Improved plasma stability and sustained release profile of gemcitabine via polypeptide conjugation

Abstract

To enhance the stability of the anticancer drug gemcitabine (2'-deoxy-2',2'-difluorocytidine), it was conjugated to poly-l-glutamic acid (PG-H) via a carbodiimide reaction. The synthesised poly-l-glutamic acid-gemcitabine (PG-G) was purified and characterised by using SDS-PAGE to estimate its molecular weight, HPLC to determine its purity and degree of drug loading, and NMR to elucidate the structure. In vitro aqueous hydrolytic studies showed that the gemcitabine release from the polymeric drug conjugate was pH dependent, and that the conjugation to PG-H improved its stability in human plasma. The release of the bound gemcitabine from PG-G in plasma was mediated by a hydrolytic process. It began with a lag phase, followed by linear release between 12 and 48h, and reached equilibrium at 72h with 51% of the gemcitabine released. In vitro cytotoxicity studies using MCF-7 and MDA-MB-231 human mammary cancer cells, as well as human dermal fibroblasts (HDF), showed that PG-G displayed a lower dose dependent cytotoxic effect with respect to the parent drug gemcitabine. On the other hand, in 4T1 mouse mammary tumour cells, PG-G and gemcitabine showed similar toxicities. Gemcitabine was more than likely released hydrolytically from PG-G and taken up by MCF-7, MDA-MB-231 and HDF, whereas both released gemcitabine and PG-G were taken up by 4T1 to mediate the observed cytotoxicities. The improved stability and extended sustained release profile may render PG-G a potential anticancer prodrug.

Renal targeting potential of a polymeric drug carrier, poly-L-glutamic acid, in normal and diabetic rats

“…The results coincided with the previous finding on the high water solubility of PG-drug conjugates (with drug loading ,10%) at physiological pH. 36 animals Male Sprague Dawley (SD) rats (body weight 250 g, 7 weeks old) were obtained from Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia. The rats were housed at 25°C in open rat cages at the Department of Pharmacology, Faculty of Medicine, University of Malaya, Malaysia, with a standard pellet diet.…”

Synthesis of Gemcitabine-(C<sub>4</sub>-<i>amide</i>)-[anti-HER2/<i>neu</i>] Utilizing a UV-Photoactivated Gemcitabine Intermediate: Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3

“…Gemcitabine has been covalently bound to biologically relevant ligands that inludes poly-L-glutamic acid (polypeptide configuration), [58] cardiolipin, [56,57] 1-dodecylthio-2-decyloxypropyl-3-phophatidic acid, [40,60] lipid-nucleosides, [76] N -(2-hydroxypropyl) methacrylamide polymer (HPMA), [21] benzodiazepine receptor ligand, [59,61] 4-( N )-valeroyl, 4-( N )-lauroyl, 4-( N )-stearoyl, [78] 1,1′,2-tris-noraqualenecarboxylic acid, [79] and the 4-fluoro [18F]-benzaldehyde derivative [77] for application as a positron-emitting radionuclide. Few if any published have described the molecular design, chemical synthesis and evaluation of the cytotoxic anti-neoplastic potency for gemcitabine immunochemotherapeutic created by generating a covalent bond at either the C 5 - methylhydroxy [36] or cytosine-like C 4 - amine groups of gemcitabine.…”

Synthesis of a covalent gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic and its cytotoxic anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma

“…Phase-II: synthesis of gemcitabine-(carbamate)-PMPI sulfhydryl-reactive intermediate Gemcitabine (10 mg/ml stock in DMSO) was combined with N-[p-maleimidophenyl]-isocyanate (PMPI) 65-67 at a 5:1 molar ratio in combination with constant gentle stirring at 25°C for 3.5 h so that the isocyanate moiety of PMPI which exclusively reacts with hydroxyl (R-OH) groups preferentially created a carbamate covalent bond at the terminal C 5 -methylhydroxy group of gemcitabine. 61,[68][69][70][71][72][73] The highly selective reaction is reportedly complete within 2 h under the applied conditions. Gemcitabine was formulated at a large molar excess to deplete un-reacted PMPI and maximize synthesis of the sulfhydryl-reactive maleimide intermediate as validated by high-performance thin-layer chromatography analysis (HP-TLC).…”

“…10,15,17,[20][21][22][23][24][25]27,28,32,19 Generation of a covalent bond at the C 5 -methylhydroxy group of gemcitabine represents a second alternative molecular strategy for synthesizing covalent gemcitabineligand conjugates. 61,[68][69][70][71][72][73] Gemcitabine has been covalently bound to a number of biologically relevant ligands. Most prominent in this regard have been poly-L-glutamic acid (polypeptide configuration), 70 Few if any reports have described the molecular design and efficacy evaluation of a covalent immunochemotherapeutic synthesized through the generation of a covalent bond structure at either the cytosine amine or C 5 -methylhydroxy groups of gemcitabine.…”

“…61,[68][69][70][71][72][73] Gemcitabine has been covalently bound to a number of biologically relevant ligands. Most prominent in this regard have been poly-L-glutamic acid (polypeptide configuration), 70 Few if any reports have described the molecular design and efficacy evaluation of a covalent immunochemotherapeutic synthesized through the generation of a covalent bond structure at either the cytosine amine or C 5 -methylhydroxy groups of gemcitabine. A common reaction scheme and identical conditions were applied for the thiolation of anti-HER2/neu monoclonal immunoglobulin fractions utilized to synthesize both gemcitabine-(carbamate)-[anti-HER2/neu] and epirubicin-(C 13 -imino)-[anti-HER2/ neu] immunochemotherapeutics.…”