DNA synthesis
Prodrug of gemcitabine. Its ultimate mechanism of action is through intracellular conversion to gemcitabine, which is subsequently phosphorylated by deoxycytidine kinase (dCK) to active metabolites (dFdCTP) that inhibit DNA/RNA synthesis. [1]

Gemcitabine elaidate (0.2 nM-1 mM; 72 h) inhibits the growth of cells that are sensitive to and resistant to gemcitabine. For L1210/L5, L4A6, BCLO, Bara-C, C26-A, C26-G, A2780, AG6000, THX, LOX, MOLT4, and MOLT4/C8 cells, the IC50s are 0.0033, 16.0, 0.0042, 13.0, 0.0015, 0.03, 0.0025, 91, 0.0040, 0.028, and 0.088 μM, respectively[1].
Gemcitabine elaidate (0.5 nM-1 μM; 72 h) increases S phase accumulation and dose-dependent cell death in A549 and WiDR cells[2].

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CP-4126 exhibited antiproliferative activity against a panel of leukemia and solid tumor cell lines, with IC50 values generally comparable to or slightly differing from gemcitabine. For example, in L1210/L5 leukemia cells, the IC50 was 0.0033 µM (CP-4126) vs. 0.0019 µM (gemcitabine); in BCLO leukemia cells, it was 0.0042 µM vs. 0.0083 µM. [1]

- In deoxycytidine kinase (dCK)-deficient cell lines (e.g., L4A6, Bara-C, AG6000), both CP-4126 and gemcitabine were inactive or showed drastically reduced activity, confirming that CP-4126 activation is dependent on dCK. [1]

- The cytotoxic activity of CP-4126 was largely independent of nucleoside transporter function. In the presence of nucleoside transport inhibitors NBMPR (100 µM) or dipyridamole (4 µg/ml), the IC50 of gemcitabine increased up to 273-fold (e.g., in MOLT4 cells), whereas the IC50 of CP-4126 increased only marginally (e.g., 1.8-fold with NBMPR in MOLT4 cells) or not at all. [1]

- In the NCI60 in vitro screen, CP-4126 showed an average IC50 of 0.1 µM, a TGI (total growth inhibition) of 12.6 µM, and an LC50 of 70.9 µM across all 60 cell lines. Leukemia, renal cancer, and non-small cell lung cancer (NSCLC) cell lines were among the most sensitive. [1]

- COMPARE analysis of the NCI60 data indicated that the growth inhibition profile of CP-4126 correlated with those of DNA antimetabolites and topoisomerase inhibitors, and showed a high correlation with the profile of gemcitabine itself, suggesting a similar mode of action. [1]

Gemcitabine elaidate (25–120 mg/kg; i.p. every 3 days for 5 doses) inhibits the growth of certain solid tumor xenografts, including fibrous histiocytoma (TAX II–1), non-small cell lung cancer (EKVX), non-classifiable sarcoma (MHMX), malignant melanoma (THX), prostate cancer (CRL–1435), and pancreatic cancer (PANC-1)[1].
Gemcitabine elaidate (10–20 mg/kg; p.o. every 3 days for 5 doses) exhibits respectable toxicity and notable antitumor activity in the colon cancer xenograft Co6044 bearing mice[1].
Gemcitabine elaidate (p.o. once daily for 5 doses) exhibits favorable toxicity and antitumor activity; however, the 15 mg/kg dose is highly toxic in the human colon cancer xenograft Co6044[1].

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Administered intraperitoneally (i.p.) every third day for five doses at its maximum tolerated dose (MTD, 40 mg/kg), CP-4126 showed antitumor efficacy comparable to gemcitabine (MTD 120 mg/kg i.p.) in a panel of human tumor xenografts in nude mice. Active antitumor effects (T/C < 25% and/or SGD > 2) were observed in models of melanoma (THX), sarcoma (MHMX, TAXII-1, OHS), lung cancer (EKVX, H-146), breast cancer (MA-11), prostate cancer (CRL-1435), and pancreatic cancer (PANC-1, MiaPaCa-2). [1]

- CP-4126 demonstrated significant oral antitumor activity, which was in stark contrast to gemcitabine (which had low/no oral bioavailability and high toxicity orally). In the human colon cancer xenograft Co6044, oral CP-4126 (administered via gavage) showed schedule- and dose-dependent antitumor activity and toxicity. For example, oral dosing at 20 mg/kg every third day for five doses (days 1,4,7,10,13) resulted in a T/C of 16% with minimal toxicity. [1]

- In the same Co6044 model, the oral antitumor activity of CP-4126 was equivalent to its intraperitoneal activity at the optimal schedules and doses. [1]

- Oral CP-4126 also showed activity in non-small cell lung cancer xenografts (EKVX and MAKSAX models). [1]

Deoxycytidine deaminase (dCDA) inhibition assay: The effect of CP-4126 on purified dCDA enzyme activity was determined. Enzyme activity was measured at 37°C using 500 µM deoxycytidine (dCyd) or gemcitabine as a substrate, in the presence or absence of 500 µM CP-4126 or gemcitabine as competitive compounds. The reaction was terminated after 15 or 25 minutes by adding trichloroacetic acid (TCA) to precipitate proteins. The supernatant was neutralized and analyzed by reversed-phase HPLC to quantify the substrate (dCyd or gemcitabine) and its deaminated product (deoxyuridine or dFdU). CP-4126 effectively inhibited the deamination of both dCyd and gemcitabine. [1]

Cell Line: A549 and WiDR cells
Concentration: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 μM
Incubation Time: 72 h
Result: Induced a G2/M and S phase accumulation.

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Chemosensitivity test for suspension cells (e.g., leukemia lines L1210, BCLO): Cells were plated in 24-well plates at densities adjusted to their doubling times. Drugs were added immediately after plating at final concentrations ranging from 2x10^-10 M to 10^-3 M. After 72 hours of continuous exposure, cells were counted using an automated cell counter. Growth inhibition curves were plotted relative to controls (cell count at plating = 0%, cell count at 72h without drug = 100%), and IC50 values were determined by interpolation. [1]

- Chemosensitivity test for adherent monolayer cells (e.g., solid tumor lines A2780, C26-A): Cells were plated in 96-well plates. After 24 hours, serial dilutions of drugs were added to achieve final concentrations ranging from 2x10^-10 M to 2x10^-3 M. Cells were exposed for 72 hours. Then, cells were fixed with cold trichloroacetic acid (TCA) and stained with sulforhodamine B (SRB). The optical density of the dissolved dye was measured, and growth inhibition curves were generated to calculate IC50 values. [1]

- Nucleoside transporter inhibition studies: Cells were plated and pre-treated with subtoxic concentrations of nucleoside transport inhibitors NBMPR (100 µM) or dipyridamole (4 µg/ml). Serial dilutions of CP-4126 or gemcitabine were then added. After 72 hours, cell viability was assessed (counting for suspension cells, SRB for adherent cells) and IC50 values were determined in the presence and absence of the inhibitors. [1]

Female BALB/c nude (nu/nu) mice (5-8 weeks; 20-27 g) were bearing tumor of EKVX, H-146, MHMX, TAX II-1, OHS, THX, MA-11, CRL-1435, PANC-1 and MiaPaCa-2, respectively
25-120 mg/kg
I.p. every 3 days for 5 doses

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Xenograft establishment: Fragments (approx. 2x2x2 mm) of human tumor xenografts were implanted subcutaneously into both flanks of female BALB/c nude mice. Treatment began when tumors reached a minimum diameter of 4 mm (volume ~32 mm³). [1]

- Intraperitoneal (i.p.) efficacy studies: Mice bearing various xenografts were treated i.p. with CP-4126 or gemcitabine every third day for a total of five doses (q3dx5 schedule). The maximum tolerated dose (MTD) for i.p. CP-4126 was determined to be 40 mg/kg, and for gemcitabine 120 mg/kg. Tumor volume was measured regularly and calculated as 0.5 x length x width². Antitumor effect was evaluated by T/C% (median tumor volume of treated/control x 100%) and Specific Growth Delay (SGD). [1]

- Oral (p.o.) efficacy and schedule studies: Mice bearing the human colon cancer xenograft Co6044 or lung cancer xenografts were treated with CP-4126 via oral gavage using different schedules: weekly (days 1 & 8), every third day for five doses (days 1,4,7,10,13), or daily for five days (days 1-5). Doses ranged from 5 mg/kg to 150 mg/kg per administration for dose-finding. Toxicity (body weight loss, deaths) and antitumor activity (T/C%) were monitored. [1]

- Drug formulation for in vivo studies: CP-4126 was supplied as a ready-to-use, sterile colloidal suspension (liposomal formulation). For in vitro studies, it was dissolved in DMSO to make a 20 mM stock solution. Gemcitabine was used in its clinical formulation. [1]

preliminary bioavailability study was conducted in two male beagle dogs. After a single intravenous injection of CP-4126 (4 mg/kg), followed by a washout period, a second oral administration of 4.3 mg/kg in capsule form was administered, and plasma and urine samples were collected. [1] - Following intravenous administration, CP-4126 itself had a very short plasma half-life (0.05–0.07 hours), while gemcitabine (released from CP-4126) had a half-life of 1.7–3.4 hours and its metabolite dFdU had a half-life of 10.8–11.2 hours. [1]
- Following intravenous (11.9–14.4 µM·h) and oral (10.7–21.8 µM·h) administration of CP-4126, the area under the curve (AUC) of gemcitabine in plasma was similar, indicating that the prodrug has oral bioavailability. [1]
- Compared with intravenous administration, oral administration of CP-4126 resulted in lower urinary excretion of the inactive metabolite dFdU, consistent with the prodrug's inhibition of dCDA-mediated deamination. [1]

In nude mice, the maximum tolerated dose (MTD) of intraperitoneal injection of CP-4126 was 40 mg/kg, administered every three days for a total of five times, defined as a weight loss of less than 10%. [1]

- Oral administration of CP-4126 showed dose- and regimen-dependent toxicity. For example, in the Co6044 colon cancer model, daily oral administration of 40 mg/kg (qd1–5) resulted in the death of 5 out of 7 mice, while oral administration of 20 mg/kg every three days (q3dx5) did not result in death, with an average weight loss of only 1%. [1]

- Conversely, oral gemcitabine showed high toxicity even at lower doses (e.g., in the Co6044 model, 40 mg/kg q3dx3 resulted in the death of all 6 mice). [1]

[1]. Antiproliferative activity, mechanism of action and oral antitumor activity of CP-4126, a fatty acid derivative of gemcitabine, in in vitro and in vivo tumor models. Invest New Drugs. 2011 Jun;29(3):456-66.

[2]. Cell cycle effects of fatty acid derivatives of cytarabine, CP-4055, and of gemcitabine, CP-4126, as basis for the interaction with oxaliplatin and docetaxel. Int J Oncol. 2010 Jan;36(1):285-94.

Gemcitabine ester is a pyrimidine 2'-deoxynucleoside. Gemcitabine ester has been used in clinical trials for the treatment of solid tumors, lung cancer, non-small cell lung cancer, and metastatic pancreatic adenocarcinoma. Gemcitabine ester is a lipophilic unsaturated fatty acid ester derivative of gemcitabine (dFdC), an anti-metabolite deoxynucleoside analog with potential antitumor activity. The prodrug gemcitabine is hydrolyzed intracellularly by esterases and then converted to the active metabolites difluorodeoxycytidine diphosphate and triphosphate (dFdCDP and dFdCTP) by deoxycytidine kinase. dFdCDP inhibits ribonucleotide reductase, thereby reducing the pool of deoxynucleotides available for DNA synthesis; dFdCTP can be incorporated into DNA, leading to DNA chain termination and apoptosis. Due to its lipophilicity, gemcitabine 5'-trans oleate exhibits increased cellular uptake and accumulation compared to gemcitabine, resulting in a higher proportion of conversion to the active metabolites. In addition, this gemcitabine formulation may not be readily deaminated and inactivated by deoxycytidine deaminase. CP-4126 is a fatty acid ester prodrug of gemcitabine, specifically, with trans-oleic acid (trans-9-octadecenoic acid) linked to the 5' position of its glycosyl group. It is designed to overcome resistance mechanisms associated with reduced nucleoside transporter activity and to promote cellular uptake and retention. [1] - It is a lipophilic compound that can cross the cell membrane independently of nucleoside transporters. Intracellularly, CP-4126 must be hydrolyzed by an esterase to release gemcitabine, which is subsequently activated by deoxycytidine cyclase (dCK). [1] - CP-4126 itself is not a substrate of deoxycytidine deaminase (dCDA) and therefore may inhibit the deamination of gemcitabine, potentially leading to increased intracellular concentrations of the active parent drug. [1]
- This study concluded that CP-4126 is a promising anticancer drug candidate because it has transporter-independent activity, good oral bioavailability, and demonstrated efficacy comparable to intravenous gemcitabine in preclinical models. [1]

C27H43N3O5F2

527.64422

527.317

C, 61.46; H, 8.21; F, 7.20; N, 7.96; O, 15.16

210829-30-4

95058-81-4; 122111-03-9 (HCl); 116371-67-6 (Gemcitabine monophosphate free acid); 1638288-31-9 (Gemcitabine monophosphate disodium salt); 840506-29-8 [Acelarin (NUC-1031) is a ProTide transformation and enhancement of the widely-used nucleoside analogue, gemcitabine]; 892128-60-8 (LY2334737, an orally bioavailable prodrug of gemcitabine)

9828310

White solid powder

1.2±0.1 g/cm3

631.4±65.0 °C at 760 mmHg

335.7±34.3 °C

0.0±4.2 mmHg at 25°C

1.536

7.7

2

7

19

37

803

3

O[C@@H](C(F)(F)[C@H](N1C(N=C(C=C1)N)=O)O2)[C@H]2COC(CCCCCCC/C=C/CCCCCCCC)=O

HESSNRGIEVBPRB-QDDPNBLJSA-N

InChI=1S/C27H43F2N3O5/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-23(33)36-20-21-24(34)27(28,29)25(37-21)32-19-18-22(30)31-26(32)35/h9-10,18-19,21,24-25,34H,2-8,11-17,20H2,1H3,(H2,30,31,35)/b10-9+/t21-,24-,25-/m1/s1

[(2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4,4-difluoro-3-hydroxyoxolan-2-yl]methyl (E)-octadec-9-enoate

Gemcitabine elaidate; CO101; CP4126; CO-101; CP-4126; CO 101; CO-101; Gemcitabine (elaidate); CP-4126 (LVT DERIVATIVE OF GEMCITABINE); CO-1.01; 231C73W7LG; CP 4126

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ≥ 100 mg/mL (~189.52 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.74 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 25.0 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: 2.5 mg/mL (4.74 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (4.74 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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