cdk2/cyclin A (IC50 = 1.5 μM); CDK2/cyclinE (IC50 = 3.6 μM); Cdk4/cyclin D1 (IC50 = 1 nM); cdk6/cyclin D3 (IC50 = 2 nM); CDK9/Cyclin T (IC50 = 28 nM); CDK5/p35 (IC50 = 0.832 μM); CDK1/cyclinB1 (IC50 = 2.4 μM); CDK7/Cyclin H/MAT1 (IC50 = 2.4 nM); Cdk5/p25 (IC50 = 1.2 nM)
CDK4/cyclin D1 (IC50 = 0.001 μM)
CDK6/cyclin D3 (IC50 = 0.002 μM)
CDK9/cyclin T (IC50 = 0.028 μM)
CDK5/p35 (IC50 = 0.832 μM)
CDK5/p25 (IC50 = 1.2 μM)
CDK2/cyclin A (IC50 = 1.5 μM)
CDK1/cyclin B1 (IC50 = 2.4 μM)
CDK7/cyclin H/Mat1 (IC50 = 2.4 μM)
CDK2/cyclin E (IC50 = 3.6 μM)
FLT3 (D835V) and FLT3 (ITD, D835V) (high potency, no wild-type inhibition)
ULK2 and NEK10 (low Kd/IC50 values) [1]

Lerociclib is the least selective member of the CDK family against CDK9/cyclin T, with a biochemical IC50 that is approximately thirty times lower than that of CDK4/cyclin D1. With an EC50 of less than 20 nM, lerociclib causes a strong and long-lasting G1 arrest in CDK4/6 dependent cells. When CDK4/6 dependent WM2664 cells are treated with Lerociclib for 24 hours, a dose-dependent increase of cells in the G1 phase of the cell cycle is seen. More than 300 times the biochemical IC50 is maintained through 300 nM, sustaining this arrest. When compared to vehicle controls, WM2664 cells treated with 30-1000 nM of Lerociclib for 24 hours show a complete inhibition of RB phosphorylation. Lerociclib treatment results in almost total inhibition of RB phosphorylation by 16 hours after treatment, with RB phosphorylation reduced within 1 hour after treatment. With EC50 concentrations as low as 23 nM, lerociclib effectively inhibits the proliferation of a wide range of tumor cell lines, including breast, melanoma, leukemia, and lymphoma[1].

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G1T38 produced a robust and sustained G1 arrest in CDK4/6-dependent cell lines (HS68, WM2664) with an EC50 of ~20 nM. This arrest was maintained through 300 nM (>300x the biochemical IC50). In CDK4/6-independent cells (A2058), the compound had no effect on the cell cycle.
In WM2664 cells, treatment with 30-1000 nM of G1T38 for 24 hours completely inhibited phosphorylation of RB at Ser807/811 sites compared to vehicle controls.
Treatment of WM2664 cells with 300 nM G1T38 reduced RB phosphorylation within 1 hour and generated near complete inhibition by 16 hours post-treatment.
G1T38 inhibited proliferation in a broad panel of CDK4/6-dependent tumor cell lines (breast, melanoma, leukemia, lymphoma) with EC50 values as low as 23 nM (e.g., MV4-11: 23 nM, MCF7: 52 nM). Inhibition in CDK4/6-independent cell lines (e.g., A2058: 2691 nM) was >2.5 μM, demonstrating a functional RB pathway is essential for activity.
A kinome screen of 468 protein kinases at 100 nM and 1 μM showed G1T38 has a high degree of selectivity, with minimal off-target activity.
G1T38 did not show a biological inhibitory effect on CDK9/cyclin T function, as measured by no loss of RNA polymerase II CTD (SER2) phosphorylation.

Lerociclib-treated mice in this HER²⁺ breast cancer model show 8% tumor regression after 21 days of treatment, whereas control animals show a 577% increase in tumor burden during the same time period. The MCF7 xenograft model exhibits tumor regression in 10 days when daily treatment with 100 mg/kg of Lerocyclib or palbociclib is administered in comparison to the mice treated with a vehicle. Tumor growth inhibition is seen in the 10, 50, and 100 mg/kg Lerociclib cohorts after 27 days of treatment (approximately 12%, 74%, and 90% inhibition, respectively). In the 10, 50, and 100 mg/kg dosage cohorts, daily oral palbociclib treatment results in an 18%, 66%, and 87% inhibition of tumor growth, respectively. It's interesting to note that lerociclib is much more effective than palbociclib at 50 mg/kg. Comparing Lerocyclib and palbociclib at the 50 mg/kg dose in the ER⁺ZR-75-1 breast cancer xenograft model yields similar results. With an overall tumor growth delay of 60% and 77% TGI, mice treated with lerociclib show that lerocyclib alone is highly effective in treating this NSCLC tumor model.

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In an ER+ MCF7 xenograft model, a single oral dose of G1T38 (100 mg/kg) led to drug concentrations in tumors that were 17-fold higher than in plasma. Tumor pRB decreased by 30% and 98% at 1 and 24 hours post-treatment, respectively, and inhibition paralleled tumor drug concentration, not plasma concentration.
In the MMTV-Neu GEMM (HER2+ breast cancer) model, mice given 100 mg/kg G1T38 in their diet for 28 days elicited 38% tumor regression, while control animals had a 577% increase in tumor burden.
In the ER+ MCF7 xenograft model, daily oral G1T38 at 10, 50, and 100 mg/kg for 28 days showed tumor growth inhibition (TGI) of approximately 12%, 74%, and 90%, respectively. At 50 mg/kg, G1T38 was significantly more efficacious than palbociclib.
In the ER+ MCF7 xenograft model, combinations of G1T38 (50 mg/kg) with tamoxifen (20 mg/kg s.c.) or fulvestrant (5 mg/mouse s.c. weekly) were significantly more effective than any single agent alone, resulting in near complete, sustained tumor regression.
Combination of G1T38 (25 or 50 mg/kg) with the oral SERD GW5638 (25 mg/kg) in the MCF7 model led to sustained tumor regression, greater than either single agent.
Combination of G1T38 (25 or 50 mg/kg) with the PI3K inhibitor GDC0941 (100 mg/kg) in the MCF7 model led to greater efficacy than any treatment alone.
In a NSCLC PDX model (CTG-0159, EGFR WT), daily oral G1T38 (100 mg/kg) for 28 days resulted in 77% TGI and a 60% tumor growth delay.
In the EGFR-mutant H1975 NSCLC xenograft model, G1T38 as a single agent (50 and 100 mg/kg) caused dose-dependent TGI (33.3% and 60.7% respectively by day 18). Combination of G1T38 (100 mg/kg) with erlotinib (70 mg/kg) resulted in 80% TGI, reversing erlotinib resistance. Combination with afatinib (20 mg/kg) increased the time to resistance.

Biochemical profiling of G1T38 against CDK family kinases was performed using microfluidic kinase detection technology. The compounds were tested in a 12-point dose response format in singlicate at the Km concentration for ATP. The phosphoacceptor substrate peptide concentration used was 1 μM. Staurosporine was used as the reference compound for all assays.

The cells in Costar 3903 96-well plates are seeded with 1000 cells per well for SupT1, Daudi, MCF7, ZR-75-1, A2058, WM2664, and H69; plated at 4000 cells per well for MV-4-11 and BV173; plated at 8,000 cells per well for Tom-1; and plated at 20,000 cells per well for NALM-1. Plates are dosed with Lerociclib (G1T38) at a nine-point dose concentration ranging from 10 μM to 1 nM after a 24-hour period. After four or six days, cell viability is assessed. GraphPad Prism 5 statistical software is used for data analysis after plates are processed on the BioTek Synergy2 multi-mode plate reader[1].

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To assess cell cycle effects, HS68, WM2664 (CDK4/6-dependent), and A2058 (CDK4/6-independent) cell lines were treated with G1T38. After treatment, cells were analyzed for cell cycle distribution.
To assess effects on RB phosphorylation, WM2664 cells were treated with varying doses of G1T38 for 24 hours or with 300 nM G1T38 for various time points. After treatment, cells were harvested and whole cell extracts were prepared. Total protein concentration was determined. Protein samples were heat denatured, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. Membranes were blocked and incubated overnight with primary antibodies against phospho-RB (Ser807/811), total RB, and α-tubulin (loading control). After incubation with fluorescent secondary antibodies, blots were imaged.
To assess anti-proliferative activity, various tumor cell lines were seeded in 96-well plates. After 24 hours, plates were dosed with G1T38 in a nine-point dose concentration series from 10 μM to 1 nM. Cell viability was determined after four or six days using a luminescent cell viability assay following the manufacturer's recommendations. Plates were read on a multi-mode plate reader and data analyzed.

Mice: Lerocyclib (G1T38) (100 mpk, medicated diet) is tested in female MMTV-NEU mice. Body composition is evaluated at the start of treatment, and weight measurements (in grams) are kept track of and used to calculate gross toxicity. NSCLC PDX CTG0159 tumors are implanted in female naked mice. After tumors reach a volume between 150 and 300 mm³, mice are randomly assigned to treatment groups, and dosing is started. Lerociclib (G1T38) at a dose of 100 mg/kg or the vehicle is taken orally for 28 days in a row. A lung adenocarcinoma model of H1975 NSC is implanted into female NCI Ath/nu mice. When tumors are 100–150 mm³ in size on average, mice are randomly assigned to treatment groups. For the duration of the study, mice are given oral doses of afatinib (20 mg/kg), erlotinib (70 mg/kg), or Lerociclib (50 or 100 mg/kg) either alone or in combination (Lerociclib+erlotinib or Lerociclib+afatinib). Up until the mice reach a tumor burden of 1500 mm³, all tumors are measured twice a week.

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MCF-7 Breast Cancer Xenograft Model: MCF-7 cells were injected into immunodeficient mice. When mean tumor volume reached approximately 150-250 mm³, G1T38 was administered orally daily at 10, 25, 50, or 100 mg/kg for the study duration. In combination studies, tamoxifen (20 mg/kg) was administered subcutaneously daily, fulvestrant (5 mg per mouse) was administered subcutaneously once a week, GW5638 (25 mg/kg) was administered orally daily, and GDC0941 (100 mg/kg) was administered orally daily.
Her2Neu Breast Cancer GEMM: Female MMTV-NEU mice were fed a diet containing G1T38 (100 mg/kg), incorporated based on the parameters of a 25g mouse eating 3.2g of food per day, for 28 days. Mice were monitored for tumor development.
CTG0159 PDX NSCLC Model: Female nude mice were implanted with NSCLC PDX CTG0159 tumor fragments. When tumors reached 150-300 mm³, mice were orally administered 100 mg/kg G1T38 or vehicle daily for 28 consecutive days. Tumors were measured twice weekly.
H1975 NSCLC Xenograft Model: Female NCI Athymic nude mice were implanted with H1975 cells. When tumors reached 100-150 mm³, mice were orally administered G1T38 (50 or 100 mg/kg), erlotinib (70 mg/kg), or afatinib (20 mg/kg) daily, either as single agents or in combination, for the study duration. Tumors were measured twice weekly.
Murine PK/PD Study: For tumor/plasma concentration comparison, MCF7 tumor-bearing mice were given a single oral dose of 100 mg/kg G1T38. Blood and tumors were harvested at various time points post-dose. For continuous oral PK analysis, C57BL/6 mice were administered daily oral doses of 10, 50, or 100 mg/kg G1T38 or palbociclib for 7 consecutive days. Blood was collected 24 hours post last dose.
Bone Marrow Proliferation (EdU) Study: C57BL/6 mice were administered daily oral doses of 10, 50, or 100 mg/kg G1T38 or palbociclib for 7 days. Eleven hours after the last dose, mice were injected intraperitoneally with EdU. Femurs were harvested 1 hour later for bone marrow cell isolation and flow cytometry analysis of EdU incorporation in myeloid progenitors.
28-Day Toxicology Study in Beagle Dogs: Beagle dogs (5/sex per group) were dosed once daily via oral gavage with G1T38 at 1, 2.5, or 5 mg/kg/day for 28 consecutive days at a dose volume of 5 mL/kg. Hematology samples were collected pre-test and at various time points during dosing and recovery periods.

In tumor-bearing mice, a single oral administration of 100 mg/kg G1T38 resulted in tumor concentrations 17 times higher than plasma concentrations. Forty-eight hours later, the drug was detectable in the tumor (approximately 65 ng/ml), while it was undetectable in plasma. G1T38 has a relatively short plasma half-life in mice, and accumulation after repeated administration is extremely low. In mice administered daily for 7 consecutive days, plasma G1T38 concentrations were approximately 11 ng/ml (approximately 22 nM) 24 hours after the last administration. In contrast, palbociclib concentrations under the same conditions were approximately 300 ng/ml (approximately 600 nM). In beagle dogs administered daily for 28 consecutive days, no accumulation of G1T38 was observed in pharmacokinetic studies.

In C57BL/6 mice treated with G1T38 daily for 28 consecutive days, a dose-dependent decrease in neutrophil count was observed, with a maximum decrease of 48% observed in the 100 mg/kg dose group after 28 days. This decrease was similar to that induced by palbociclib. In the same mouse study, regardless of dose, G1T38 did not cause a reduction in myeloid progenitor cell (Mac1+ Gr1+) proliferation (as determined by the EdU incorporation assay) 12 hours after 7 days of treatment, while palbociclib caused a reduction in myeloid progenitor cell proliferation of more than 50% at doses of 50 and 100 mg/kg. In a 28-day repeated-dose GLP toxicology study in beagle dogs, oral administration of G1T38 resulted in a dose-dependent decrease in hematopoietic function, including myelopenia and lymphocyte depletion. However, at clinically relevant doses, these effects were insufficient to limit the duration of administration to 28 days. No increased incidence of infection or bleeding was reported. The hematopoietic suppression was reversible after discontinuation of the drug. In the first 14 days of G1T38 treatment in beagles, the neutrophil count showed a rapid, dose-dependent decline, but reached a stable state between days 14 and 25, and recovered rapidly after discontinuation of the drug.

[1]. Preclinical development of G1T38: A novel, potent and selective inhibitor of cyclin dependent kinases 4/6 for use as an oral antineoplastic in patients with CDK4/6 sensitive tumors. Oncotarget. 2017 Jun 27;8(26):42343-42358

Lerociclib is being investigated in the clinical trial NCT02983071 (G1T38, a CDK 4/6 inhibitor, used in combination with fulvestrant for the treatment of hormone receptor-positive, Her2-negative locally advanced or metastatic breast cancer). Lerociclib is an orally bioavailable inhibitor of cyclin-dependent kinases (CDKs) type 4 (CDK4) and type 6 (CDK6) with potential antitumor activity. After administration, lerociclib selectively inhibits CDK4 and CDK6, thereby inhibiting phosphorylation of retinoblastoma protein (Rb) in early G1 phase, preventing CDK-mediated G1-S phase transition, leading to cell cycle arrest. This can inhibit DNA replication and reduce tumor cell proliferation. CDK4 and CDK6 are serine/threonine kinases upregulated in many tumor cell types and play a crucial role in regulating the progression of the cell cycle from G1 to S phase and in tumor cell proliferation. G1T38 is a novel, highly effective, selective, and orally bioavailable CDK4/6 small molecule inhibitor. This drug is designed to minimize targeted myelosuppression, potentially enabling continuous daily dosing without the need for treatment intervals required with palbociclib. G1T38 has demonstrated high selectivity in a broad kinaseome screening and exhibited potent antiproliferative activity in CDK4/6-dependent tumor models, including breast cancer and non-small cell lung cancer. This compound has completed a Phase 1a study in healthy volunteers (NCT02821624) and is currently being evaluated in a Phase 1b/2a clinical trial in combination with fulvestrant in ER+/HER2- breast cancer patients (NCT02983071).

C26H34N8O

474.6012

474.29

C, 65.80; H, 7.22; N, 23.61; O, 3.37

1628256-23-4

Lerociclib dihydrochloride;2097938-59-3

86269224

Solid powder

3.4

2

7

4

35

750

0

YPJRHEKCFKOVRT-UHFFFAOYSA-N

InChI=1S/C26H34N8O/c1-18(2)32-10-12-33(13-11-32)20-6-7-22(27-16-20)30-25-28-15-19-14-21-24(35)29-17-26(8-4-3-5-9-26)34(21)23(19)31-25/h6-7,14-16,18H,3-5,8-13,17H2,1-2H3,(H,29,35)(H,27,28,30,31)

4-[[5-(4-propan-2-ylpiperazin-1-yl)pyridin-2-yl]amino]spiro[1,3,5,11-tetrazatricyclo[7.4.0.02,7]trideca-2,4,6,8-tetraene-13,1'-cyclohexane]-10-one

Lerociclib; G1T38; G1T-38; G1T 38; G1-T38; G1 T-38; G1 T38

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.

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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).

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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)

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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).

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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

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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.

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