K-Ras(G12C)

MRTX849's impact on cell viability is assessed using 2D (3-day adherent cells) and 3D (12-day spheroids) cell growth conditions on a panel of 17 KRAS^G12C-mutant and three non-KRAS^G12C-mutant cancer cell lines in order to assess the range of its activity. IC50 values of MRTX849 range from 10 nM to 973 nM in 2D format and from 0.2 nM to 1042 nM in 3D format, indicating that it potently inhibits cell growth in the majority of KRAS^G12C-mutant cell lines[1].

At the 15th study day, animals in the 30 mg/kg and 100 mg/kg cohorts show signs of a full response, and rapid tumor regression is seen at the earliest posttreatment tumor measurement. Four mice in the 100 mg/kg cohort and two out of seven mice in the 30 mg/kg cohort remain tumor-free until study day 70. The dose is stopped at study day 16.[1]

KRASG12C Target Engagement[2]
Tumor fragments were homogenized in 6 M guanidine–HCl, 50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (pH 7.5), and 5 mM TCEP. Following centrifugation, the protein concentration of the supernatant was determined using a Bradford assay. An internal standard (13C15N recombinant KRASG12C) and 20 mM iodoacetamide were added to 200 μg of tumor protein in 200 μL of lysis buffer, and samples were incubated at 37 °C for 30 min in the dark. Following alkylation, 100 μL of the reaction was exchanged into 1 M guanidine–HCl, 50 mM HEPES (pH 7.5), using a 96-well Zeba spin plate. Proteins were digested with 1 μg of trypsin/Lys-C mix at 37 °C for 18 h. Peptides were desalted using a C18 spin plate, and the solvent was removed by evaporation. Peptides were solubilized in 0.1% formic acid, 5% acetonitrile, 95% water, for LCMS analysis. A targeted method on a Sciex TripleTOF instrument was used to monitor the Cys-12-containing KRASG12C peptide, an internal reference peptide, as well as the corresponding isotope-labeled peptides. KRASG12C engagement was calculated as previously reported.[2]

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Measurement of kinact/KI[2]
Recombinant KRASG12C “Lite” (C51S/C80L/C118S) was reacted with a range of MRTX849 concentrations in 25 mM HEPES (pH 7.0), 150 mM NaCl, 5 mM MgCl2, 10 mM octyl β-glucopyranoside, and 0.5 mM TCEP, for 0–45 s, at room temperature. At each time-point, the reaction was quenched with 50 mM HCl, and 0.25 μg of pepsin was added. KRASG12C was digested for 4 h at 37 °C, and the resulting Cys-12-containing peptide was analyzed by LCMS. The percent of modified KRASG12C at each time-point was calculated from the 0 s control sample for each concentration of MRTX849, and kobs was subsequently calculated from the slope of the ln(POC) versus time data. Rate versus concentration data fit the Michaelis–Menten equation.

All cell lines were kept in a humidified incubator with 5% CO2 at 37 °C, and their mycoplasma levels were routinely examined. Cell viability was assessed using the CellTiter-Glo assay on seven KRAS G12C-mutant cell lines and three non-KRAS G12C-mutant cell lines that were grown in either 3D conditions using 96-well ULA plates in a 12-day assay or 2D tissue culture conditions in a 3-day assay.

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Cell Viability Assay[1]

Cell Types: MIA PaCa-2, H1373, H358, H2122, SW1573, H2030, KYSE-410 cells (G12C); H1299 (WT); A549 (G12S), HCT116 (G13D) cells
Tested Concentrations: 0.1, 1, 10, 100, 1000, 10000 nM
Incubation Duration: 24 h
Experimental Results: Inhibits cell growth in the vast majority of KRAS G12C-mutant cell lines with IC₅₀ values ranging between 10 and 973 nM in the 2D format and between 0.2 and 1042 nM in the 3D format.

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Western Blot Analysis[1]

Cell Types: MIA PaCa-2 cells
Tested Concentrations: 0.24, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 nM
Incubation Duration: 24 h
Experimental Results: Inhibits KRAS-dependent signaling targets including ERK1/2 phosphorylation (Thr202/Tyr204 ERK1; pERK), S6 phosphorylation (RSK-dependent Ser235/236; pS6) and expression of the ERK-regulated DUSP6, each with IC₅₀s in the single-digit nanomolar range in cell lines.

Animal/Disease Models: MIA PaCa-2 model (6-8-week-age, female, athymic nude-Foxn1 nu mice)[1]
Doses: 1, 3, 10, 30, 100 mg/kg
Route of Administration: p.o., for 16 days, daily
Experimental Results: Rapid tumor regression was observed at the earliest posttreatment tumor measurement and animals in the 30 and 100 mg/kg cohorts exhibited evidence of a complete response at study Day 15. Dosing was stopped at study Day 16 and all 4 mice in the 100 mg/kg cohort and 2 out of 7 mice in the 30 mg/kg cohort remained tumor-free through study Day 70.

Absorption, Distribution and Excretion
The AUC and Cmax of adagrasib increase in a dose-proportional manner between 400 mg and 600 mg (0.67 to 1 times the approved recommended dose). At the recommended dose, adagrasib reached steady-state within 8 days, with a 6-fold accumulation. The Tmax of adagrasib is approximately 6 hours. The administration of a high-fat and high-calorie meal (900-1000 calories, 50% from fat) did not have a clinically significant effect on the pharmacokinetics of adagrasib. Adagrasib has high oral bioavailability and is able to penetrate the central nervous system.
Adagrasib is eliminated through feces and urine. In patients given a single dose of radiolabeled adagrasib, 75% of the dose was recovered in feces (14% as unchanged), while 4.5% was recovered in urine (2% as unchanged).
Adagrasib has an apparent volume of distribution of 942 L.
Adagrasib has an apparent oral clearance (CL/F) of 37 L/h.

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Metabolism / Metabolites
Following single-dose administration, adagrasib is mainly metabolized by CYP3A4. However, since adagrasib inhibits CYP3A4 following multiple dosing, other enzymes such as CYP2C8, CYP1A2, CYP2B6, CYP2C9, and CYP2D6 contribute to its metabolism at steady-state.

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Biological Half-Life
Adagrasib has a terminal elimination half-life of 23 hours.

Hepatotoxicity
In the prelicensure clinical trials of adagrasib in patients with solid tumors harboring KRAS G12C mutations, liver test abnormalities were frequent although usually self-limited and mild. Some degree of ALT elevations arose in 28% to 46% of adagrasib treated patients and elevations above 5 times the upper limit of normal (ULN) were seen in 5% to 7%. In these trials that enrolled approximately 366 patients, adagrasib was discontinued early due to increased AST or ALT in 8% of patients. In addition, a small proportion of patients developed clinically apparent hepatotoxicity requiring adagrasib discontinuation. The liver test abnormalities had a median onset of 3 weeks after initiation of therapy. While serum aminotransferase elevations were occasionally quite high (5 to 20 times ULN), there were no accompanying elevations in serum bilirubin and no patient developed clinically apparent liver injury with jaundice. The product label for adagrasib recommends monitoring for routine liver tests before, at 3 week intervals during the first 3 months of therapy, and thereafter as clinically indicated.
Likelihood score: D (possible but infrequent cause of clinically apparent liver injury).

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Protein Binding
_In vitro_, adagrasib has a human plasma protein binding of 98%.

[1]. Cancer Discov. 2020 Jan;10(1):54-71.

[2]. J Med Chem. 2020 Jul 9;63(13):6679-6693.

Adagrasib (MRTX849) is an oral, small-molecule KRAS inhibitor developed by Mirati Therapeutics. KRAS mutations are highly common in cancer and account for approximately 85% of all RAS family mutations. However, the development of KRAS inhibitors has been challenging due to their high affinity for guanosine triphosphate (GTP) and guanosine diphosphate (GDP), as well as the lack of a clear binding pocket. Adagrasib targets KRASG12C, one of the most common KRAS mutations, at the cysteine 12 residue and inhibits KRAS-dependent signalling. In a phase I/IB clinical study that included patients with KRASG12C-mutated advanced solid tumors (NCT03785249), adagrasib exhibited anti-tumor activity. The phase II of the same study showed that in patients with KRASG12C-mutated non-small-cell lung cancer (NSCLC), adagrasib was efficient without new safety signals. In February 2022, the FDA accepted a new drug application (NDA) for adagrasib for the treatment of patients with previously treated KRASG12C–positive NSCLC. In December 2022, the FDA granted accelerated approval to adagrasib for the treatment of KRASG12C-mutated locally advanced or metastatic NSCLC who have received at least one prior systemic therapy. Adagrasib joins [sotorasib] as another KRASG12C inhibitor approved by the FDA.
Adagrasib is a small molecule inhibitor of the KRAS G12C mutant protein which is found in up to 13% of refractory cases of non-small cell lung cancer. Serum aminotransferase elevations are common during therapy with adagrasib, and a proportion of patients develop clinically apparent liver injury that can be severe.
Adagrasib is an orally available, small molecule inhibitor that targets the oncogenic KRAS substitution mutation, G12C, with potential antineoplastic activity. Upon oral administration adagrasib covalently binds to cytosine 12 within the switch II pocket of GDP-bound KRAS G12C, thereby inhibiting mutant KRAS-dependent signaling. KRAS, a member of the RAS family of oncogenes, serves an important role in cell signaling, division and differentiation. Mutations of KRAS may induce constitutive signal transduction leading to tumor cell growth, proliferation, invasion, and metastasis.

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Drug Indication
Adagrasib is indicated for the treatment of adult patients with KRAS G12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC), as determined by an FDA-approved test, who have received at least one prior systemic therapy. This indication is approved under accelerated approval based on objective response rate (ORR) and duration of response (DOR). Continued approval for this indication may be contingent upon verification and description of a clinical benefit in a confirmatory trial(s).
Treatment of all solid and haematological malignancies

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Mechanism of Action
In normal cells, KRAS is activated by binding to guanosine triphosphate (GTP), and this promotes the activation of the MAP kinase pathway and intracellular signal transduction. When GTP is hydrolyzed to guanosine diphosphate (GDP), KRAS is inactivated. This mechanism works as an "on"/"off" system that regulates cell growth. The substitution of Gly12 by cysteine in KRAS (KRAS^G12C) impairs GTP hydrolysis, and maintains KRAS in its active form. Therefore, the presence of this mutation leads to uncontrolled cellular proliferation and growth, as well as malignant transformation. Adagrasib is a covalent inhibitor of KRAS^G12C that irreversibly and selectively binds and locks KRAS^G12C in its inactive, guanosine diphosphate–bound state. Therefore, the use of adagrasib inhibits tumor cell growth and viability in cancers with KRAS^G12C mutations with minimal off-target activity.

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Pharmacodynamics
The exposure-response relationship and pharmacodynamic response time course of adagrasib have not been elucidated. The use of adagrasib can cause QTc interval prolongation. The increase in QTc is concentration-dependent. In patients given 600 mg of adagrasib twice daily, the mean QTcF change from baseline (ΔQTcF) was 18 ms at the mean steady-state maximum concentration. The use of adagrasib can also lead to severe gastrointestinal adverse reactions, hepatotoxicity and interstitial lung disease/pneumonitis.

C32H35CLFN7O2

604.1174

603.25

C, 63.62; H, 5.84; Cl, 5.87; F, 3.14; N, 16.23; O, 5.30

2326521-71-3

MRTX849 analog 24; 2490716-96-4; LC-2; 2502156-03-6

138611145

White to yellow solid powder

From > 262 mg/mL to < 0.010 mg/mL

5

0

9

7

43

1060

2

CN1CCC[C@H]1COC2=NC3=C(CCN(C3)C4=CC=CC5=C4C(=CC=C5)Cl)C(=N2)N6CCN([C@H](C6)CC#N)C(=O)C(=C)F

PEMUGDMSUDYLHU-ZEQRLZLVSA-N

InChI=1S/C32H35ClFN7O2/c1-21(34)31(42)41-17-16-40(18-23(41)11-13-35)30-25-12-15-39(28-10-4-7-22-6-3-9-26(33)29(22)28)19-27(25)36-32(37-30)43-20-24-8-5-14-38(24)2/h3-4,6-7,9-10,23-24H,1,5,8,11-12,14-20H2,2H3/t23-,24-/m0/s1

2-[(2S)-4-[7-(8-chloronaphthalen-1-yl)-2-[[(2S)-1-methylpyrrolidin-2-yl]methoxy]-6,8-dihydro-5H-pyrido[3,4-d]pyrimidin-4-yl]-1-(2-fluoroprop-2-enoyl)piperazin-2-yl]acetonitrile

Adagrasib; MRTX 849; MRTX849; KRAZATI; Kras G12C inhibitor MRTX849; 8EOO6HQF8Y; Adagrasib [USAN]; MRTX-849; brand name: Krazati

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)

DMSO: 25~100 mg/mL (41.4~165.5 mM)
Ethanol: ~100 mg/mL

Solubility in Formulation 1: ≥ 2.62 mg/mL (4.34 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (4.14 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 3: ≥ 2.5 mg/mL (4.14 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.
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 4: in 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (8.28mM) (add these co-solvents sequentially from left to right, and one by one),

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