BET (IC50 = 32.5-42.5 nM)[1]

Molibresib (I-BET 762) showed the strongest affinity interaction with BET. Molibresib binds to the tandem bromodomains of BET with great affinity (dissociation constant Kd of 50.5-61.3 nM). Molibresib displaces, with high efficacy (half-maximum inhibitory concentration IC50 of 32.5-42.5 nM), a tetra-acetylated H4 peptide that had been pre-bound to tandem bromodomains of BET[1]. Molibresib exhibits high affinity for BD1/BD2 domain of BRD2/3/4 proteins. Molibresib therapy leads to a reduction in the recruitment of all three proteins to chromatin[2]. Molibresib inhibits OPM-2 cell growth with IC50 of 60.15 nM[3].

Next, we tested the antimyeloma activity of I-BET762 dosed orally in an in vivo systemic xenograft model generated by injecting OPM-2 cells into NOD-SCID mice. Daily oral doses of I-BET762 up to 10 mg/kg and 30 mg/kg given every other day were well tolerated with no clear impact on body weight compared with vehicle control (Figure 6B). We found that plasma hLC concentration was significantly reduced in mice treated with I-BET762 (Figure 6C). Specifically, as disease progressed, hLC concentration in the blood of myeloma-bearing mice increased precipitously. As expected, in vehicle-treated animals, levels of hLC continued to increase until termination, consistent with progressive myeloma. Although an increase in hLC levels was found in mice treated with I-BET762, mice treated with the 3 highest doses showed a significant reduction (P ≤ .001) in the hLC concentration at all 4 time points studied (Figure 6C). Human CD38+ BM cells were 10% in vehicle-treated animals, while they were <1% in animals treated with the 3 highest doses (P ≤ .001) (Figure 6D; supplemental Figure 4A). Similarly, histopathologic analysis of vertebrae at the time of euthanasia shows significantly lower OPM-2 cell infiltration in I-BET762–treated animals (supplemental Figure 4B). Finally, pharmacokinetic sampling 30 minutes after dose in this study was consistent with anticipated concentrations based on studies of intravenous or oral administration at 3 and 30 mg/kg in BALB/c mice (supplemental Methods and supplemental Table 2). This considerable antimyeloma activity resulted in a significant (P ≤ .002) survival advantage observed in all 4 I-BET762–treated groups of mice, with median survival not reached in animals treated with the 3 highest doses of I-BET762 (Figure 6E), notably including the groups of mice dosed at 20 to 30 mg/kg per day (that had a dosing holiday during the study) and those at 30 mg/kg every other day (Figure 6E). These data represent the first example of an orally active BET inhibitor significantly delaying myeloma progression in vivo.[3]

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We then examined the necessity of the cell death modulated by Bim for the anticancer function of GEM and I-BET762 in xenograft mice. In Panc-1 tumor-bearing mice, GEM and I-BET762 decreased the tumor weight and volume. The combination of GEM and I-BET762 triggered a remarkable decline in tumor weight and volume compared with that of either agent alone (Fig. 6A). TUNEL and Ki67 assays indicated that I-BET762 and GEM induced less apoptosis when used alone than did the combination treatment (Fig. 6B and C). In contrast, compared with the parental tumors, Bim-KD tumors showed noticeably weaker growth suppression in response to the combination therapy (Fig. 6A–C). Furthermore, to evaluate the toxicity effects of I-BET762 and the combination of I-BET762 and GEM on mice, we measured ALT, AST and BUN levels after treatment. We found that I-BET762 did not influence the ALT or AST in serum samples or their GEM-induced elevation. BUN was not affected by any therapy mentioned above (Fig. 6D).[5]

Binding activity was assessed in BRD2, BRD3 and BRD4 fluorescence anisotropy (FP) assays as previously described [J. Med. Chem., 54 (2011), p. 3827]. Analogues of the isoxazoloquinolines competed with the FP ligand for binding to the bromodomains with sub-micromolar IC50’s, as shown in Table 1. A 1.8 Å resolution X-ray crystal structure of compound 1 was obtained by soaking into crystals of the BRD2 N-terminal bromodomain,6 revealing its binding mode (Fig. 1A)[4].

For in vitro cell proliferation and apoptosis assays, myeloma cell lines were cultured by using RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, penicillin 500 IU/mL, and streptomycin 500 μg/mL. Cells were placed in 96-well U-bottom plates at final concentration of 0.2 × 106 cells per milliliter in a humidified incubator with 5% CO2 at 37°C. For stroma vs nonstroma experiments, myeloma cells were placed in flat-bottom 96-well plates with MS5 cells at >90% confluence or in wells without stroma. Compounds (ie, I-BET151, I-BET762, the inactive isomer I-BET768, and JQ1) were serially diluted into media and added to the cultures at the indicated concentrations, starting from a 10-mM dimethylsulfoxide (DMSO) stock solution. Primary myeloma cells were cultured in flat-bottom 96-well plates in the presence of MS5 stroma cells by using complete medium as above, supplemented with interleukin-6 (IL-6) at 5 ng/mL.[3]

Xenotransplantation experiments[3]
The antimyeloma efficacy of orally administered I-BET762 was tested in a systemic xenograft myeloma model. For this purpose, sublethally irradiated (200 cGy) NOD/SCID mice age 9 to 11 weeks were given 107 OPM-2 myeloma cells via tail vein injection. On day 15 following inoculation, animals were started on oral treatment with I-BET762 at escalating doses or vehicle (1% methylcellulose and 0.2% sodium lauryl sulfate), which was continued up to day 83. Specifically, we treated 1 group of mice with vehicle and 4 groups with different dosing schedules of I-BET762: 3 mg/kg per day; 10 mg/kg per day; 30 mg/kg on alternate days; and 30 to 20 mg/kg per day (ie, 30 mg/kg per day for 14 days, followed by 2 weeks [days 15 to 31] off treatment [drug was withheld due to a decline in body weight until animals had regained weight], followed by 20 mg/kg per day until termination of the experiment [days 43 to 82]). Blood samples (∼70 μL) were removed at 0.5 hours after oral administration of I-BET762 on day 15 (treatment initiation); days 27, 45, and 82 (3, 10, and 20 to 30 mg/kg once per day groups only); and day 83 (30 mg/kg once every other day group only). The blood was centrifuged to obtain 20 μL plasma and stored at −20°C prior to analysis for I-BET762 by using a specific liquid chromatography/mass spectrometry/mass spectrometry assay. Serum human λ light chain (hLC) was measured with enzyme-linked immunosorbent assay, and the frequency of BM CD38+ human myeloma cells was measured by flow cytometry and by histologic examination (in euthanized animals).

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BALB/c nude mice were subcutaneously injected with pancreatic cancer cells in their right flanks. When the tumor volume reached 150–200 mm3, 24 tumor-bearing mice were randomly divided into 4 groups (I-BET762, GEM, both, and control). The mice in the GEM group were injected with GEM (25 mg/kg/day) through the caudal vein every 3 days for 13 days, and those in the I-BET762 group received an intraperitoneal injection of I-BET762 (30 mg/kg/day) daily for 13 days. The mice in the combination group were treated with both I-BET762 (30 mg/kg/day) and GEM (25 mg/kg/day). In the control group, mice were treated with an equivalent amount of vehicle. Changes in body weight were monitored throughout the experiment. Tumor growth was measured every other day according to the following formula: tumor volume = length × width2/2. Mice were sacrificed on day 22 of the treatment. The tumors were excised and weighed, and the tumor volume was measured. Finally, 0.5 ml of blood was drawn from every mouse by cardiac puncture and was sent to clinical laboratories to evaluate the hepatic and renal activities.[5]

Pharmacokinetics [2] In Part II of the study, the median plasma concentrations of the total active ingredient (TOI) 0.5–2.0 hours after administration were 2960 nM (range: 64.5–8990.0 nM; n = 95) in Week 1 and 2622.8 nM (range: 110.8–6234.5 nM; n = 43) in Week 4. The median TOI plasma concentrations in Week 1 (0.5–2.0 hours after administration) and Week 4 (before administration and 0.5–2.0 hours after administration) were similar across the tumor cohorts (Table S7). These concentration-time data were also analyzed using a population pharmacokinetic approach to obtain individual pharmacokinetic parameters for patients with limited pharmacokinetic sampling in Part II, the methods and results of which have been published separately.

Safety[2]
Overall, the safety profile of the second population was similar to that of the total study population and largely consistent across different tumor types (Tables 2 and S2), and consistent with the results of the first study²⁴. The proportion of patients in the second population who discontinued their dose due to adverse events was higher than that in the total study population (83% vs. 71%, respectively).
Table 2 summarizes the most frequently reported adverse events and their highest toxicity levels. The most frequently reported treatment-related adverse events during the second study included thrombocytopenia (n = 65 [64%]), nausea (n = 44 [43%]), decreased appetite (n = 38 [n = 37%]), diarrhea (n = 33 [32%]), dysgeusia (n = 33 [32%]), and anemia (n = 32 [31%]). The most common treatment-related serious adverse events (SAEs) were thrombocytopenia (n = 22 [22%]), anemia (n = 6 [6%]), vomiting (n = 5 [5%]), nausea (n = 4 [4%]), and factor VII deficiency (n = 3 [3%]). The most common adverse events (AEs) leading to dose reduction were thrombocytopenia (n = 19 [19%]), asthenia (n = 5 [5%]), decreased appetite (n = 4 [4%]), and fatigue (n = 3 [3%]); the most common adverse events leading to dose interruption were thrombocytopenia (n = 40 [39%]), asthenia (n = 11 [11%]), and nausea (n = 11 [11%]). The most common adverse events leading to permanent discontinuation were thrombocytopenia (n = 6 [6%]), asthenia (n = 4 [4%]), and fatigue (n = 3 [3%]; Table S3). Overall, 37% of patients (n = 38) required dose reduction for any reason, and 88% of patients (n = 90) required discontinuation of treatment (Tables S4 and S5); the median duration of discontinuation (any reason) was 8 days (range: 1–41 days, by tumor type). During the second part of the study, a total of 79 patients (77%) died, the majority of whom (n = 63 [62%]) died more than 28 days after their last dose. One 52-year-old woman with triple-negative breast cancer (TNBC) experienced a fatal pulmonary embolism 15 days after starting treatment, an event considered related to the study treatment. In Part II, Grade 3 thrombocytopenic purpura events were observed from Week 2 to Week 45, with an incidence ranging from 1% (n = 1/94) in Week 2 to 33% (n = 1/30) in Week 41. Grade 4 thrombocytopenic purpura events were observed from Week 3 to Week 17, with an incidence ranging from 2% (n = 1/44) in Week 9 to 6% in Week 3 (n = 5/85) and Week 17 (n = 1/16). Analysis of platelet levels over time showed that the lowest platelet levels in patients receiving moribuzin (Part II population) occurred on average 37 days after the start of treatment, representing a mean decrease of 69% from baseline (absolute platelet count, mean ± standard deviation: 84.4 ± 75.1 × 10⁹/L). Except for patients with gastrointestinal stromal tumors (GIST), the lowest platelet count (LSC) was generally consistent across other tumor types, with most patients having LSCs ranging from 25 to 200 × 10⁹/L (Figure 1; Table S6). However, we noted that LSCs may be lower in patients with castration-resistant prostate cancer (CRPC) and small cell lung cancer (SCLC), with most patients having LSCs ranging from 10 to 75 × 10⁹/L (Figure 1). For GIST patients, LSCs ranged from 66 to 279 × 10⁹/L (Figure 1). Clinical laboratory assessments showed that 6 patients (6%) had bilirubin levels ≥2 × ULN, and 3 patients (3%) had ALT levels ≥3 × ULN. In addition, 1 patient (1%) had hepatocellular injury. Grade 2 changes in serum creatinine were observed in weeks 2 to 5, with an incidence of 1% to 4% (n = 1–3). One of the two patients assessed at week 49 (50%) experienced another Grade 2 change. One patient (2%) was observed to have a Grade 3 change in creatinine at week 9 (n = 1/44). In the second part of the study, 12 patients (12%) experienced QTcF interval prolongation after baseline, of any grade. Absolute changes in left ventricular ejection fraction from baseline (n = 86) showed that 44 patients (51%) experienced an absolute decrease greater than 0% to less than 10%, 14 patients (16%) experienced an absolute decrease of 10% to 19%, and one patient (1%) experienced an absolute decrease greater than or equal to 20%. With the exception of one patient (ER+ breast cancer, week 4, mean [range] 0.6195 [0-6.294] μ/L), the mean troponin I level remained below 0.1 μ/L for all tumor types before week 9. In non-cancerous breast cancer patients, the mean troponin I level slightly increased between weeks 13 and 21; in small cell lung cancer patients, the mean troponin I level slightly increased between weeks 9 and 29, but remained below 0.16 μ/L.

[1]. Suppression of inflammation by a synthetic histone mimic. Nature. 2010 Dec 23;468(7327):1119-23.

[2]. Safety, pharmacokinetic, pharmacodynamic and clinical activity of molibresib for the treatment of nuclear protein in testis carcinoma and other cancers: Results of a Phase I/II open-label, dose escalation study. Int J Cancer. 2022 Mar 15;150(6):993-1006.

Moribucib benzylsulfonate is the benzylsulfonate salt of moribucib, a small molecule inhibitor that inhibits bromodomain proteins of the BET (bromodomain and terminal extradomain) family, possessing potential antitumor activity. Upon administration, moribucib binds to the acetylated lysine recognition motif on the bromodomain of the BET protein, thereby preventing the interaction between the BET protein and acetylated histone peptides. This disrupts chromatin remodeling and gene expression. Inhibition of the expression of certain growth-promoting genes may lead to suppression of tumor cell growth. BET proteins are characterized by tandem repeat sequences of the N-terminal bromodomain, including BRD2, BRD3, BRD4, and BRDT, which are transcriptional regulators that play important roles in development and cell growth.

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The interaction between pathogens and immune system cells leads to the activation of inflammatory gene expression. Although this response is crucial for immune defense, it is often detrimental to the host due to the excessive production of inflammatory proteins. The extent of the inflammatory response reflects the activation state of upstream signaling proteins of inflammatory genes and the signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Nuclear proteins recognize post-translational modified histones, initiate mRNA transcription, and support mRNA elongation, which are key steps in gene expression regulation. This paper proposes a new pharmacological approach to target inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and terminal domain (BET) protein family. We describe a synthetic compound (I-BET) that “mimics” acetylated histones to disrupt the chromatin complex responsible for the expression of key inflammatory genes in activated macrophages, thereby protecting the body from lipopolysaccharide-induced endotoxin shock and bacterial-induced sepsis. Our results suggest that synthetic compounds that specifically target and recognize post-translational modified histones can serve as a new generation of immunomodulatory drugs. [1]

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Molibresib is a small molecule inhibitor of BET protein with high oral bioavailability and selectivity. Results from a first-in-human study in solid tumors have determined that a 75 mg once-daily dose of molibresib is the recommended dose for Phase II (RP2D). This article presents the results of the second part of our study, which aimed to investigate the safety, pharmacokinetics, pharmacodynamics, and clinical activity of moribut at RP2D doses against nucleoproteins in testicular cancer (NC), small cell lung cancer, castration-resistant prostate cancer (CRPC), triple-negative breast cancer, estrogen receptor-positive breast cancer, and gastrointestinal stromal tumors. The primary safety endpoint was the incidence of adverse events (AEs) and serious adverse events (SAEs); the primary efficacy endpoint was the overall response rate. Secondary endpoints included plasma concentrations and gene set enrichment analysis (GSEA). No unexpected toxicities were observed with once-daily 75 mg moribut treatment. The most common treatment-related adverse events (of any grade) were thrombocytopenia (64%), nausea (43%), and decreased appetite (37%); 83% of patients required discontinuation of treatment due to adverse events, and 29% required dose reduction. Antitumor activity was observed in normal control (NC) and castration-resistant prostate cancer (CRPC) (one confirmed partial response and tumor volume reduction were observed in each), but the pre-specified clinically significant response rate was not achieved in any tumor type. The median plasma concentration of the total active ingredient was similar in all tumor cohorts after single and repeated administration. GSEA analysis showed that changes in gene expression induced by moribuz varied by patient, response status, and tumor type. It is necessary to investigate combination therapy regimens using BET inhibitors to eliminate resistance to other targeted therapies. [2]

C28H28CLN5O5S

582.070424079895

581.149

C, 57.78; H, 4.85; Cl, 6.09; N, 12.03; O, 13.74; S, 5.51

1895049-20-3

Molibresib;1260907-17-2

133082230

White to off-white solid powder

2

8

6

40

823

1

CCNC(=O)C[C@H]1C2=NN=C(N2C3=C(C=C(C=C3)OC)C(=N1)C4=CC=C(C=C4)Cl)C.C1=CC=C(C=C1)S(=O)(=O)O

UQGMFOYDYUZADE-FERBBOLQSA-N

InChI=1S/C22H22ClN5O2.C6H6O3S/c1-4-24-20(29)12-18-22-27-26-13(2)28(22)19-10-9-16(30-3)11-17(19)21(25-18)14-5-7-15(23)8-6-147-10(8,9)6-4-2-1-3-5-6/h5-11,18H,4,12H2,1-3H3,(H,24,29)1-5H,(H,7,8,9)/t18-/m0./s1

2-((4S)-6-(4-Chlorophenyl)-8-methoxy-1-methyl-4H-(1,2,4)triazolo(4,3-a)(1,4)benzodiazepin-4-yl)-N-ethylacetamide monobenzenesulfonate salt

GSK 525762C; GSK 525762A; IBET762; GSK525762; GSK525762A; IBET762; GSK525762; GSK525762; GSK525762A; IBET 762; Molibresib besylate; 1895049-20-3; Molibresib (besylate); GSK525762C; K04D7I4BCH; UNII-K04D7I4BCH; GSK-525762C; benzenesulfonic acid;2-[(4S)-6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide; Molibresib.

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 (~42.95 mM)

Solubility in Formulation 1: ≥ 2.67 mg/mL (4.59 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 26.7 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: ≥ 2.67 mg/mL (4.59 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 26.7 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: ≥ 2.67 mg/mL (4.59 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 26.7 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.)