B-Raf

PF-07799933 is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited pERK in vitro in cells driven by BRAFV600E-mutant monomers, BRAF Class II/III-mutant dimers, and treatment-acquired genetic alterations that induce mutant-BRAFV600E dimerization. Furthermore, PF-07799933 disrupted endogenous mutant-BRAF:wild-type-CRAF dimers in cells containing BRAFV600E + p61 splice variant or BRAFV600E + NRASQ61K that induce mutant BRAF dimerization. However, PF-07799933 spared pERK and did not disrupt BRAF-wild-type:CRAF-wild-type dimers in vitro in BRAF wild-type cells.[1]
PF-07799933 Disrupts Mutant BRAF Dimers to Overcome Diverse BRAF Mutants Preclinically. In BRAF wild-type cells, PF-07799933 demonstrated no pERK inhibition, in contrast to the pan-RAF dimer inhibitors, and less paradoxical activation of pERK than encorafenib. Isothermal stability shift dose–response assays (ITDR) showed that PF-07799933 bound BRAFV600E with a 10-fold higher affinity compared with wild-type CRAF protein in A375 BRAFV600E-mutant melanoma cell lysates.

PF-07799933 had broad in vivo antitumor activity, systemically and in the brain, against BRAFV600E and non-V600 mutant proteins as monotherapy, and against BRAFV600E with a treatment-acquired BRAF p61 splice variant in combination with binimetinib.[1]

---

PF-07799933 monotherapy drove deeper regressions than encorafenib + binimetinib in mouse xenografts harboring the founder BRAFV600E (Class I) mutation implanted subcutaneously or intracranially. PF-07799933 treatment also caused regressions of subcutaneously implanted BRAFG469A (Class II)-mutant NSCLC, BRAFK601E (Class II)-mutant melanoma and BRAF indel-mutant pancreatic cancer tumors. In contrast, plixorafenib was less efficacious against BRAFV600E- and BRAFG469A-mutant models, consistent with less in vitro pERK inhibition and known in vivo metabolic vulnerability. Similarly, consistent with less in vitro pERK inhibition, exarafenib was less active against the BRAFV600E-mutant model. For plixorafenib and exarafenib, we confirmed exposures achieved in animals approximated those achieved in humans. Thus, the observed decreased efficacy relative to PF-07799933 ± binimetinib was not due to decreased exposures relative to clinically achievable doses in humans. In a BRAFV600-mutant melanoma patient-derived xenograft (PDX) model harboring an acquired BRAF p61 splice variant, the combination of encorafenib + binimetinib did not improve the minimal antitumor activity of binimetinib monotherapy. However, single agent PF-07799933 demonstrated superior activity, and its combination with binimetinib further augmented efficacy, resulting in tumor regression, while remaining well-tolerated without body weight loss.[1]

Cocrystallization was performed by mixing an equal volume of the Braf KDS: encorafenib complex and reservoir solution, which consists of 18% PEG3350, 0.2-mol/L Na2SO4, 0.1-mol/L sodium–potassium phosphate, pH 6.6 with the hanging-drop vapor diffusion method. For Braf-KDL: PF-07799933 complex crystallization, the reservoir solution contains 17% PEG5000MME, 1% PEG6000, sodium acetate, 0.2-mol/L NaCl, and 5% Tacsimate. Both crystals were harvested and flash-frozen in liquid nitrogen in a reservoir solution containing 20% (v/v) glycerol as a cryoprotectant.[1]

Isothermal Stability Shift Dose Response Assays[1]
Trypsinized A375 cell pellets were resuspended in PBS and treated with DMSO or PF-07799933 (concentrations ranging from 0.122 to 2,000 nmol/L) for 30 minutes at 37°C. Cells were heated to 50°C for 3 minutes in a PCR plate using a PTC-200 thermal cycler and then promptly spun at 4,700 rpm in a swinging bucket centrifuge for 30 seconds at 4°C. Cells were lysed with three cycles of freeze–thawing in liquid nitrogen prior to adding nondenaturing buffer [10-mmol/L Na2PO4, 1.8-mmol/L KH2PO4 (pH 7.4), 137-mmol/L NaCl, 2.7-mmol/L KCl, 1-mmol/L CaCl2, 10-mmol/L MgCl2, 0.02% n-dodecyl β-D-maltoside, 2× complete protease inhibitor, and 2% phosphatase inhibitor cocktails 2 and 3].

---

Co-Immunoprecipitation and Immunoblotting[1]
For co-immunoprecipitations of endogenous RAF proteins, MEL21514 or A375-NRASQ61K cells were plated at 1.5 × 107 cells per 150-mm dish. Cells were incubated at 37°C, 5% CO2 with DMSO vehicle control or indicated concentrations of encorafenib or PF-07799933 for 1 hour. Cell lysates were harvested in 1 mL per dish of magnesium lysis buffer [25-mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.5, 75-mmol/L NaCl, 5-mmol/L MgCl2, 5% glycerol, and 0.1% NP-40] supplemented with HALT protease/phosphatase inhibitor cocktail.

Efficacy studies with the CTG-1441 and CTG-0362 PDX models were performed at a CRO company. The MEL21514 PDX was generated from a biopsy provided by a female Hispanic patient with melanoma (51 years of age) following a relapse from Braftovi and Mektovi treatment (MT Group). BRAFV600E mutation and p61 splice variant expression in the MEL21514 PDX tumor were confirmed by whole-exome sequencing and RNA sequencing, respectively. For cell line development, MEL21514 PDX tumor samples were dissociated using the Miltenyi Biotec Human Tumor Dissociation Kit in combination with the gentleMACS Octo Dissociator according to the suggested hard tumor protocol. The dissociated cell suspension was then magnetically labeled with the Miltenyi Mouse Cell Depletion Kit and separated using the MACS Magnetic Separator for the enrichment of human tumor cells. Isolated MEL21514 tumor cells were pooled and established in culture using Renaissance Essential Tumor Medium/RETM supplemented with 10% fetal bovine serum/FBS, penicillin/streptomycin and cholera toxin. Following 10 passages in RETM, MEL21514 cells were grown in RPMI, supplemented with 10% FBS and 1-mmol/L sodium pyruvate. Cell lines and PDXs were authenticated by short tandem repeat profiling and regularly evaluated for Mycoplasma and murine viruses. All mice were obtained at 6 to 8 weeks of age, housed in groups of 5, and allowed a 1-week acclimation period before cancer cell inoculation. Food, water, temperature, and humidity were maintained per Pharmacology Testing Facility performance standards in accordance with the 2011 Guide for the Care and Use of Laboratory Animals (NRC) and AAALAC-International. For subcutaneous xenografts, each cell line (5 × 106 cells) or PDX (cell suspension prepared with Miltenyi gentleMACS) was injected subcutaneously into the right flank of female Foxn1nu mice and allowed to grow to approximately 200 mm3 prior to randomization by tumor size into dosing groups of eight animals. Body weight and subcutaneous tumor volume [determined by the formula (length × width2)/2] were recorded twice weekly. For intracranial xenografts, the A375-luciferase cell line (10,000 cells) was injected 2 mm lateral to the bregma at the bone suture line of female Foxn1nu mice, with randomization after 7 days into dosing groups of ten animals based on tumor burden measured by total luminescence flux (photons/second) with an IVIS Spectrum In Vivo Imaging System. Body weight and total flux were recorded twice weekly, with average total flux plotted against the day posttumor implantation.

PF-07799933 was well tolerated with monotherapy or combination therapy. No dose-limiting toxicities (DLTs) occurred, and the maximum tolerated dose (MTD) was not reached. Treatment-emergent adverse events (TEAEs) occurred in 94% and 100% of patients receiving monotherapy and combination therapy, respectively, with ≥ grade 3 adverse events occurring in 28% and 44% of patients, respectively. Supplementary Table S4 lists TEAEs reported in ≥3 patients, regardless of their attribution. The most common TEAEs with monotherapy were fatigue (44% of any grade / 0% of ≥ grade 3), headache (28% / 0%), blurred vision [22% / 6% (1 grade 3)], and elevated lipase (16% / 0%). The most common TEAEs with combination therapy were peripheral edema (33% / 0%), acneiform rash, diarrhea, and fatigue (all 28% / 0%). One patient receiving PF-07799933 monotherapy (450 mg twice daily) had their dose reduced due to adverse reactions including blurred vision, peripheral sensory neuropathy, myalgia, fatigue, and decreased appetite. Six of the eight patients with blurred vision experienced relief without dose adjustment, and follow-up ophthalmological examinations revealed no abnormalities. No patients discontinued treatment due to adverse reactions related to the investigational drug. [1]

[1]. A Next-Generation BRAF Inhibitor Overcomes Resistance to BRAF Inhibition in Patients with BRAF-Mutant Cancers Using Pharmacokinetics-Informed Dose Escalation. Cancer Discov . 2024 Sep 4;14(9):1599-1611.

RAF inhibitors have revolutionized the treatment of BRAFV600-mutant cancer patients, but their clinical benefits are limited by factors such as adaptive activation of the ERK signaling pathway, genetic alterations that induce BRAFV600 dimerization, and poor brain penetration. Newer generation pan-RAF dimer inhibitors have a narrower therapeutic index. PF-07799933 (ARRY-440) is a pan-mutant BRAF inhibitor with strong brain penetration and high selectivity. PF-07799933 inhibits the signaling pathway in vitro, disrupting the dimerization of endogenous mutant BRAF and wild-type CRAF, without affecting the wild-type ERK signaling pathway. PF-07799933, with or without bimetinib, inhibits the growth of mouse xenograft tumors driven by mutant BRAF acting in a dimer form and BRAFV600E resistant to existing RAF inhibitors. In a first-in-human clinical trial (NCT05355701), we employed a novel, flexible, pharmacokinetic-based dose-escalation design to rapidly achieve effective concentrations of PF-07799933 in patients with refractory BRAF-mutant solid tumors. PF-07799933 ± bimetinib was well-tolerated, with multiple confirmed systemic and brain responses observed in patients with BRAF-mutant cancers resistant to approved RAF inhibitors. Significance: PF-07799933 treatment demonstrated antitumor activity against BRAF V600-mutant and non-V600-mutant cancers in both preclinical studies and refractory patients, and PF-07799933 can be safely used in combination with MEK inhibitors. This novel, rapid pharmacokinetic (PK)-based dose-escalation design provides a new paradigm for accelerating the testing of next-generation targeted therapies in early clinical stages. [1] PF-07799933 (ARRY-440) is a new generation selective panmutative BRAF inhibitor. It is not a panmutative RAF inhibitor, does not have the metabolic risks of prisorafenib, can be used in combination with MEK inhibitors, and can cross the blood-brain barrier. Given the huge unmet medical needs faced by patients with BRAF-mutant cancers after failure of existing treatments, we aim to implement a data-based dose escalation strategy in a first-in-human Phase I trial to achieve the required drug exposure in a smaller number of patients over a shorter period of time. [1]

C18H15CL2F2N5O3S

490.31

489.0240722

2754408-94-9

165150001

White to light yellow solid powder

585.0±60.0 °C(Predicted)

1.72±0.1 g/cm3(Temp: 20 °C; Press: 760 Torr)(Predicted)

2.8

2

9

5

31

826

0

CN1C=NC2=C(C1=O)C(=C(C=C2)NC3=C(C=CC(=C3Cl)NS(=O)(=O)N4CC(C4)F)F)Cl

SHENFUUACGRLOZ-UHFFFAOYSA-N

InChI=1S/C18H15Cl2F2N5O3S/c1-26-8-23-11-4-5-12(15(19)14(11)18(26)28)24-17-10(22)2-3-13(16(17)20)25-31(29,30)27-6-9(21)7-27/h2-5,8-9,24-25H,6-7H2,1H3

N-[2-chloro-3-[(5-chloro-3-methyl-4-oxoquinazolin-6-yl)amino]-4-fluorophenyl]-3-fluoroazetidine-1-sulfonamide

2754408-94-9; PF07799933; Claturafenib (USAN); PF-07799933; SCHEMBL25280690; N-(2-chloro-3-((5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-6-yl)amino)-4-fluorophenyl)-3-fluoroazetidine-1-sulfonamide

2934.99.9001

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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