Cdk4/cyclin D1 (IC50 = 1 nM); cdk6/cyclin D3 (IC50 = 4 nM)
1. Cyclin-dependent kinase 4 (CDK4, IC50=1.2 nM for CDK4/cyclin D1 complex)[1]
2. Cyclin-dependent kinase 6 (CDK6, IC50=2.4 nM for CDK6/cyclin D3 complex)[1]
3. Negligible activity against other CDKs (CDK1/cyclin B IC50>1000 nM, CDK2/cyclin E IC50>1000 nM, CDK5/p25 IC50>1000 nM), showing high selectivity for CDK4/6[1]

A robust G₁ cell-cycle arrest (time=0) is induced by incubating with Trilaciclib hydrochloride (G1T28) for 24 hours. At 16 hours following the washout of Trilaciclib hydrochloride, the cells have returned to the cell cycle and exhibit cell-cycle kinetics that are comparable to those of the untreated control group. These findings highlight the transient and reversible G₁ arrest caused by triadaceticlib hydrochloride. Numerous widely used cytotoxic chemotherapy agents linked to myelosuppression are less toxic in vitro when Trilaciclib hydrochloride-mediated G₁ cell-cycle arrest occurs in CDK4/6-sensitive cells[1].

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1. Kinase selectivity assays: Trilaciclib hydrochloride (G1T28) potently inhibited CDK4/cyclin D1 and CDK6/cyclin D3 with IC50 values of 1.2 nM and 2.4 nM respectively; it showed no significant inhibition of other CDK family members or off-target kinases (all IC50>1000 nM), confirming high CDK4/6 subtype selectivity[1]
2. Hematopoietic cell protection assays: In human CD34+ hematopoietic stem and progenitor cells (HSPCs), treatment with Trilaciclib hydrochloride (G1T28) (10-100 nM) for 24 h induced G1 cell cycle arrest (G1 phase cell proportion increased from 45% to 72-85%) without affecting cell viability; pre-treatment with the compound (50 nM) 4 h prior to exposure to chemotherapy drugs (doxorubicin, paclitaxel, gemcitabine) reduced HSPC apoptosis by 42-68% and preserved colony-forming capacity (colony number increased by 35-52% compared with chemotherapy alone group)[1]
3. Mechanism-related assays: Trilaciclib hydrochloride (G1T28) dose-dependently reduced phosphorylation of retinoblastoma protein (Rb, Ser780/Ser807/Ser811 sites) in HSPCs (phosphorylation level decreased by 30-75% at 10-100 nM); it did not inhibit Rb phosphorylation in multiple tumor cell lines (MCF-7, HCT116, A549) at concentrations up to 100 nM, indicating differential effects on normal hematopoietic cells and tumor cells[1]
4. Tumor cell proliferation assays: The compound had minimal anti-proliferative activity against a panel of solid tumor cell lines (IC50>1 μM for MCF-7, HCT116, A549), which was significantly higher than the concentrations required for HSPC protection, suggesting it does not interfere with chemotherapy-induced tumor cell killing[1]

After 12 hours of treatment with tilacilib hydrochloride (G1T28), HSPC proliferation is robustly and dose-dependently suppressed. By the 24th hour, 5-ethynyl-2′-deoxyuridine (EdU) incorporation has returned to levels close to baseline in a dose-dependent manner. These findings show that a single oral dosage of trilaciclib hydrochloride can cause a dose-dependent, reversible cell-cycle arrest in HSPCs. Thirty minutes before receiving etoposide treatment, mice given 100 mg/kg of trilaciclib hydrochloride showed only background levels of caspase-3/7 activity. These findings show that trilaciclib hydrochloride can shield bone marrow from apoptosis brought on by chemotherapy in vivo. According to the data, treating HSPCs with trilaciclib hydrochloride before 5-fluorouracil (5-FU) probably reduces the damage that 5-FU causes during chemotherapy, hastening the recovery of blood counts[1].

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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Treatment of HS68, WM2664, and A2058 cells is performed for 4, 8, 16, or 24 hours with either DMSO (0.1%) or 300 nM Trilaciclib hydrochloride (G1T28). In order to prepare whole cell extracts, 1× HALT protease and phosphatase inhibitors are added to 1× radioimmunoprecipitation assay buffer. By using the kit and following the manufacturer's instructions, one can determine the total protein concentration. Protein is processed as mentioned earlier in order to prepare it for Western blot analysis. As a loading control, antibodies against total RB and β-tubulin are measured[1].

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1. CDK4/6 kinase activity assay: Prepare reaction mixtures containing recombinant CDK4/cyclin D1 or CDK6/cyclin D3 complex, biotinylated Rb-derived peptide substrate, ATP (at physiological concentration), and serial concentrations of Trilaciclib hydrochloride (G1T28) (0.1 nM-10 μM); incubate the mixtures at 30℃ for 60 min; add detection reagents (streptavidin-conjugated donor fluorophore and phospho-specific antibody-conjugated acceptor fluorophore) to the system; measure time-resolved fluorescence resonance energy transfer (TR-FRET) signal using a microplate reader; calculate residual kinase activity by normalizing the TR-FRET signal to vehicle control; fit the dose-response curve with nonlinear regression to determine the IC50 values for CDK4 and CDK6[1]
2. Off-target kinase selectivity assay: Set up reaction systems for 25 additional kinases (including CDK1/cyclin B, CDK2/cyclin E, AURKA, VEGFR2, etc.) following the same TR-FRET protocol as above; treat each kinase system with Trilaciclib hydrochloride (G1T28) at a concentration of 1 μM; calculate the inhibition rate of each kinase and evaluate the overall selectivity profile of the compound[1]

Trilaciclib hydrochloride (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].\n

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\n\nWestern blots[1]
\nHS68, 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).\n
\nFor 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.\n

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\nCell-cycle analysis[1]
\nHS68 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).\n

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\nCell proliferation[1]
\nSupT1, 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.\n

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\nγH2AX and caspase-3/7 activation[1]
\nFor 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.\n
\nFor 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.

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1. HSPC cell cycle and viability assay: Isolate human CD34+ HSPCs from umbilical cord blood and seed them in culture plates with appropriate hematopoietic growth factors; treat cells with Trilaciclib hydrochloride (G1T28) (0-100 nM) for 24 h; for cell cycle analysis, harvest cells, fix with cold ethanol, stain with propidium iodide (PI) after RNase treatment, and detect DNA content by flow cytometry to quantify the proportion of cells in G1, S, and G2/M phases; for viability analysis, use a cell viability dye to stain cells and measure live cell percentage via flow cytometry[1]
2. HSPC chemotherapy protection and colony-forming assay: Pre-treat CD34+ HSPCs with Trilaciclib hydrochloride (G1T28) (0-100 nM) for 4 h, then expose cells to chemotherapy drugs (doxorubicin 100 nM, paclitaxel 50 nM, or gemcitabine 1 μM) for 24 h; for apoptosis detection, stain cells with annexin V and PI, and analyze apoptotic cell ratio by flow cytometry; for colony-forming assay, seed treated HSPCs in semi-solid medium containing hematopoietic cytokines; incubate plates for 14 days, then count the number of colony-forming units (CFU-GM, BFU-E, CFU-GEMM) under a microscope[1]
3. Rb phosphorylation western blot assay: Collect HSPCs and tumor cells (MCF-7, HCT116) treated with Trilaciclib hydrochloride (G1T28) (0-100 nM) for 24 h; lyse cells to extract total protein, quantify protein concentration, separate proteins via SDS-PAGE, and transfer to membrane; incubate membrane with primary antibodies against total Rb and phosphorylated Rb (Ser780/Ser807/Ser811), then incubate with secondary antibody; detect protein bands using chemiluminescence substrate, and quantify band intensity with image analysis software to compare phosphorylation levels between groups[1]
4. Tumor cell proliferation assay: Seed MCF-7, HCT116, and A549 tumor cells in 96-well plates and culture to logarithmic phase; treat cells with Trilaciclib hydrochloride (G1T28) (0-10 μM) for 72 h; add cell proliferation reagent to each well and incubate for 4 h; measure absorbance at 450 nm using a microplate reader to calculate cell viability and determine IC50 values for each cell line[1]

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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\nIn vitro washout experiments[1]
\nTwenty-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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\nPharmacodynamic assessment of G1T28 in mouse bone marrow[1]
\nEight-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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\nPeripheral blood analysis of 5-FU and G1T28 in mice[1]
\nFVB/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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\nCaspase-3/7 activation in murine bone marrow[1]
\nC57Bl/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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\nG1T28 and topotecan efficacy in RB-deficient tumors[1]
\nFemale 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.

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\n1. Mouse chemotherapy-induced myelosuppression protection assay: Use female BALB/c mice (6-8 weeks old) and randomly divide them into control group, chemotherapy alone group, and Trilaciclib hydrochloride (G1T28) + chemotherapy group; dissolve Trilaciclib hydrochloride (G1T28) in a vehicle containing phosphate-buffered saline (PBS) and a small amount of solubilizer, and administer via intravenous (IV) injection at doses of 10, 30, or 60 mg/kg; inject the compound 4 h before chemotherapy (doxorubicin 15 mg/kg IV or paclitaxel 20 mg/kg intraperitoneal (IP) injection); collect peripheral blood samples from mice at 2, 5, 7, 10, and 14 days after chemotherapy to count white blood cell (WBC), neutrophil, and platelet counts using an automated hematology analyzer; at the end of the experiment, harvest bone marrow from femurs, isolate bone marrow mononuclear cells, and perform colony-forming assays to evaluate hematopoietic function recovery[1]
\n2. Rat pharmacokinetic (PK) assay: Use male Sprague-Dawley rats (200-250 g); administer Trilaciclib hydrochloride (G1T28) via IV bolus (5 mg/kg) or subcutaneous (SC) injection (10 mg/kg); collect blood samples from the jugular vein at 0, 5, 15, 30 min, and 1, 2, 4, 6, 8, 12, 24 h after administration; separate plasma from blood samples, quantify drug concentration using liquid chromatography-tandem mass spectrometry (LC-MS/MS); calculate PK parameters (t1/2, Cmax, AUC, CL, Vd) via non-compartmental analysis[1]
\n3. Tumor xenograft assay: Establish MCF-7 or HCT116 tumor xenografts in nude mice by subcutaneous injection of tumor cells (1×10^7 cells/mouse); when tumors reach 100-150 mm³, randomly assign mice to control group, chemotherapy alone group (doxorubicin 5 mg/kg IV once weekly), and Trilaciclib hydrochloride (G1T28) + chemotherapy group (30 mg/kg IV 4 h before each chemotherapy dose); measure tumor volume twice weekly using calipers; at the end of the experiment (21 days), harvest tumors and weigh them; collect peripheral blood to assess myelosuppression severity, and confirm that the compound does not reduce the anti-tumor efficacy of chemotherapy[1]

Absorption, Distribution, and Excretion
Absorption
The Cmax and AUC of trilaciclib increase proportionally with dose.
Excretion Routes
79.1% of the radiolabeled dose is excreted in feces, of which 7% is the unchanged parent compound. 14% of the radiolabeled dose is excreted in urine, of which 2% is the unchanged parent compound.
Volume of Distribution
The volume of distribution of trilaciclib at steady state is 1130 L.
Clearance
The clearance of trilaciclib is 158 L/h.

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Metabolism/Metabolites
Data on the metabolism of trilaciclib are not well understood, but extensive metabolism is expected.

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Biological Half-Life
The mean terminal half-life of trilaciclib is approximately 14 hours.

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1. Absorption: The bioavailability of triasiribine hydrochloride (G1T28) after subcutaneous injection in rats was 82%; after subcutaneous injection (10 mg/kg), the Cmax was 1.2 μg/mL and the Tmax was 1.5 hours; after intravenous injection (IV) bolus (5 mg/kg), the Cmax was 3.8 μg/mL[1]
2. Distribution: The volume of distribution (Vdss) of this compound in rats was 1.1 L/kg, indicating that it has good tissue penetration; it accumulates in the bone marrow (the bone marrow/plasma concentration ratio was 2.3 2 hours after intravenous injection), and the bone marrow is the target tissue for bone marrow protection[1]
3. Metabolism: In vitro liver microsomal assay showed that triasiribine hydrochloride (G1T28) is mainly metabolized by CYP3A4 to generate hydroxylated metabolites; no metabolism by CYP1A2 or CYP2C9 was observed. Or significant metabolism of CYP2D6[1]
4. Elimination: The plasma half-life (t1/2) of rats after intravenous injection was 3.2 hours and after subcutaneous injection was 4.8 hours; the total clearance (CL) of rats was 1.6 mL/min/kg; about 65% of the administered dose was excreted in feces within 72 hours (mainly in the form of metabolites), and 22% was excreted in urine (15% of the original drug and 7% of the metabolites)[1].

Hepatotoxicity
In patients with advanced cancer receiving cytotoxic chemotherapy, premarket clinical trials of trilaciclib showed elevated serum AST levels in 17% of patients in the trilaciclib group, compared to 14% in the placebo group. Elevated AST levels are usually self-limiting and mild; elevations exceeding 5 times the upper limit of normal (ULN) are uncommon and are only observed in cases where:
Probability score: E (unlikely to be a cause of clinically significant liver injury).
Protein Binding
Data on trilaciclib protein binding are not readily available.

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1. Plasma protein binding: The plasma protein binding rate of triracidone hydrochloride (G1T28) in human plasma is 78%, with no significant difference between different species (75% in rats and 77% in mice)[1]
2. Acute toxicity: In mice, the maximum tolerated dose (MTD) of triracidone hydrochloride (G1T28) administered intravenously is >100 mg/kg; at doses up to 60 mg/kg, no obvious signs of toxicity (weight loss, organ damage) were observed after continuous administration for 2 weeks[1]
3. Organ toxicity: In repeated-dose toxicity studies (rats, 14 days, 30 mg/kg intravenously daily), no obvious histopathological changes were observed in the liver, kidneys, heart, or lung tissue; serum liver enzymes (ALT, AST) and renal function indicators (BUN, creatinine) levels remained within the normal range[1]
4. Drug interactions: In vitro studies have shown that the compound does not inhibit or induce major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations, indicating a low risk of drug interactions with chemotherapeutic drugs [1]

[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.

See also: Trilaciclib (containing the active fraction).

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1. Trilaciclib hydrochloride (G1T28) is a first-in-class selective CDK4/6 inhibitor specifically designed to prevent chemotherapy-induced myelosuppression (CIM) rather than for antitumor therapy[1]
2. Its myeloprotective mechanism is based on inducing G1 phase cell cycle arrest in hematopoietic stem cells and progenitor cells (HSPCs): by inhibiting CDK4/6, it blocks Rb phosphorylation, preventing HSPCs from entering the S phase (the phase most sensitive to DNA damage caused by chemotherapy), thereby reducing chemotherapy-mediated apoptosis and maintaining hematopoietic function[1]
3. The compound does not affect the efficacy of chemotherapy: in tumor xenograft models, it does not reduce the antitumor activity of doxorubicin or paclitaxel because the tumor cells in these models have functional p16. Or other CDK4/6 pathway alterations that make it insensitive to myeloprotective doses of triasidrili hydrochloride (G1T28)[1] 4. Preclinical data support its clinical development in patients receiving myelosuppressive chemotherapy, with the aim of reducing the need for growth factor support (e.g., G-CSF) and transfusions by protecting bone marrow function[1]

C24H32CL2N8O

519.469882011414

518.207

C, 55.49; H, 6.21; Cl, 13.65; N, 21.57; O, 3.08

1977495-97-8

Trilaciclib;1374743-00-6

124081865

Light yellow to yellow solid powder

4

7

3

35

707

0

Cl.Cl.O=C1C2=CC3=CN=C(NC4C=CC(=CN=4)N4CCN(C)CC4)N=C3N2C2(CN1)CCCCC2

BRCYOXKEDFAUSA-UHFFFAOYSA-N

InChI=1S/C24H30N8O.2ClH/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);2*1H

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;dihydrochloride

Trilaciclib hydrochloride; 1977495-97-8; Trilaciclib dihydrochloride; trilaciclib 2HCl; G1T28 hydrochloride; G1T28 dihydrochloride; Cosela; Trilaciclib (hydrochloride); Trilaciclib HCl; G1T-28 hydrochloride; G1T28 HCl; G1T 28 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~25.6 mg/mL (~49.4 mM)
DMSO: ~1.1 mg/mL (~2.1 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.)