and demonstrated clinical benefit, including objective responses, in patients with HR-deficient tumors. The compound's high selectivity and favorable PK properties distinguish it from first-generation PARP inhibitors, potentially offering a wider therapeutic window and reduced toxicity. The mechanism of action, known as "synthetic lethality," exploits the fact that cancer cells with HR deficiency rely on PARP-mediated base excision repair for survival. Inhibiting PARP causes the accumulation of DNA damage that these cells cannot repair, leading to their selective death. Mefuparib is not yet approved for sale in any region.

C17H16CLFN2O2

334.772546768188

334.088

1449746-00-2

1392502-82-7; 1449746-00-2 (HCl);

117734810

White to off-white solid powder

3

4

4

23

398

0

Cl.FC1C=C(C(N)=O)C2=C(C=1)C=C(C1C=CC(CNC)=CC=1)O2

GPFWTAVHQKERKY-UHFFFAOYSA-N

InChI=1S/C17H15FN2O2.ClH/c1-20-9-10-2-4-11(5-3-10)15-7-12-6-13(18)8-14(17(19)21)16(12)22-15;/h2-8,20H,9H2,1H3,(H2,19,21);1H

5-fluoro-2-[4-(methylaminomethyl)phenyl]-1-benzofuran-7-carboxamide;hydrochloride

Mefuparib HClCVL-218 CVL 218 CVL218

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~25 mg/mL (~74.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.47 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: ≥ 2.5 mg/mL (7.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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