Gemcitabine-vitamin E conjugates: Synthesis, characterization, entrapment into nanoemulsions, and in-vitro deamination and antitumor activity

Abstract

Gemcitabine is the first line therapy for pancreatic cancer. It is, however, extensively metabolized to the inactive form by deamination enzymatic reaction. Conjugation of gemcitabine with fatty acids on its 4-amino group was found to protect it from deamination deactivation reaction. The objective of the present study was to test the in-vitro anticancer activity of gemcitabine conjugated to the γ-tocotrienol isomer of vitamin E against pancreatic tumor cells. This objective was based on reported studies in which it was demonstrated that free tocotrienol isomers of vitamin E can potentiate the anticancer activity of gemcitabine. To accomplish this objective, a full synthesis scheme for gemcitabine conjugation to fatty acids (stearic and linoleic) and the tocopherol and tocotrienol isomers of vitamin E (α-T and γ-T3) was presented. The conjugates were characterized by ¹H NMR and mass spectrometry analysis and tested for their susceptibility to deamination. Also discussed is the impact of entrapping the conjugates into nanoemulsions on the physiochemical properties of the delivery system and the in vitro anticancer activity of gemcitabine against Bx-PC-3 and PNAC-1 pancreatic cancer cells. In-vitro enzymatic deamination study showed that the γ-T3 conjugate of gemcitabine was least affected by deamination deactivation reaction when compared with the free and conjugated gemcitabine in solution. Furthermore, in-vitro cytotoxicity study demonstrated that entrapment of gemcitabine-lipid conjugates into nanoemulsions significantly enhanced their anticancer activity when compared to the free drug. It was concluded that conjugation to the γ-T3 isomer is a viable option for gemcitabine delivery and is worthy of further investigation.

Keywords: (1)H NMR characterization; Enzymatic deamination; Gemcitabine; In-vitro anticancer activity; Lipid-conjugation; Nanoemulsions; Synthesis; Tocopherols; Tocotrienols; Vitamin E.

Fig. 1

Synthesis scheme of the Bocylated gemcitabine

Fig. 2

Synthesis scheme of the gemcitabine-α-tocopherol (Gem-α-T) conjugate

Fig. 3

Synthesis scheme of the gemcitabine-γ-tocotrienol (Gem-γ-T3) conjugate

Fig. 4

Synthesis scheme of the gemcitabine-stearic acid (Gem-SA) conjugate

Fig. 5

Synthesis scheme of the gemcitabine- linoleic acid (Gem-LA) conjugate

Fig. 6

¹H-NMR in CDCl₃ and ESI mass spectra of gemcitabine-lipid conjugates; (A) Gemcitabine-α-tocopherol (Gem-α-T), (B) Gemcitabine-γ-tocotrienol (Gem-γ-T3), (C) Gemcitabine-stearic acid (Gem-SA) and (D) Gemcitabine-linoleic acid (Gem-LA)

Fig. 7

Size and zeta potential of the nanoemulsions loaded with the gemcitabine-lipid conjugates in water. Each value represents the mean ± SD for triplicate samples

Fig. 8

In-vitro enzymatic deamination activity of the gemcitabine-lipid conjugates. Human recombinant cytidine deaminase (CDA) was used in the current study. 100 μM of free gemcitabine HCL or equivalents from gemcitabine-lipid conjugates were incubated with 0.25 μg of CDA at 37°C and the absorbance was recorded at λ = 220 nm for 60 min. the decrease in the absorbance intensity after CDA addition indicated that deamination reaction had taken place [12]. * P value < 0.05: indicates that Gem-γ-T3 was significantly less affected by deamination than the free gemcitabine HCL

Fig. 9

In-vitro anticancer activity of gemcitabine-lipid conjugates nanoemulsions against pancreatic cancer cell lines; (A) Bx-PC-3 and (B) PANC-1. Cells were treated for 72 hr. Values reported are the mean ± SD for triplicate samples. * P value < 0.05: indicates that gemcitabine-lipid conjugates nanoemulsions had significantly higher anticancer activity than the gemcitabine-lipid conjugates in DMSO against both cell lines. ** P value < 0.05: indicates that the Gem-γ-T3 conjugate in DMSO had significantly higher anticancer activity than the other Gem-lipid conjugates in DMSO against both cell lines