Novel formulation of a methotrexate derivative with a lipid nanoemulsion

Figures & data

Figure 1 Esterification reaction of methotrexate in the presence of cesium carbonate.

Figure 2 Transmission electron microscopy of (A) lipid nanoemulsion and (B) LDE-ddMTX.

Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Figure 3 In vitro release profile of ddMTX from LDE in dialysis against human plasma at 37°C (mean ± standard deviation, n = 5).

Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Table 1 IC₅₀ values of methotrexate, ranging from 2.25 to 22.5 mM and of LDE-dMTX, ranging from 0.1 to 2 mM in K562 and HL60 cell lines. Cells were incubated for 48 hours at 37°C

Formulations	IC50 (mM)
	K562	HL60
LDE-ddMTX	1.6	0.2
MTX	18.2	26

Abbreviations: ddMTX, didodecyl methotrexate; MTX, methotrexate; LDE, lipid nanoemulsion; IC₅₀, 50% inhibitory concentration.

Figure 4 Cytotoxicity of (A) LDE (o), LDE-ddMTX (●), and methotrexate (■) in (B) 562 (I) and HL60 (II) cell lines. The cells were incubated with the formulations (final concentration range 0.1–2.0 mM) for 4 hours at 37°C, and survival was quantified by enzyme-linked immunosorbent assay. Results are presented the mean ± standard error of the mean of three experiments.

Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Figure 5 Uptake of LDE-ddMTX (●) and methotrexate (○) 4 hours by (A) K562 and (B) HL60 cell lines. Cells were incubated with the formulations (final concentration range 0.15–2.0 mM) for 4 hours at 37°C, lysed, and the drugs were then quantified by high-performance liquid chromatography. Results are presented the mean ± standard error of the mean of three experiments.

Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion.

Table 2 Maximum tolerated dose and 50% lethal dose values of methotrexate and LDE-ddMTX intravenously administered as a single dose in Balb/c mice

Formulations	Single dose
	MTD	LD50
LDE-ddMTX	47 mg/kg	115 mg/kg
MTX	94 mg/kg	470 mg/kg

Abbreviations: ddMTX, didodecyl methotrexate; MTX, methotrexate; LDE, lipid nanoemulsion; LD₅₀, 50% lethal dose; MTD, maximum tolerated dose.

Figure 6 (A) Red cell count, (B) platelet count, (C) leukocyte count, and (D) leukocyte differential count in mice after 24 hours of intravenous LDE-ddMTX or methotrexate (both at 180 mg/kg dose) administration and after intravenous LDE without methotrexate.

Notes: ns indicates p > 0.05; *** indicates p < 0.001.

Abbreviations: ddMTX, didodecyl methotrexate; LDE, lipid nanoemulsion; LYMPH, lymphocytes; NEUTRO, neutrophyls; EOSINO, eosinophyls; MONO, monocytes; LYMPHOBL, lymphoblast.