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

体外活性：PD 0332991 对其他蛋白激酶包括 EGFR、FGFR、PGFR、IR 几乎没有影响。 PD 0332991 是 Cdk4 的非 ATP 竞争性抑制剂。 PD 0332991 抑制 MDA-MB-435 乳腺癌细胞，IC50 为 66 nM，这是由于 Ser780 处的 Rb 磷酸化减少所致。 PD 0332991 抑制胸苷掺入 Rb 阳性人乳腺癌、结肠癌、肺癌以及人白血病的 DNA，IC50 值范围为 0.04-0.17 μM。 PD 0332991 在 Rb 阴性细胞中没有显示出活性。 PD 0332991 导致 MDA-MB-453 乳腺癌细胞和 Colo-205 癌细胞中 G1 期细胞积聚。 PD 0332991 还在 5T33MM 骨髓瘤细胞（免疫活性模型）中显示出活性，并使细胞对硼替佐米的杀伤敏感。 PD 0332991 抑制 luminal ER 阳性以及 HER2 扩增的乳腺癌细胞系，包括 MDA-MB-175、ZR-75-30、CAMA-1、MDA-MB-134、HCC-202 和 UACC-893。 PD 0332991 增强这些细胞系中他莫昔芬和曲妥珠单抗的活性。 PD 0332991 增强 MCF7 他莫昔芬耐药细胞中他莫昔芬的敏感性。最近的一项研究表明，PD 0332991 可以抑制恶性横纹肌瘤 (MRT) 细胞系，包括 MP-MRT-AN、KP-MRT-RY、G401、KP-MRT-NS，且 MRT 细胞系对 PD 0332991 的敏感性呈反比。与p16的表达相关。激酶测定：在 DMSO 中制备 PD0332991 的储备溶液。 CDK 测定在 96 孔过滤板中进行。所有CDK-细胞周期蛋白激酶复合物均通过杆状病毒感染在昆虫细胞中表达并纯化。检测的底物是与 GST 融合的 pRb 片段（氨基酸 792-928）（GST·RB-Cterm）。每孔总体积为 0.1 mL，含有终浓度 20 mM Tris-HCl、pH 7.4、50 mM NaCl、1 mM 二硫苏糖醇、10 mM MgCl2、25 μM ATP（对于 CDK4-细胞周期蛋白 D1、CDK6-细胞周期蛋白 D2、和 CDK6-细胞周期蛋白 D3）或 12 μM ATP（对于 CDK2-细胞周期蛋白 E、CDK2-细胞周期蛋白 A 和 CDC2-细胞周期蛋白 B），含 0.25 μCi [γ-32P]ATP、20 ng 酶、1 μg GST·RB -Cterm 和 PD 0332991 (0.001-0.1μM)。除 [γ-32P]ATP 外的所有组分均添加至孔中，并将板置于板混合器上 2 分钟。通过添加 [γ-32P]ATP 开始反应，并将板在 25 °C 下孵育 15 分钟。通过添加 0.1 mL 20% 三氯乙酸终止反应，并将板在 4 °C 下保持至少 1 小时，以使底物沉淀。然后用 0.2 mL 10% 三氯乙酸洗涤孔 5 次，并用 β 板计数器测定放射性掺入。细胞测定：细胞（肿瘤细胞系包括 MDA-MB-435、ZR-75-1、T-47D、MCF-7、H1299、Colo-205、MDA-MB-468、H2009、CRRF-CEM 和 K562）以每孔 2 × 104 的密度接种到 96 孔板中并孵育过夜。将 PD 0332991 (0.01-1 μM) 添加到孔中，并在 37 °C 下再孵育 24 小时。将[14C]胸苷(0.1μCi)添加到每个孔中，并允许放射性标记的掺入持续72小时。用β板计数器测定掺入的放射性。

PD 0332991 表明 150 mg/kg 的 MDA-MB-435 异种移植物中肿瘤完全停滞。 PD 0332991 还通过消除肿瘤组织中的磷酸化 Rb 和增殖标记物 Ki-67 以及在 E2F 转录控制下下调基因，在多个人类肿瘤异种移植物中显示出广谱抗肿瘤活性。

CDK 测定在 96 孔滤板中进行，用于动力学分析和 IC50 计算。通过用杆状病毒感染昆虫细胞，所有 CDK-细胞周期蛋白激酶复合物都得到表达和纯化。与 GST 融合的 pRb 的一部分（氨基酸 792-928）用作测定的底物 (GST•RB-Cterm)。 20 mM Tris-HCl，pH 7.4，50 mM NaCl，1 mM 二硫苏糖醇，10 mM MgCl2，25 μM ATP（用于 CDK4-细胞周期蛋白 D1、CDK6-细胞周期蛋白 D2 和 CDK6-细胞周期蛋白 D3）、0.25 μCi [γ-32P ]ATP、20 ng 酶、1 μg GST•RB-Cterm 和适当稀释的抑制剂包含在 0.1 mL 的总反应体积中。将除 [γ-32P]ATP 之外的所有成分添加到孔中后，将它们放在平板混合器上两分钟。添加 [γ-32P]ATP 启动反应，然后在 25°C 下孵育 15 分钟。为了使底物沉淀，通过添加 0.1 mL 20% 三氯乙酸并将板在 4°C 下保持至少 1 小时来终止反应。用 0.2 mL 10% 三氯乙酸洗五次孔后，使用 β 板计数器测量放射性掺入。

在 96 孔板中，每孔接种 2 × 10⁴ 细胞并孵育一整晚。添加到孔中后，PD 0332991 (0.01-1 μM) 在 37 °C 下再孵育 24 小时。将[¹⁴C]胸苷 (0.1 μCi) 添加到每个孔中，并允许放射性标记的掺入持续 72 小时。使用β板计数器，可以测定掺入的放射性。

Advanced stage human tumor xenografts including Colo-205, MDA-MB-435 breast, SF-295 glioblastoma, ZR-75-1 breast, PC-3 prostate, H125 lung, SW-620 colon, H23 lung and MDA-MB-468 breast (Rb negative) are established in severe combined immunodeficient mice.
0-150 mg/kg
Given by gavage

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Multiple myeloma (MM) remains incurable partly because no effective cell cycle-based therapy has been available to both control tumor cell proliferation and synergize with cytotoxic killing. PD 0332991 is an orally active small molecule that potently and specifically inhibits Cdk4 and Cdk6. It has been shown to induce rapid G(1) cell cycle arrest in primary human myeloma cells and suppress tumor growth in xenograft models. To improve therapeutic targeting of myeloma progression, we combined tumor suppression by PD 0332991 with cytotoxic killing by bortezomib, a proteasome inhibitor widely used in myeloma treatment, in the immunocompetent 5T33MM myeloma model. We show that 5T33MM tumor cells proliferate aggressively in vivo due to expression of cyclin D2, elevation of Cdk4, and impaired p27(Kip1) expression, despite inhibition of Cdk4/6 by p18(INK4c) and the maintenance of a normal plasma cell transcription program. PD 0332991 potently inhibits Cdk4/6-specific phosphorylation of Rb and cell cycle progression through G(1) in aggressively proliferating primary 5T33MM cells, in vivo and ex vivo. This leads to tumor suppression and a significant improvement in survival. Moreover, induction of G(1) arrest by PD 0332991 sensitizes 5T33MM tumor cells to killing by bortezomib. Inhibition of Cdk4/6 by PD 0332991, therefore, effectively controls myeloma tumor expansion and sensitizes tumor cells to bortezomib killing in the presence of an intact immune system, thereby representing a novel and promising cell cycle-based combination therapy.[2]

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]. Mol Cancer Ther . 2004 Nov;3(11):1427-38.

[2]. Cancer Res . 2008 Jul 15;68(14):5519-23.

[3]. Breast Cancer Res . 2009;11(5):R77.

[4]. Biochem Biophys Res Commun. 2011 Sep 16;413(1):62-8.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC(50) values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r=0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p<0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.[4]

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Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.[3]

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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.

C24H29N7O2.HCL

483.99

483.214

C, 59.56; H, 6.25; Cl, 7.33; N, 20.26; O, 6.61

827022-32-2

Palbociclib;571190-30-2;Palbociclib hydrochloride;571189-11-2;Palbociclib-d8;1628752-83-9;Palbociclib isethionate;827022-33-3;Palbociclib dihydrochloride;Palbociclib orotate;2757498-64-7

11431660

Yellow solid powder

727ºC at 760 mmHg

393.5ºC

4.234

105.04

3

8

5

34

775

0

Cl.O=C1N(C2CCCC2)C2C(=CN=C(NC3C=CC(N4CCNCC4)=CN=3)N=2)C(C)=C1C(C)=O

STEQOHNDWONVIF-UHFFFAOYSA-N

InChI=1S/C24H29N7O2.ClH/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);1H

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

Palbociclib; PD-332991; PD332991; PD 332991; PD0332991; PD-0332991; PD0332991 HCl, PD-0332991 hydrochloride; Palbociclib HCl; Palbociclib (hydrochloride); PD 0332991 HCl; Palbociclib (PD-0332991) HCl; PD-0332991 hydrochloride; BKC4F3Q5XL; PD 0332991; Palbociclib HCl; Trade name: Ibrance

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 0.54 mg/mL (1.12 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 5.4 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.54 mg/mL (1.12 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.4mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: Saline: 20 mg/mL

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配方 4 中的溶解度: 20 mg/mL (41.32 mM) in 0.5%HPMC 1%Tween80 (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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配方 5 中的溶解度: 4.17 mg/mL (8.62 mM) in Lactic acid buffer (50 mM, pH 4.0) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。