DYRK1A 2000 nM (IC50) MAPK 8000 nM (IC50) Cdk4/cyclin D3 9 nM (IC50) Cdk4/cyclin D1 11 nM (IC50) Cdk6/cyclin D2 16 nM (IC50)

Palbociclib hydrochloride (0–1 μM, 24 hours) has an IC50 value of 0.063 μM that suppresses retinoblastoma phosphorylation at Ser795 in MDA-MB-435 cells. It also has comparable effects on both Ser780 and Ser795 phosphorylation in colon cancer Colo-205[1]. Only MDA-MB-453 cells in the G1 phase are arrested by palbociclib hydrochloride (0–10 μM, 24 h)[1]. In MDA-MB-453 and MDA-MB-361 cells, palbociclib (500 nM, 7 days) hydrochloride enhances the expression of homologous genes (H2d1, H2k1, and B2m)[2]. With IC50 values ranging from 4 nM to 1 μM, palbociclib (0-1 μM, 6 days) hydrochloride inhibits the development of many luminal ER-positive and HER2-amplified breast cancer cell lines[3]. Palbociclib hydrochloride (0-1 μM, 3 days) produces a reversible cell cycle arrest and suppresses the growth of human liver cancer cell lines with IC50 values ranging from 0.01 μM to 3.49 μM[4].

Palbociclib hydrochloride, administered orally at 75 or 150 mg/kg every day for 14 days, causes tumors to develop more slowly and rapidly regress[1]. Tumor-free mice with reduced Treg counts and the Treg:CD8 ratio in the spleen and lymph nodes are treated with oral administration of palbociclib hydrochloride (90 mg/kg, daily for 12 days), indicating tumor-independent effects[2]. Palbociclib hydrochloride (oral dosing, 100 mg/kg, daily for 1 week) exhibits strong antitumor effects in a mosaic mouse model of genetically altered liver cancer[4].

CDK assays are run in 96-well filter plates for kinetic analysis and IC50 calculations. By infecting insect cells with baculovirus, all CDK-cyclin kinase complexes are expressed and purified. A portion of pRb fused to GST (amino acids 792–928) serves as the substrate for the assays (GST•RB-Cterm). 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2, 25 μM ATP (for CDK4-cyclin D1, CDK6-cyclin D2, and CDK6-cyclin D3), 0.25 μCi of [γ-32P]ATP, 20 ng of enzyme, 1 μg of GST•RB-Cterm, and suitable dilutions of inhibitor are included in the overall reaction volume of 0.1 mL. After adding all the ingredients to the wells—aside from the [γ-32P]ATP—they are put on a plate mixer for two minutes. Addition of [γ-32P]ATP initiates the reaction, which is then incubated for 15 minutes at 25°C. In order to allow the substrate to precipitate, the reaction is stopped by adding 0.1 mL of 20% trichloroacetic acid and keeping the plate at 4°C for at least an hour. After five well washes with 0.2 mL of 10% trichloroacetic acid, radioactive incorporation is measured using a β plate counter.

Cell Proliferation Assay[3]
Cell Types: ER-positive as well as HER2-amplified breast cancer cell lines (MDA- MB-175, ZR-75-30, CAMA-1, etc.)
Tested Concentrations: 0-1 μM
Incubation Duration: 6 days
Experimental Results: Inhibited growth of luminal ER-positive as well as HER2-amplified breast cancer cell lines.

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Cell Cycle Analysissup>[1]
Cell Types: MDA-MB-453 cells
Tested Concentrations: 0-1 μM
Incubation Duration: 24 h
Experimental Results: Arrested MDA-MB-453 cells in G1.

Animal/Disease Models: Mice bearing Colo-205 colon carcinoma xenografts (p16 deleted)[1]
Doses: 75, 150 mg/kg
Route of Administration: Oral administration; daily for 14 days
Experimental Results: Produced rapid tumor regressions and a corresponding tumor growth delay of ~50 days.

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Animal/Disease Models: Tumor-free female FVB mice[2]
Doses: 90 mg/kg
Route of Administration: Oral administration; daily for 12 days
Experimental Results: diminished total thymic mass and immature CD4+ and CD8+ double-positive thymocytes, and increased the fractions of CD4+ and CD8+ single-positive thymocytes.

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Animal/Disease Models: Genetically engineered mosaic mouse model of liver cancer (Myc;p53 -sgRNA)[4]
Doses: 100 mg/kg
Route of Administration: Oral administration; daily for 1 week
Experimental Results: diminished the luminescence signal in liver and delayed tumor growth.

Absorption, Distribution and Excretion
Palbociclib presents a linear pharmacokinetic profile and its peak plasma concentration was observed 6-12 hours after oral administration. The oral bioavailability is reported to be of 46% with a steady-state reached after 8 days and a median accumulation ratio of 2.4. The absorption of palbociclib is significantly reduced under fasting conditions and hence, food intake is recommended when this drug is administered.
The main route of elimination of palbociclib is through feces after hepatic metabolism while renal clearance seems to play a minor role accounting only for 17.5% of the eliminated dose.
The mean apparent distribution of palbociclib is 2583 L which suggests that palbociclib penetrates extensively into peripheral tissues.
The mean apparent oral clearance of palbociclib is of 63.1 L/h.

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Metabolism / Metabolites
Palbociclib is mainly hepatically transformed. the metabolism is mainly performed by the activities of the cytochrome P450 isoenzyme 3A and the sulfotransferase 2A1. The metabolism of palbociclib is represented mainly by reactions of oxidation and sulfonation followed by acylation and glucuronidation as minor reactions. After its metabolism, palbociclib forms mainly inactive glucuronide and sulfamic acid conjugates. The major circulating metabolite, accounting for 1.5% of the dose in excreta is is the glucuronide conjugate.

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Biological Half-Life
The mean plasma elimination half-life of palbociclib is 29 hours.

Hepatotoxicity
In the large clinical trials, adverse events were common and led to dose reductions in one-third of patients and discontinuation in 8%. Publications on the efficacy and safety of palbociclib rarely mentioned serum ALT elevations or hepatotoxicity. In a study of women with refractory, metastatic breast cancer, serum ALT elevations occurred in 6% [2% over 5 times ULN] receiving palbociclib and fulvestrant compared to 3% [none over 5 times ULN] on fulvestrant alone. Since its approval and more widescale use, there have been several reports of prominent ALT elevations arising after 2 or 3 cycles of palbociclib, that improved on discontinuation and recurred rapidly when restarted. Serum bilirubin and alkaline phosphatase levels were normal and symptoms were not mentioned. In addition, there have been rare reports of patients with refractory metastatic breast cancer who developed pseudocirrhosis within 2 to 3 months of starting palbociclib presenting with fatigue, jaundice and ascites with only modest elevations in serum aminotransferase and alkaline phosphatase levels. Imaging revealed a severely nodular liver, but liver histology showed desmoplastic changes in areas of necrotic metastatic tumor without cirrhosis. The liver also had vascular changes suggestive of sinusoidal obstruction syndrome, changes possibly caused by the dramatic involution of the metastatic tumor tissue combined with vascular damage. Pseudocirrhosis has been reported with other highly successful antineoplastic therapies of cancer metastatic to the liver, but the frequency is rare.
Likelihood score: C (probable rare cause of clinically apparent liver injury that may represent pseudocirrhosis from nodular transformation of the liver in response to necrosis of hepatic metastases).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of palbociclib during breastfeeding. Because palbociclib is 85% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 29 hours and it might accumulate in the infant. It is also given in combination with letrozole or fulvestrant, which may increase the risk to the infant. The manufacturer recommends that breastfeeding be discontinued during palbociclib therapy and for 3 weeks after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Binding of palbociclib to human plasma proteins in vitro accounts for approximately 85% of the administered dose.

[1]. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther. 2004 Nov;3(11):1427-38.

[2]. CDK4/6 inhibition triggers anti-tumour immunity. Nature. 2017 Aug 24;548(7668):471-475.

[3]. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast Cancer Res. 2009;11(5):R77.

[4]. Palbociclib (PD-0332991), a selective CDK4/6 inhibitor, restricts tumour growth in preclinical models of hepatocellular carcinoma. Gut. 2017 Jul;66(7):1286-1296.

Palbociclib is a member of the class of pyridopyrimidines that is 2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}pyrido[2,3-d]pyrimidin-7-one bearing additional methyl, acetyl and cyclopentyl substituents at positions 5, 6 and 8 respectively. It is used in combination with letrozole for the treatment of metastatic breast cancer. It has a role as an EC 2.7.11.22 (cyclin-dependent kinase) inhibitor and an antineoplastic agent. It is a pyridopyrimidine, an aminopyridine, a secondary amino compound, a member of piperidines, an aromatic ketone, a member of cyclopentanes and a tertiary amino compound.
Palbociclib is a piperazine pyridopyrimidine that acts in the cell cycle machinery. It is a second generation cyclin-dependent kinase inhibitor selected from a group of pyridopyrimidine compounds due to its favorable physical and pharmaceutical properties. Palbociclib was developed by Pfizer Inc after the discovery that identified the cyclin-dependent kinases as key regulators of cell growth. It was originally FDA approved on March 2015 for the treatment of HR-positive, HER2-negative advanced or metastatic breast cancer and its indications were updated in April 2019 to include male patients based on findings from postmarketing reports and electronic health records demonstrating safety and clinical efficacy.
Palbociclib is a Kinase Inhibitor. The mechanism of action of palbociclib is as a Kinase Inhibitor, and Cytochrome P450 3A Inhibitor.
Palbociclib is a unique cyclin-dependent kinase inhibitor that is used in combination with aromatase inhibitors in the treatment of postmenopausal women with metastatic breast cancer. Palbociclib is associated with transient and usually mild elevations in serum aminotransferase during therapy and to an unusual form of liver injury called pseudocirrhosis caused by shrinkage of tumor metastases in the liver combined with desmoplastic changes and vascular damage, that can be severe, progressive and even fatal.
Palbociclib is an orally available cyclin-dependent kinase (CDK) inhibitor with potential antineoplastic activity. Palbociclib selectively inhibits cyclin-dependent kinase 4 (CDK4) and 6 (CDK6), thereby inhibiting retinoblastoma (Rb) protein phosphorylation early in the G1 phase leading to cell cycle arrest. This suppresses DNA replication and decreases tumor cell proliferation. CDK4 and 6 are serine/threonine kinases that are upregulated in many tumor cell types and play a key role in the regulation of cell cycle progression.
See also: Palbociclib Isethionate (is active moiety of).

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Drug Indication
Palbociclib is indicated in combination with [letrozole] as initial endocrine-based therapy for the treatment of human epidermal growth factor receptor type 2 (HER2)-negative and hormone receptor(HR)-positive tumors in adult patients with advanced/metastatic breast cancer. It is as well approved in combination with [fulvestrant] in patients with disease progression with prior endocrine therapy. In the official labeling, the use of palbociclib should be accompanied with either an aromatase inhibition, no restricted to letrozole, as initial endocrine-based therapy in postmenopausal women or in man. The breast cancer starts as a group of cancer cells that grow into and destroy the nearby breast tissue. This growth can spread into other parts of the body which is called metastasis. According to the location of the cancer cells, it can be categorized in ductal carcinoma and lobular carcinoma. However, other types of breast cancer include inflammatory breast cancer, Paget disease of the breast, triple negative breast cancer non-Hodgkin lymphoma and soft tissue sarcoma. In males, breast cancer is usually treated as the cases of postmenopausal women and almost all the cases are ductal carcinoma.
FDA Label
Ibrance is indicated for the treatment of hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative locally advanced or metastatic breast cancer : in combination with an aromatase inhibitor; in combination with fulvestrant in women who have received prior endocrine therapy. In pre- or perimenopausal women, the endocrine therapy should be combined with a luteinizing hormone releasing hormone (LHRH) agonist.
Treatment of Ewing sarcoma
Treatment of breast malignant neoplasms

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Mechanism of Action
Palbociclib is a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor that acts by binding to the ATP pocket with an IC50 in the range of 9-15 nmol/L. It is important to consider that it presents low to absent activity against other kinases. The CDK4/6 kinase is involved, with coregulatory partner cyclin D, in the G1-S transition. Hence, inhibition of this step prevents cell cycle progression in cells in whose this pathway is functioning. This step includes the pathways of the phosphorylation of retinoblastoma protein and the E2F family of transcription factors.

C24H30CLN7O2

514.99

519.192

571189-11-2

Palbociclib;571190-30-2;Palbociclib monohydrochloride;827022-32-2;Palbociclib-d8;1628752-83-9;Palbociclib dihydrochloride;Palbociclib orotate;2757498-64-7

60167560

Light yellow to yellow solid powder

5.036

4

8

5

35

775

0

YORIVNBSTFKZTO-UHFFFAOYSA-N

InChI=1S/C24H29N7O2.2ClH/c1-15-19-14-27-24(28-20-8-7-18(13-26-20)30-11-9-25-10-12-30)29-22(19)31(17-5-3-4-6-17)23(33)21(15)16(2)32;;/h7-8,13-14,17,25H,3-6,9-12H2,1-2H3,(H,26,27,28,29);2*1H

6-acetyl-8-cyclopentyl-5-methyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one;dihydrochloride

Palbociclib 2HCl; 571189-11-2; PD 0332991 hydrochloride; 6-Acetyl-8-cyclopentyl-5-methyl-2-[[5-(1-piperazinyl)-2-pyridinyl]amino]pyrido[2,3-d]pyrimidin-7(8H)-one hydrochloride; PD-0332991 hydrochloride; 1831842-69-3; 6-acetyl-8-cyclopentyl-5-methyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one;dihydrochloride; PALBOCICLIB DIHYDROCHLORIDE;

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 : 2 mg/mL (3.88 mM)
DMSO : 1.25 mg/mL (2.43 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.)