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 2 of the study, the 0.5 to 2.0 hours postdose total active moiety median plasma concentration was 2960 nM at Week 1 (range: 64.5-8990.0 nM; n = 95) and 2622.8 nM at Week 4 (range: 110.8-6234.5 nM; n = 43). Both Week 1 (0.5-2.0 hours postdose) and Week 4 (predose and 0.5-2.0 hours post dose) total active moiety median plasma concentrations were similar across individual tumor cohorts (Table S7). These concentration-time data were also analyzed using population PK methodology to obtain individual PK parameters for patients in Part 2 whose PK sampling was limited, the methodology and results of which are published separately.

Safety [2]
Overall, the safety profile of the Part 2 population was similar to the total study population and broadly consistent across tumor types (Tables 2 and S2) and with the findings from Part 1 of the study.24 The proportion of patients with AEs leading to dose interruption was numerically greater in the Part 2 population compared to the total study population (83% and 71%, respectively).
The most commonly reported AEs are summarized by maximum toxicity grade in Table 2. The most commonly reported treatment-related AEs during Part 2 of the study were thrombocytopenia (n = 65 [64%]), nausea (n = 44 [43%]), decreased appetite (38 [n = 37%]), diarrhea (n = 33 [32%]), dysgeusia (n = 33 [32%]) and anemia (n = 32 [31%]). The most commonly reported treatment-related SAEs were thrombocytopenia (n = 22 [22%]), anemia (n = 6 [6%]), vomiting (n = 5 [5%]), nausea (n = 4 [4%]) and a decrease in factor VII (n = 3 [3%]). The most commonly reported AEs leading to dose reductions were thrombocytopenia (n = 19 [19%]), asthenia (n = 5 [5%]), decreased appetite (n = 4 [4%]) and fatigue (n = 3 [3%]); the most common AEs leading to dose interruptions were thrombocytopenia (n = 40 [39%]), asthenia (n = 11 [11%]) and nausea (n = 11 [11%]); and the most common AEs leading to permanent treatment discontinuation were thrombocytopenia (n = 6 [6%]), asthenia (n = 4 [4%]) and fatigue (n = 3 [3%]; Table S3). Overall, 37% of patients (n = 38) required a dose reduction for any cause and 88% (n = 90) required dose interruptions (Tables S4 and S5); the median duration of dose interruptions (any cause) was 8 days (range: 1-41 days across tumor types). In total, 79 patients (77%) died during Part 2 of the study, with the time from the last dose of study medication to death greater than 28 days in most patients (n = 63 [62%]). One 52-year-old female with TNBC experienced a fatal pulmonary embolism 15 days after beginning treatment, which was considered related to the study treatment.
In Part 2, Grade 3 thrombocytopenia events were observed from Weeks 2 to 45, with incidences ranging from 1% (n = 1/94) at Week 2 to 33% (n = 1/30) at Week 41. Occurrences of Grade 4 thrombocytopenia were observed from Weeks 3 to 17, with incidences ranging from 2% (n = 1/44) at Week 9 to 6% at Week 3 (n = 5/85) and Week 17 (n = 1/16). Analysis of platelet levels over time showed that the lowest levels in patients receiving molibresib (Part 2 population) occurred at a mean of 37 days after the start of treatment and decreased by a mean of 69% from baseline (absolute platelet count, mean ± SD: 84.4 ± 75.1 × 109 platelets/L). With the exception of patients with GIST, minimum platelet levels were broadly consistent across tumor types, with the majority ranging from 25 to 200 × 109 platelets/L (Figure 1; Table S6). However, a potential trend for lower minimum platelet levels in patients with CRPC and SCLC was noted, with most minimum platelet levels ranging from 10 to 75 × 109 platelets/L (Figure 1). For patients with GIST, minimum platelet levels ranged from 66 to 279 × 109 platelets/L (Figure 1). Clinical laboratory assessments showed that six patients (6%) experienced bilirubin levels ≥2 × ULN and three patients (3%) experienced ALT levels ≥3 × ULN. In addition, hepatocellular injury was detected in one patient (1%). Grade 2 changes in serum creatinine were observed from Weeks 2 to 5, with incidences ranging from 1% to 4% (n = 1-3) across those weeks and another Grade 2 change in 1/2 patients assessed at Week 49 (50%). A single Grade 3 creatinine change was observed at Week 9 (2%; n = 1/44).
Twelve patients (12%) had a postbaseline QTcF prolongation of any grade during Part 2 of the study. For left ventricular ejection fraction absolute change from baseline (n = 86), 44 patients (51%) had an absolute decrease of >0 to <10%, 14 patients (16%) had an absolute decrease of 10% to 19% and one patient (1%) had a decrease of ≥20%. Mean troponin I levels were consistently below 0.1 μ/L for all tumor types up to Week 9 with one exception (ER + BC, Week 4, mean [range] 0.6195 [0-6.294] μ/L). A slight increase in the mean troponin I levels was observed from Weeks 13 to 21 in patients with NC and Weeks 9 to 29 in patients with SCLC, but remained ≤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.

Molibresib Besylate is the besylate salt of molibresib, a small molecule inhibitor of the BET (Bromodomain and Extra-Terminal) family of bromodomain-containing proteins with potential antineoplastic activity. Upon administration, molibresib binds to the acetylated lysine recognition motifs on the bromodomain of BET proteins, thereby preventing the interaction between the BET proteins and acetylated histone peptides. This disrupts chromatin remodeling and gene expression. Prevention of the expression of certain growth-promoting genes may lead to an inhibition of tumor cell growth. Characterized by a tandem repeat of bromodomain at the N-terminus, BET proteins, comprising of BRD2, BRD3, BRD4 and BRDT, are transcriptional regulators that play an important role during development and cellular growth.

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Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.[1]

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Molibresib is an orally bioavailable, selective, small molecule BET protein inhibitor. Results from a first time in human study in solid tumors resulted in the selection of a 75 mg once daily dose of the besylate formulation of molibresib as the recommended Phase 2 dose (RP2D). Here we present the results of Part 2 of our study, investigating safety, pharmacokinetics, pharmacodynamics and clinical activity of molibresib at the RP2D for nuclear protein in testis carcinoma (NC), small cell lung cancer, castration-resistant prostate cancer (CRPC), triple-negative breast cancer, estrogen receptor-positive breast cancer and gastrointestinal stromal tumor. The primary safety endpoints were incidence of adverse events (AEs) and serious AEs; the primary efficacy endpoint was overall response rate. Secondary endpoints included plasma concentrations and gene set enrichment analysis (GSEA). Molibresib 75 mg once daily demonstrated no unexpected toxicities. The most common treatment-related AEs (any grade) were thrombocytopenia (64%), nausea (43%) and decreased appetite (37%); 83% of patients required dose interruptions and 29% required dose reductions due to AEs. Antitumor activity was observed in NC and CRPC (one confirmed partial response each, with observed reductions in tumor size), although predefined clinically meaningful response rates were not met for any tumor type. Total active moiety median plasma concentrations after single and repeated administration were similar across tumor cohorts. GSEA revealed that gene expression changes with molibresib varied by patient, response status and tumor type. Investigations into combinatorial approaches that use BET inhibition to eliminate resistance to other targeted therapies are warranted. [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.)