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].

---

- Anti - proliferative Activity: Adagrasib potently inhibits the growth of KRAS G12C - mutant cancer cell lines. In a panel of 17 KRAS G12C - mutant and 3 non - KRAS G12C - mutant cancer cell lines, its IC50 values range between 10 nM and 973 nM in 2D (3 - day, adherent cells) culture, and between 0.2 nM and 1042 nM in 3D (12 - day, spheroids) culture, indicating strong anti - proliferative activity against KRAS G12C - mutant cells.

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]

---

Antitumor Efficacy: Adagrasib can cause rapid tumor regression in animal models. In the experiment, animals in the 30 mg/kg and 100 mg/kg dosage cohorts showed signs of 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, suggesting its significant in - vivo antitumor effect.

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]

---

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.

---

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.

---

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.

---

- Cell Viability Assay: Cultivate 17 KRAS G12C - mutant and 3 non - KRAS G12C - mutant cancer cell lines, and set up 2D (adherent cells cultured for 3 days) and 3D (spheroids cultured for 12 days) culture systems. Add different concentrations of Adagrasib to the culture medium, and after the specified culture time, use appropriate methods to detect cell viability, so as to evaluate the inhibitory effect of Adagrasib on the growth of KRAS G12C - mutant cancer cells.

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.

---

Administer Adagrasib to animal models by gavage (the specific solvent and formulation are not described in the literature). Set up 30 mg/kg and 100 mg/kg dosage groups, and observe the tumor volume change of animals at different time points. The results show that rapid tumor regression can be observed at the earliest post - treatment tumor measurement, and some animals have a complete response, and part of them can maintain a tumor - free state for a long time after the drug is stopped.

Absorption, Distribution and Excretion
The AUC and Cmax of adagrasibel increase dose-proportionally between 400 mg and 600 mg (0.67 to 1 times the approved recommended dose). At the recommended dose, adagrasibel reaches steady state within 8 days, accumulating 6-fold. The Tmax of adagrasibel is approximately 6 hours. Consuming a high-fat, high-calorie meal (900–1000 calories, 50% of which are from fat) has no clinically significant effect on the pharmacokinetics of adagrasibel. Adagarasibel has high oral bioavailability and can penetrate the central nervous system. Adagarasibel is primarily excreted via feces and urine. In patients receiving a single dose of radiolabeled adagrasibel, 75% of the dose is recovered in feces (14% unchanged) and 4.5% in urine (2% unchanged). The apparent volume of distribution of adagrasibel is 942 liters.
The apparent oral clearance (CL/F) of adagrasibu is 37 L/h.
Metabolism/Metabolites

After a single dose, adagrasibu is primarily metabolized via CYP3A4. However, due to the inhibition of CYP3A4 by repeated doses, other enzymes such as CYP2C8, CYP1A2, CYP2B6, CYP2C9, and CYP2D6 also participate in its metabolism under steady state.
Biological Half-Life

The terminal elimination half-life of adagrasibu is 23 hours.

Hepatotoxicity
Liver function abnormalities were common in pre-marketing clinical trials of adagrasib in patients with KRAS G12C-mutant solid tumors, but were usually self-limiting and mild. Between 28% and 46% of patients treated with adagrasib experienced varying degrees of ALT elevation, with 5% to 7% experiencing ALT elevations exceeding 5 times the upper limit of normal (ULN). In these trials, which included approximately 366 patients, 8% discontinued adagrasib treatment prematurely due to elevated AST or ALT. Furthermore, a small number of patients experienced clinically significant hepatotoxicity, requiring discontinuation of adagrasib treatment. The median time to onset of liver function abnormalities was 3 weeks after treatment initiation. Although serum transaminases occasionally elevated to 5 to 20 times the ULN, serum bilirubin was not elevated, and no patients developed clinical liver injury with jaundice. The product information for adagrasibu recommends routine liver function tests every 3 weeks before treatment and during the first 3 months of treatment, followed by tests as needed clinically. Probability score: D (may cause, but is rare, clinical liver injury). Protein binding: In vitro studies show that adagrasibu has a 98% protein binding rate in human plasma.

[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 very common in cancer, accounting for approximately 85% of all RAS family mutations. However, the development of KRAS inhibitors has been challenging due to the high affinity of KRAS for guanosine triphosphate (GTP) and guanosine diphosphate (GDP) and the lack of a well-defined binding pocket. Adagrasib targets the 12th cysteine residue of KRASG12C (one of the most common KRAS mutations), thereby inhibiting the KRAS-dependent signaling pathway. In a phase I/IB clinical trial (NCT03785249) enrolling patients with KRASG12C-mutant advanced solid tumors, adagrasib demonstrated antitumor activity. Phase II results from the same study showed significant efficacy of adagrasib in patients with KRASG12C-mutant non-small cell lung cancer (NSCLC) without identifying any new safety signals. In February 2022, the FDA accepted the New Drug Application (NDA) for adagrasib for the treatment of previously treated KRASG12C-positive NSCLC patients. In December 2022, the FDA granted accelerated approval to adagrasib for the treatment of patients with KRASG12C-mutant locally advanced or metastatic NSCLC who have received at least one prior systemic therapy. Adagrasib is the second KRASG12C inhibitor to receive FDA approval, following sotorasib. Adagrasib is a small molecule inhibitor of the KRAS G12C mutant protein, which is present in up to 13% of refractory non-small cell lung cancer cases. Elevated serum transaminases are common during adagrasib treatment, and some patients may experience clinically significant liver damage, which can be quite severe. Adagrasib is an oral small molecule inhibitor that targets the oncogenic KRAS gene G12C mutation and has potential anti-tumor activity. After oral administration, adagrasib covalently binds to cytosine 12 in the type II switching pocket of KRAS G12C, which binds to GDP, thereby inhibiting mutant KRAS-dependent signaling. KRAS is a member of the RAS oncogene family and plays an important role in cell signaling, division, and differentiation. Mutations in the KRAS gene can induce constitutive signal transduction, leading to tumor cell growth, proliferation, invasion, and metastasis.

---

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

---

Mechanism of Action
In normal cells, KRAS is activated by binding to guanosine triphosphate (GTP), thereby promoting the activation of the MAP kinase pathway and intracellular signal transduction. KRAS is inactivated when GTP is hydrolyzed to guanosine diphosphate (GDP). This mechanism acts as an "on/off" system, regulating cell growth. The substitution of Gly12 with cysteine in KRAS (KRAS^G12C) inhibits GTP hydrolysis, keeping KRAS in an active state. Therefore, this mutation leads to uncontrolled cell proliferation and growth, as well as malignant transformation. Adagrasib is a covalent inhibitor of KRAS^G12C that irreversibly and selectively binds to KRAS^G12C and locks it in an inactive state bound to guanosine diphosphate. Therefore, adagrasibel inhibits the growth and survival of tumor cells carrying KRAS G12C mutations with extremely low off-target activity.

---

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

---

- Mechanism of Action: Adaggrasibel is a potent, selective covalent KRAS G12C inhibitor. It selectively modifies mutant cysteine 12 in GDP-binding KRAS G12C, thereby inhibiting KRAS-dependent signaling and exerting an anti-tumor effect. - Therapeutic efficacy: It has shown some efficacy in treating KRAS G12C-mutant non-small cell lung cancer. In clinical trials, it can induce partial remission in some patients, but the remission is not durable and acquired resistance may occur. For example, a patient with KRAS G12C-mutant non-small cell lung cancer initially had a partial remission with adagrasib, but later developed resistance associated with the occurrence of secondary KRAS Y96D mutation, which affects the binding of the drug to the KRAS switch II pocket. - Indications: Primarily used to treat adult patients with KRAS G12C-mutant locally advanced or metastatic non-small cell lung cancer who have received at least one prior systemic therapy.

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.

---

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.

---

View More

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.

---

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),

---

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