CDK4 (IC50 = 1 nM); CDK6 (IC50 = 40 nM)
Trilaciclib targets CDK4 and CDK6, two key regulators of the cell cycle. It is a highly potent, selective, and reversible inhibitor that binds to the ATP-binding pocket of these kinases. The compound has IC50 values of 1 nM for CDK4/cyclin D1 and 4 nM for CDK6/cyclin D3. By inhibiting CDK4 and CDK6, trilaciclib prevents the phosphorylation of the retinoblastoma (Rb) protein, thereby blocking the G1-to-S phase transition of the cell cycle. This cell cycle arrest is transient and reversible, allowing hematopoietic stem and progenitor cells to be protected during chemotherapy exposure while tumor cells, which are often dependent on CDK4/6 for proliferation, may also be directly inhibited.

G1T28 is a highly effective and specific CDK4/6 inhibitor that prevents RB from being phosphorylated and causes an exclusive, reversible G1 arrest. G1T28 shields RB competent cells from chemotherapy-induced damage (measured by γH2AX) and apoptosis via activation of caspase 3/7 both in vitro and in vivo. [1]
In vitro, trilaciclib has been shown to be a potent inhibitor of CDK4/6 kinase activity. It effectively inhibits the proliferation of various cancer cell lines by inducing a G1 cell cycle arrest. In cell-based assays, the compound demonstrates its ability to protect hematopoietic progenitor cells from the cytotoxic effects of chemotherapeutic agents. This cytoprotective effect is a key feature of its mechanism of action, as it allows for the preservation of the bone marrow during chemotherapy, reducing the incidence of myelosuppression.

G1T28 modulates HSPC proliferation in vivo in a dose-and time-dependent manner in both mouse and canine bone marrow. Complete blood counts (CBCs) can recover from chemotherapy more quickly in mice that have received G1T28 pretreatment. Furthermore, rather than shielding RB-deficient tumors from chemotherapy, G1T28 strengthens the anti-tumor effect.[1]
In vivo, trilaciclib has been shown to reduce the hematological toxicity caused by chemotherapy in animal models and clinical trials. It induces a robust G1 cell-cycle arrest in hematopoietic stem and progenitor cells, protecting them from the damaging effects of chemotherapy. This results in a reduction in the severity and duration of neutropenia, anemia, and thrombocytopenia. The compound is administered intravenously prior to chemotherapy. Its short-acting nature allows for the rapid recovery of normal cell cycle progression after the compound is cleared from the body.

Nanosyn CDK in vitro assay[1]
Compounds were tested in CDK2-CYCLIN A, CDK2-CYCLIN E, CDK4-CYCLIN D1, CDK6-CYCLIN D3, CDK5-p25, CDK5-p35, CDK7-CYCLIN H-MAT1, and CDK9-CYCLIN T kinase assays by Nanosyn, Inc. The assays were completed using microfluidic kinase detection technology. The compounds were tested in 12-point dose–response format in singlicate at the Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μmol/L and staurosporine was used as the reference compound for all assays.

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KINOMEscan primary screen and Kd determination[1]
G1T28 was profiled at DiscoveRx using their KINOMEscan and scanMAX screening technology. Briefly, G1T28 was tested at 100 and 1,000 times the biochemical IC50 as described in Table 1. All target kinases that responded to greater than 90% inhibition were tested as individuals for Kd determination.

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Treatments for HS68, WM2664, and A2058 cells include 300 nM Trilaciclib (G1T28) or DMSO (0.1%) for 4, 8, 16, or 24 hours. Using 1× HALT protease and phosphatase inhibitors in 1× radioimmunoprecipitation assay buffer, whole cell extracts are made. In accordance with the manufacturer's instructions, the kit is used to determine the total protein concentration. Protein is prepared as previously mentioned for Western blot analysis. Antibodies against both total RB and β-tubulin are evaluated as a loading control [1].
In vitro enzyme or receptor binding (non-cell) assays for trilaciclib involve measuring its inhibition of CDK4 and CDK6 kinase activity. These assays use purified recombinant CDK4/cyclin D1 and CDK6/cyclin D3 complexes and a peptide substrate in the presence of ATP. The phosphorylation of the substrate is measured using a radioactive (e.g., 33P-ATP) or fluorescent method. The compound is incubated with the enzyme and substrate at various concentrations, and the IC50 values are determined. For trilaciclib, IC50 values of 1 nM for CDK4/cyclin D1 and 4 nM for CDK6/cyclin D3 have been reported.

Trilaciclib (G1T28) at final concentrations of 10, 30, 100, 300, 1,000, or 3,000 nM is applied to HS68 cells for a duration of 24 hours. After harvesting, cells are preserved in ice-cold methanol. PBS-CMF (calcium magnesium free)+1% BSA, Fraction V, 20 μg propidium iodide, and 50 μg RNAse A are used to stain fixed cells. Software is used to finish the cell-cycle analysis after samples are processed on a Cyan ADP Analyzer[1].

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Western blots[1]
HS68, WM2664, and A2058 cells were treated with 300 nmol/L G1T28 or DMSO (0.1%), for 4, 8, 16, or 24 hours. Whole cell extracts were prepared using 1× radioimmunoprecipitation assay buffer containing 1x HALT protease and phosphatase inhibitors. Total protein concentration was determined by using the bicinchoninic acid (BCA) Protein Assay Kit, according to the manufacturer's instructions. Fifteen micrograms of protein was heat denatured for 10 minutes at 70°C and resolved by Novex NuPAGE SDS–PAGE gel system and transferred to 0.45 μm nitrocellulose membrane by electroblotting. Membranes were blocked in LiCor Membrane Blocking Buffer and incubated overnight with rabbit anti-pRb (Ser807/811) antibody at a 1:1,000 dilution and mouse anti-MAPK antibody at a 1:2,000 dilution, as a loading control. Secondary antibodies were Goat anti-rabbit (680RD) and Goat anti-mouse (800CW) at a 1:15,000 dilution. Blots were incubated for 1 hour, washed and imaged using LiCor ImageStudio software (Version 4.0.21).
For H69, MCF7, SupT1, and ZR75-1 Western blot analysis, protein was processed as described previously. Antibodies to total RB and β-tubulin run as a loading control were assessed. A goat anti-rabbit secondary antibody was utilized at a dilution of 1:15,000.

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Cell-cycle analysis[1]
HS68 cells were treated for 24 hours with G1T28 at 10, 30, 100, 300, 1,000, or 3,000 nmol/L final concentration. Cells were harvested and fixed in ice-cold methanol. Fixed cells were stained with 20 μg propidium iodide, 50 μg RNAse A in PBS-CMF (calcium magnesium free) + 1% BSA, Fraction V (Fisher Scientific). Samples were processed on Cyan ADP Analyzer, and cell-cycle analysis was completed using FlowJo software (Version 10.0.8; Tree Star).

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Cell proliferation[1]
SupT1, MCF7, ZR-75-1, A2058, and H69 cells were seeded at 1,000 cells per well in Costar 3903 96-well plates. After 24 hours, plates were dosed with G1T28 at a nine-point dose concentration from 10 μmol/L to 1 nmol/L. Cell viability was determined after 4 or 6 days using the CellTiter-Glo assay following the manufacturer's recommendations. Plates were processed on BioTek Synergy2 multimode plate reader and data analyzed using GraphPad Prism 5 statistical software.

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γH2AX and caspase-3/7 activation[1]
For the γH2AX assay, 30,000 HS68 cells were plated per well in 12-well plates and incubated for 24 hours at 37°C. Cells were incubated with 10, 30, 100, 300, or 1,000 nmol/L G1T28 or dimethyl sulfoxide as vehicle control for 16 hours. Plates were subsequently dosed with chemotherapy [5 μmol/L etoposide, 1 μmol/L doxorubicin, 100 μmol/L carboplatin, 156 nmol/L camptothecin, or 250 nmol/L paclitaxel]. For γH2AX, cells were harvested for analysis 8 hours after exposure to chemotherapy. Cells were fixed and stained using the H2AX Phosphorylation Assay Kit by the manufacturer's instruction. γH2AX-positive HS68 cells were quantified using FACSCalibur Flow Cytometer and FlowJo analysis software.
For the in vitro caspase-3/7 assays, HS68, H69, and SHP77 cells were seeded at 1,000 cells per well in Costar 3903 96-well plates. Cells were incubated with 10, 30, 100, 300, or 1,000 nmol/L G1T28 or dimethyl sulfoxide as vehicle control for 16 hours. Plates were subsequently dosed with chemotherapy as previously described and were analyzed directly in the plates 48 hours after chemotherapy treatment. Caspase-3/7 induction was measured using Caspase-Glo 3/7 Assay System by following the manufacturer's recommended instructions.
In vitro cell-based assays for trilaciclib are performed using hematopoietic progenitor cells and cancer cell lines. Hematopoietic progenitor cells, such as CD34+ cells, are treated with the compound in the presence or absence of chemotherapeutic agents. Cell viability, apoptosis, and colony-forming ability are assessed to evaluate the cytoprotective effects. Cancer cell lines are treated with the compound to assess its direct anti-proliferative effects. Cell cycle analysis is performed using flow cytometry to evaluate the G1 arrest. The compound's effects on cell proliferation are measured using standard assays such as MTT or CCK-8.

Mice：H69 cells are inserted into female athymic nude mice, who are then watched until treatment starts. When tumors are 150 mm³ in size, mice are given different doses of Trilaciclib (100 mg/kg) and topotecan five days a week for four weeks. Up to 60 days following treatment, tumors are measured. All mice are humanely put to death if their tumor burden reaches an excessive level before 60 days. The amounts of topotecan and trilaciclib in the blood plasma from mice given either topotecan or trilaciclib hydrochloride are processed and examined utilizing accepted techniques.

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After implanting H69 cells, female athymic nude mice are observed until the start of treatment. When the tumors are large enough (150 mm³), mice are given different doses of topotecan and trilaciclib hydrochloride (G1T28) five days a week for four weeks. A maximum of 60 days following treatment are spent measuring tumors. If a mouse's tumor burden becomes too great before 60 days, it is humanely put down. Utilizing established procedures, the levels of topotecan and Trilaciclib hydrochloride in the blood plasma from mice treated with either or both of these agents are processed and examined[1].

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In vitro washout experiments[1]
Twenty-four hours after seeding on 60-mm dishes, HS68 cells were treated with G1T28 at a 300 nmol/L final concentration for 24 hours. Wells were washed twice with PBS-CMF, and then replenished with fresh culture medium. The cells were further incubated for a series of time points (t = 16, 24, 40, 48 hours after washout). At the conclusion of the experiment, cells were harvested, fixed, and stained for cell-cycle analysis as described previously.

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Pharmacodynamic assessment of G1T28 in mouse bone marrow[1]
Eight-week-old female FVB/N mice were given a single oral dose of vehicle alone (20% Solutol, Sigma-Aldrich) or G1T28 at 50, 100, or 150 mg/kg, followed 11 or 23 hours later by a single intraperitoneal injection of 100 μg 5-ethynyl-2′-deoxyuridine (EdU). Mice were euthanized 1 hour after EdU injection (i.e., total G1T28 treatment of 12 or 24 hours), and Lineage-negative cells (Lin−) were isolated using biotin anti-mouse lineage panel and anti-biotin microbeads (Miltenyi Biotec). Lin− cells were stained for EdU following the manufacturer's instructions.

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Peripheral blood analysis of 5-FU and G1T28 in mice[1]
FVB/N female mice were given single oral doses of vehicle or G1T28 at 150 mg/kg, followed 30 minutes later by a single intraperitoneal dose of 5-fluorouracil (5-FU) at 150 mg/kg. CBCs were measured every 2 days starting on day 6. Data reported are from day 6 (Platelets), day 10 [white blood cells (WBC), neutrophils (Neu), lymphocytes (Lymph)], or day 16 [red blood cells (RBC)].

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Caspase-3/7 activation in murine bone marrow[1]
C57Bl/6 female mice were given single oral doses of vehicle, 50 mg/kg or 100 mg/kg of G1T28 followed 30 minutes later by a single intraperitoneal dose of etoposide at 2 mg/kg. Six hours after treatment, mice were euthanized and bone marrow harvested. Caspase-3/7 activation was assessed using 100,000 bone marrow cells per well as previously described.

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G1T28 and topotecan efficacy in RB-deficient tumors[1]
Female athymic nude mice were implanted with H69 cells and monitored until treatment initiation. Once tumors reached an acceptable size (150 mm3), mice were dosed in various combinations of G1T28 and topotecan for 5 days per week for 4 weeks. Tumors were measured for up to 60 days after treatment. All mice that reached excessive tumor burden before 60 days were humanely euthanized. All protocols were IACUC approved and experiments were completed at South Texas Accelerated Research Treatments (START). Topotecan and G1T28 levels in blood plasma from the mice treated with G1T28 and/or topotecan were processed and analyzed using established methods at Bioanalytical Systems, Inc.
In vivo animal experiments for trilaciclib are conducted using mouse models of chemotherapy-induced myelosuppression. Mice are treated with chemotherapeutic agents, such as topotecan or platinum-based drugs, with or without prior administration of trilaciclib. Hematological parameters, including white blood cell, red blood cell, and platelet counts, are monitored over time. Bone marrow cellularity and progenitor cell content are assessed. Tumor growth is also evaluated in xenograft models to confirm that the cytoprotective effects do not compromise the anti-tumor efficacy of chemotherapy.

Absorption, Distribution and Excretion
The Cmax and AUC of trilaciclib increase proportionally with dose. 79.1% of the radiolabeled dose is recovered in feces, of which 7% is the unchanged parent compound. 14% of the radiolabeled dose is recovered in urine, of which 2% is the unchanged parent compound. The steady-state volume of distribution of trilaciclib is 1130 L. The clearance rate of trilaciclib is 158 L/h. Metabolism/Metabolites Data on the metabolism of trilaciclib are not well understood, but extensive metabolism is expected. Biological Half-Life The mean terminal half-life of trilaciclib is approximately 14 hours.
Pharmacokinetic (PK) properties of trilaciclib are characterized by its short half-life and rapid clearance. The compound has a molecular weight of 446.55 and a molecular formula of C24H30N8O. It is slightly soluble in water and DMSO. The compound is administered intravenously, and its pharmacokinetic profile is characterized by a rapid distribution and elimination. The short half-life is a key feature of its design, allowing for transient cell cycle arrest and rapid recovery of normal cell function after drug clearance. The powder is stable when stored at -20°C for up to 3 years.

Hepatotoxicity
In premarket clinical trials of trilaciclib in patients with advanced cancer receiving cytotoxic chemotherapy, 17% of patients in the trilaciclib group experienced elevated serum AST, compared to 14% in the placebo group. Elevated AST is usually self-limiting and mild; exceeding the upper limit of normal (ULN) by 5 times is uncommon. Probability score: E (unlikely to be the cause of clinically significant liver injury). Protein Binding Currently, there are no data regarding protein binding of trilaciclib.
Toxicology (toxicology) data for trilaciclib indicate that it is generally well-tolerated. The most common adverse effects are related to its mechanism of action and include fatigue, nausea, and headache. As a CDK4/6 inhibitor, it may also cause neutropenia, although its primary purpose is to reduce chemotherapy-induced myelosuppression. The compound's short-acting nature and reversibility contribute to its favorable safety profile. Long-term safety data are being evaluated in ongoing clinical trials.

[1]. Preclinical Characterization of G1T28: A Novel CDK4/6 Inhibitor for Reduction of Chemotherapy-Induced Myelosuppression. Mol Cancer Ther. 2016 May;15(5):783-93.

Pharmacodynamics
Trilaciclib is indicated to reduce the probability of chemotherapy-induced myelosuppression in patients with extensive-stage small cell lung cancer prior to treatment with platinum-based chemotherapy regimens and etoposide or topotecan. It has a short duration of action, approximately 16 hours, and a narrow therapeutic index. Patients should be informed of the risks of injection site reactions, hypersensitivity reactions, and interstitial lung disease.
Other information: Trilaciclib is marketed under the brand name Cosela. It is a first-in-class therapy indicated to reduce the incidence of chemotherapy-induced myelosuppression in adult patients with extensive-stage small cell lung cancer when administered prior to a platinum/etoposide-containing regimen or a topotecan-containing regimen. It is also being investigated for its potential to protect the bone marrow in other cancer types and to enhance immune system function. The compound is also known as G1T28.

C24H30N8O

446.55

446.254

C, 64.55; H, 6.77; N, 25.09; O, 3.58

1374743-00-6

Trilaciclib hydrochloride;1977495-97-8

68029831

White to yellow solid powder

1.5±0.1 g/cm3

1.765

0.87

2

7

3

33

707

0

N1(CCN(C)CC1)C1C=NC(NC2N=C3C(=CN=2)C=C2C(NCC4(CCCCC4)N23)=O)=CC=1

PDGKHKMBHVFCMG-UHFFFAOYSA-N

InChI=1S/C24H30N8O/c1-30-9-11-31(12-10-30)18-5-6-20(25-15-18)28-23-26-14-17-13-19-22(33)27-16-24(7-3-2-4-8-24)32(19)21(17)29-23/h5-6,13-15H,2-4,7-12,16H2,1H3,(H,27,33)(H,25,26,28,29)

4-[[5-(4-methylpiperazin-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

G1T-28; G1T28; Trilaciclib; 1374743-00-6; G1T28; 2'-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one; Trilaciclib [USAN]; U6072DO9XG; UNII-U6072DO9XG; 4-[[5-(4-methylpiperazin-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; G1T 28; Cosela

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: 2~6.8 mg/mL (4.5~15.3 mM)

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.)