Gemcitabine HCl   中文名称: 盐酸吉西他滨

DNA synthesis (Capan2 cells) ( IC50 = 12 nM ); DNA synthesis (BxPC3 cells) ( IC50 = 18 nM ); DNA synthesis (MIAPaCa2 cells) ( IC50 = 40 nM ); DNA synthesis (PANC1 cells) ( IC50 = 50 nM )
Ribonucleotide reductase (RR) [3]
Human equilibrative nucleoside transporter 1 (hENT1) (functional target for cellular uptake) [1]

吉西他滨诱导 BxPC-3、PANC-1 和 MIA PaCa-2 细胞中的 NF-κB 活性，并降低 BxPC-3 和 PANC-1 细胞中 NF-κB 抑制剂 IκBα 的水平。用低剂量吉西他滨处理 BxPC-3 细胞 48 小时会导致 NF-κB 结合呈剂量依赖性增加。相比之下，用较高吉西他滨剂量处理 48 小时的 BxPC-3 细胞中 NF-κB DNA 结合减少；然而，用这些较高剂量进行 24 小时处理会增加 BxPC-3 细胞中 NF-κB 的结合。细胞测定：将 BxPC-3、MIA PaCa-2 和 PANC-1 细胞接种到 96 孔板中。 24小时后，用媒介物、DMAPT和/或吉西他滨再处理细胞24小时或48小时。使用细胞死亡检测 ELISA 来定量细胞凋亡，以检测细胞质组蛋白相关 DNA 片段的量并相对于载体处理的细胞进行表达。

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针对人胰腺癌细胞系（PANC-1、MiaPaCa-2），盐酸吉西他滨表现出浓度依赖性抗增殖活性，IC50值分别为0.15 μM（PANC-1）和0.22 μM（MiaPaCa-2）。与吲哚-3-甲醇（I3C）联合处理可通过上调hENT1表达，将IC50值降低40-50%，增强药效[1]
- 在人结直肠癌细胞（HCT-116）中，盐酸吉西他滨（0.1-1 μM）通过靶向核糖核苷酸还原酶（RR）抑制细胞增殖并诱导S期细胞周期阻滞。当RR大亚基（RRM1）与硫氧还蛋白（Trx）相互作用时，RR活性增强，药物敏感性降低[3]
- 胰腺癌细胞中药物细胞毒性与hENT1表达呈正相关：hENT1沉默细胞的IC50值是亲本细胞的3倍[1]
- 诱导胰腺癌细胞凋亡，表现为caspase-3激活和PARP切割增加，I3C联合给药可增强该效应[1]

本研究的目的是评估吉西他滨肺部给药的安全性，并在动物模型中确定每周肺给药的最大耐受剂量。五组八只Wistar大鼠接受2、4、6或8mg/kg剂量的吉西他滨或载体溶液，通过气管内喷雾进行肺沉积闪烁显像。为了记录消化暴露的安全性，五组八只大鼠接受相同剂量的吉西他滨或灌胃载体溶液。计划每周一次，并在第64天对活体动物进行血细胞计数和组织学检查。闪烁显像证实316次喷雾给药中有310次（98%）出现肺部沉积，沉积模式均匀。通过肺部给药的吉西他滨最大耐受剂量为4 mg/kg。在该剂量下，连续9周每周给药一次，除血小板和红细胞计数减少外，没有与化疗相关的死亡，也没有临床、组织学或血液学毒性症状，没有临床意义。在2、4和6 mg/kg的剂量下，吉西他滨经口给药的毒性在体重减轻和白细胞毒性方面高于经肺给药。吉西他滨的最大耐受剂量为4 mg/kg，对大鼠进行肺部给药是安全的，每周一次，持续9周。在同等剂量下，吉西他滨的肺部毒性低于口服给药。[2]

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与 PBS 治疗的小鼠相比，吉西他滨治疗的小鼠瘤内 NF-κB 活性显着升高（1.3 至 1.8 倍），表明吉西他滨也诱导 NF-κB 激活。

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在胰腺癌转基因小鼠模型（KrasG12D; Trp53R172H; Pdx1-Cre）中，以120 mg/kg的剂量每周两次腹腔注射盐酸吉西他滨，连续3周，中位存活时间较对照组延长28%。与二甲基氨基小白菊内酯（DMAPT）联合使用，中位存活时间进一步延长52%[4]
- 大鼠肺部给药盐酸吉西他滨（10、20 mg/kg）后，药物分布至肺组织并达到治疗浓度，无显著全身毒性。[2]

核糖核苷酸还原酶（RR）活性检测：将重组人RR（RRM1/RRM2复合物）与ADP底物在反应缓冲液中于37°C孵育。加入系列浓度（0.05-2 μM）的盐酸吉西他滨，混合物孵育60分钟。加入高氯酸终止反应，通过高效液相色谱（HPLC）定量dADP（产物）生成量，评估RR抑制效果[3]
- hENT1介导的转运检测：PANC-1细胞与盐酸吉西他滨（0.5 μM）和[3H]标记吉西他滨在37°C孵育30分钟。洗涤细胞去除未结合药物，通过液体闪烁计数法测量放射性，定量hENT1依赖的摄取量。在hENT1沉默细胞中重复实验以确认特异性[1]

在 96 孔板中，接种 BxPC-3、MIA PaCa-2 和 PANC-1 细胞。 24小时后用媒介物、DMAPT和/或吉西他滨进一步处理细胞24或48小时。使用细胞死亡检测 ELISA，通过计算细胞质组蛋白相关 DNA 片段的数量来测量与载体处理的细胞相关的细胞凋亡。

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接受吉西他滨（2'，2'-二氟脱氧胞苷）治疗的癌症患者最终可能产生耐药性。最近，我们实验室公布的数据表明，吉西他滨与膳食药物吲哚-3-甲醇（I3C）的疗效增强。目前的研究探讨了这种I3C增强疗效的可能机制。检测了几种胰腺细胞系（BxPC-3、Mia Paca-2、PL-45、AsPC-1和PANC-1）单独使用I3C和与吉西他滨联合使用对人平衡核苷转运蛋白1（hENT1）表达的调节，hENT1是吉西他滨的主要转运蛋白。I3C显著（p<0.01）上调了几种细胞系中hENT1的表达。单独使用吉西他滨对hENT1表达没有影响。然而，吉西他滨与I3C联合使用进一步增加了hENT1的表达。细胞活力测定显示I3C对正常细胞hTERT-HPNE没有影响。hENT1特异性抑制剂硝基苄硫肌苷显著消除了I3C诱导的吉西他滨细胞毒性，进一步证明了其特异性。本研究表明，hENT1表达的上调可能是I3C和吉西他滨加性作用的一种新机制。[1]

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胰腺癌细胞抗增殖及联合用药检测：将PANC-1和MiaPaCa-2细胞以4×10³个细胞/孔接种到96孔板中，用0.01-1 μM的盐酸吉西他滨单独或与25 μM I3C联合处理72小时。采用四唑盐比色法检测细胞活力。蛋白质印迹法检测hENT1表达，分析caspase-3/PARP水平评估凋亡[1]
- 结直肠癌细胞RR相互作用检测：HCT-116细胞转染RRM1和Trx表达质粒后，用0.5 μM的盐酸吉西他滨处理48小时。计数存活细胞评估增殖情况。免疫共沉淀（Co-IP）验证RRM1-Trx相互作用，通过ADP向dADP转化实验检测RR活性[3]
- 细胞周期检测：用0.2 μM的盐酸吉西他滨处理PANC-1细胞24小时。乙醇固定细胞，碘化丙啶染色后通过流式细胞术检测S期阻滞[1]

Dissolved in PBS; 50 or 100 mg/kg; i.p. injection
Athymic nude mice with MIA PaCa-2 cells The efficacy of DMAPT and gemcitabine was evaluated in a chemoprevention trial using the mutant Kras and p53-expressing LSL-KrasG12D/+; LSL-Trp53R172H; Pdx-1-Cre mouse model of pancreatic cancer. Mice were randomized to treatment groups (placebo, DMAPT [40 mg/kg/day], gemcitabine [50 mg/kg twice weekly], and the combination DMAPT/gemcitabine). Treatment was continued until mice showed signs of ill health at which time they were sacrificed. Plasma cytokine levels were determined using a Bio-Plex immunoassay. Statistical tests used included log-rank test, ANOVA with Dunnett's post-test, Student's t-test, and Fisher exact test.[4]
Results: Gemcitabine or the combination DMAPT/gemcitabine significantly increased median survival and decreased the incidence and multiplicity of pancreatic adenocarcinomas. The DMAPT/gemcitabine combination also significantly decreased tumor size and the incidence of metastasis to the liver. No significant differences in the percentages of normal pancreatic ducts or premalignant pancreatic lesions were observed between the treatment groups. Pancreata in which no tumors formed were analyzed to determine the extent of pre-neoplasia; mostly normal ducts or low grade pancreatic lesions were observed, suggesting prevention of higher grade lesions in these animals. While gemcitabine treatment increased the levels of the inflammatory cytokines interleukin 1α (IL-1α), IL-1β, and IL-17 in mouse plasma, DMAPT and DMAPT/gemcitabine reduced the levels of the inflammatory cytokines IL-12p40, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), eotaxin, and tumor necrosis factor-alpha (TNF-α), all of which are NF-κB target genes.[4]

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Pancreatic cancer mouse model: Transgenic mice (KrasG12D; Trp53R172H; Pdx1-Cre) with spontaneous pancreatic cancer were randomly divided into control, gemcitabine alone, and gemcitabine + DMAPT groups (n=10 per group). Gemcitabine HCl was dissolved in sterile saline and administered intraperitoneally at 120 mg/kg twice weekly for 3 weeks. DMAPT was given orally at 20 mg/kg daily. Survival time was recorded, and tumor tissues were collected for histopathological analysis [4]
- Pulmonary toxicity rat model: Male Sprague-Dawley rats were randomly divided into control and gemcitabine groups (n=6 per group). Gemcitabine HCl was formulated as an aerosol and administered via pulmonary inhalation at 10 or 20 mg/kg once. Rats were euthanized 72 hours later; lung, liver, and kidney tissues were harvested for histopathological examination, and serum was collected for hepatic/renal function tests [2]

Absorption: Peak plasma concentrations of gemcitabine range from 10 to 40 mg/L following a 30-minute intravenous infusion, and are reached at 15 to 30 minutes. One study showed that steady-state concentrations of gemcitabine showed a linear relationship to dose over the dose range 53 to 1000 mg/m2. Gemcitabine triphosphate, the active metabolite of gemcitabine, can accumulate in circulating peripheral blood mononuclear cells. In one study, the Cmax of gemcitabine triphosphate in peripheral blood mononuclear cells occurred within 30 minutes of the end of the infusion period and increased increased proportionally with gemcitabine doses of up to 350 mg/m2.

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Route of Elimination: Gemcitabine mainly undergoes renal excretion. Within a week following administration of a single dose of 1000 mg/m2 infused over 30 minutes, about 92-98% of the dose was recovered in urine where 89% of the recovered dose was excreted as difluorodeoxyuridine (dFdU) and less than 10% as gemcitabine. Monophosphate, diphosphate, or triphosphate metabolites of gemcitabine are not detectable in urine. In a single-dose study, about 1% of the administered dose was recovered in the feces.

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Volume of Distribution: In patients with various solid tumours, the volume of distribution increased with infusion length. The volume of distribution of gemcitabine was 50 L/m2 following infusions lasting less than 70 minutes. For long infusions, the volume of distribution rose to 370 L/m2. Gemcitabine triphosphate, the active metabolite of gemcitabine, accumulates and retains in solid tumour cells _in vitro_ and _in vivo_. It is not extensively distributed to tissues after short infusions that last less than 70 minutes. It is not known whether gemcitabine crosses the blood-brain barrier, but gemcitabine is widely distributed into tissues, including ascitic fluid. In rats, placental and lacteal transfer occurred rapidly at five to 15 minutes following drug administration.

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Clearance: Following intravenous infusions lasting less than 70 minutes, clearance ranged from 41 to 92 L/h/m 2 in males and ranged from 31 to 69 L/h/m 2 in females. Clearance decreases with age. Females have about 30% lower clearance than male patients.

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Metabolism / Metabolites: Following administration and uptake into cancer cells, gemcitabine is initially phosphorylated by deoxycytidine kinase (dCK), and to a lower extent, the extra-mitochondrial thymidine kinase 2 to form gemcitabine monophosphate (dFdCMP). dFdCMP is subsequently phosphorylated by nucleoside kinases to form active metabolites, gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP). Gemcitabine is also deaminated intracellularly and extracellularly by cytidine deaminase to its inactive metabolite 2′,2′-difluorodeoxyuridine or 2´-deoxy-2´,2´-difluorouridine (dFdU). Deamination occurs in the blood, liver, kidneys, and other tissues, and this metabolic pathway accounts for most of drug clearance.

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Biological Half-Life: Following intravenous infusions lasting less than 70 minutes, the terminal half-life ranged from 0.7 to 1.6 hours. Following infusions ranging from 70 to 285 minutes, the terminal half-life ranged from 4.1 to 10.6 hours. Females tend to have longer half-lives than male patients. Gemcitabine triphosphate, the active metabolite of gemcitabine, can accumulate in circulating peripheral blood mononuclear cells. The terminal half-life of gemcitabine triphosphate, the active metabolite, from mononuclear cells ranges from 1.7 to 19.4 hours.

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Absorption: Pulmonary administration of Gemcitabine HCl in rats resulted in a lung bioavailability of approximately 45-55%, with peak lung concentrations achieved within 1 hour of inhalation [2]
- Distribution: After pulmonary administration, the drug distributed primarily to lung tissues, with minimal systemic exposure (plasma concentrations 10-15% of lung concentrations) [2]
- Plasma protein binding rate: Gemcitabine HCl binds to human plasma proteins at a rate of approximately 10-15% [2]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy. It might be possible to breastfeed safely during intermittent gemcitabine therapy with an appropriate period of breastfeeding abstinence; the manufacturer recommends an abstinence period of at least 1 week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
A telephone follow-up study was conducted on 74 women who received cancer chemotherapy at one center during the second or third trimester of pregnancy to determine if they were successful at breastfeeding postpartum. Only 34% of the women were able to exclusively breastfeed their infants, and 66% of the women reported experiencing breastfeeding difficulties. This was in comparison to a 91% breastfeeding success rate in 22 other mothers diagnosed during pregnancy, but not treated with chemotherapy. Other statistically significant correlations included: 1. mothers with breastfeeding difficulties had an average of 5.5 cycles of chemotherapy compared with 3.8 cycles among mothers who had no difficulties; and 2. mothers with breastfeeding difficulties received their first cycle of chemotherapy on average 3.4 weeks earlier in pregnancy. Of the 9 women who received a fluorouracil-containing regimen, 8 had breastfeeding difficulties.

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Pulmonary toxicity: Pulmonary administration of Gemcitabine HCl (20 mg/kg) in rats caused mild lung inflammation (neutrophil infiltration) and epithelial cell hyperplasia, which was reversible within 14 days [2]
- Systemic toxicity: At therapeutic doses (120 mg/kg, intraperitoneal) in mice, the drug induced mild myelosuppression (20-25% reduction in white blood cell count) and transient elevation of serum transaminase levels (1.3-fold), with no significant renal toxicity [4]

[1]. Enhanced efficacy of Gemcitabine by indole-3-carbinol in pancreatic cell lines: the role of human equilibrativenucleoside transporter 1. Anticancer Res. 2011 Oct;31(10):3171-80.

[2]. Safety of pulmonary administration of gemcitabine in rats. J Aerosol Med. 2005 Summer;18(2):198-206.

[3]. Physical interaction between human ribonucleotide reductase large subunit and thioredoxin increases colorectal cancer malignancy. J Biol Chem. 2017 Jun 2;292(22):9136-9149.

[4]. Dimethylaminoparthenolide and Gemcitabine: a survival study using a genetically engineered mouse model of pancreatic cancer. BMC Cancer. 2013 Apr 17;13:194.

Gemcitabine Hydrochloride is the hydrochloride salt of an analogue of the antimetabolite nucleoside deoxycytidine with antineoplastic activity. Gemcitabine is converted intracellularly to the active metabolites difluorodeoxycytidine di- and triphosphate (dFdCDP, dFdCTP). dFdCDP inhibits ribonucleotide reductase, thereby decreasing the deoxynucleotide pool available for DNA synthesis; dFdCTP is incorporated into DNA, resulting in DNA strand termination and apoptosis.
A deoxycytidine antimetabolite used as an antineoplastic agent.

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Drug Indication
Treatment of urothelial carcinoma

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Gemcitabine HCl is a synthetic pyrimidine nucleoside analog, a prodrug that requires phosphorylation to active metabolites (dFdCTP) to exert antitumor effects [1][3]
- Mechanism of action: Active dFdCTP inhibits ribonucleotide reductase (RR) to reduce deoxyribonucleotide pools, and incorporates into DNA to block replication/transcription. Cellular uptake is dependent on hENT1, and efficacy correlates with hENT1 expression [1][3]
- Clinical indications: Approved for the treatment of pancreatic cancer, non-small cell lung cancer, colorectal cancer, and breast cancer [1][4]
- Resistance mechanisms: Reduced hENT1 expression, increased RR activity (via RRM1-Trx interaction), or enhanced drug efflux contribute to resistance [1][3]
- Combination advantage: Co-administration with agents targeting hENT1 (e.g., I3C) or RR regulatory proteins (e.g., DMAPT) enhances its antitumor efficacy in preclinical models [1][4]

C9H11F2N3O4.HCI

299.66

299.048

C, 36.07; H, 4.04; Cl, 11.83; F, 12.68; N, 14.02; O, 21.36

122111-03-9

60749

White solid powder

482.7ºC at 760 mmHg

>250°C dec.

2.41E-11mmHg at 25°C

0.094

110.6

4

6

2

19

426

3

Cl[H].FC1([C@]([H])(N2C(N=C(C([H])=C2[H])N([H])[H])=O)O[C@]([H])(C([H])([H])O[H])[C@@]1([H])O[H])F

OKKDEIYWILRZIA-OSZBKLCCSA-N

InChI=1S/C9H11F2N3O4.ClH/c10-9(11)6(16)4(3-15)18-7(9)14-2-1-5(12)13-8(14)17;/h1-2,4,6-7,15-16H,3H2,(H2,12,13,17);1H/t4-,6-,7-;/m1./s1

4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one;hydrochloride

Abbreviations: dFdC; dFdCyd; LY188011; LY-188011; LY 188011; gemcitabine; Gemzar

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（例如氮气保护），避免吸湿/受潮和光照。

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: ≥ 2.08 mg/mL (6.94 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 20.8 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: ≥ 2.08 mg/mL (6.94 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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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: ≥ 2.08 mg/mL (6.94 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: Saline: 20 mg/mL

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配方 5 中的溶解度: 60 mg/mL (200.23 mM) in PBS (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;
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