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 )

The purpose of this research was to evaluate the safety of pulmonary administration of gemcitabine and to determine the maximum tolerated dose by weekly pulmonary administrations in an animal model. Five groups of eight Wistar rats received gemcitabine at doses of 2, 4, 6, or 8 mg/kg or the vehicle solution by endotracheal spray with scintigraphic imaging of lung deposition. In order to document the safety of digestive exposure, five groups of eight rats received gemcitabine at the same dosages or the vehicle solution by gavage. Nine weekly sessions were planned, and blood cell counts and histological examinations were performed in live animals at day 64. Scintigraphic imaging confirmed pulmonary deposition in 310 of 316 spray administrations (98%) with homogeneous pattern of deposition. The maximum tolerated dose of gemcitabine by pulmonary administration was 4 mg/kg. At this dosage, administered once a week for 9 consecutive weeks, there were no chemotherapy-related deaths and no clinical, histological, or hematological signs of toxicity except for a decrease in platelet and red blood cell counts, with no clinical significance. The toxicity of gemcitabine was higher via oral than lung delivery in terms of weight loss and white blood cell toxicity at dosages of 2, 4, and 6 mg/kg. Pulmonary administration of gemcitabine is safe in rats at a maximum tolerated dose of 4 mg/kg once a week for 9 weeks. At an equivalent dosage, the toxicity of gemcitabine is lower by lung than oral administration.[2]

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Gemcitabine Hydrochloride can be supplied to rats via endotracheal spray once a week for nine weeks without causing noticeable toxicity, up to a maximum tolerated dose of 4 mg/kg. At doses of 2, 4, and 6 mg/kg, gemcitabine is less toxic when administered by lung than when taken orally[2]. The median survival time is increased by more than 30 days when compared to the placebo group in the LSL-KrasG12D/+, LSL-Trp53R172H, and Pdx-1-Cre mice treated with either gemcitabine (50 mg/kg, i.p.) or the combination DMAPT/Gemcitabine Hydrochloride[3].

In a 96-well plate, BxPC-3, MIA PaCa-2, and PANC-1 cells are seeded. Cells are treated for a further 24 or 48 hours with vehicle, DMAPT, and/or Gemcitabine after 24 hours. Using the Cell Death Detection ELISA, apoptosis is measured in relation to vehicle-treated cells by counting the quantity of cytoplasmic histone-associated DNA fragments.

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Pancreatic cancer patients treated with gemcitabine (2',2'-difluorodeoxycytidine) can eventually develop resistance. Recently, published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C). The current study examined the possible mechanism for this I3C-enhanced efficacy. Several pancreatic cell lines (BxPC-3, Mia Paca-2, PL-45, AsPC-1 and PANC-1) were examined for modulation of human equilibrative nucleoside transporter 1 (hENT1) expression, the major transporter for gemcitabine, by I3C alone and combined with gemcitabine. I3C significantly (p<0.01) up-regulated hENT1 expression in several cell lines. Gemcitabine alone showed no effect on hENT1 expression. However, combining gemcitabine with I3C further increased hENT1 expression. Cell viability assays revealed no effect of I3C on normal cells, hTERT-HPNE. hENT1-specific inhibitor, nitrobenzylthioinosine, significantly abrogated I3C-induced gemcitabine cytotoxicity, further demonstrating its specificity. This study demonstrates that up-regulation of hENT1 expression may be a novel mechanism involved in the additive effect of I3C and gemcitabine.[1]

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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Dissolved in PBS; 50 or 100 mg/kg; i.p. injection

Athymic nude mice with MIA PaCa-2 cells

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/m² in males and ranged from 31 to 69 L/h/m² 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.

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.

[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

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

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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 20 mg/mL

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Solubility in Formulation 5: 60 mg/mL (200.23 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)