Pharmaceutical characterization of novel tenofovir liposomal formulations for enhanced oral drug delivery: in vitro pharmaceutics and Caco-2 permeability investigations

Figures & data

Figure 1 Chemical structure of (A) tenofovir and (B) tenofovir disoproxil fumarate.

Table 1 System suitability data of six injections of 2 µg/mL tenofovir and 2 µg/mL ddC as a resolution standard

USP criteria	Specification	Day 1	Day 2	Day 3	Pass/fail
Retention time	≤2.0%	0.248%	0.249%	0.085%	Pass
Capacity factor k’	>0.3	0.623	0.670	0.698	Pass
Symmetry	>0.50	0.612	0.619	0.630	Pass
Area	≤2.0%	0.328%	0.288%	0.237%	Pass
Theoretical plates	>3000	22,361	22,402	22,027	Pass
USP tailing	>1.75	1.609	1.587	1.557	Pass
Resolution	>3.0	15.119	15.024	14.895	Pass

Abbreviations: ddC, dideoxy-cytosine; USP, United States Pharmacopeial Convention.

Figure 2 HPLC chromatograms for (A) blank, (B) system suitability (6 µg/mL tenofovir and 6 µg/mL ddC), (C) EF sample of formulation A, (D) EF sample of formulation B, (E) Caco-2 study sample of formulation A, and (F) Caco-2 study sample of formulation B. Formulations were prepared using 50 mg cholesterol, either 7.5% (formulation A) or 15% (formulation B) stearylamine as a positive charge imparting agent and an amount of phospholipon 100H to make a total lipid pool of 150 mg.

Abbreviations: HPLC, high-performance liquid chromatography; EF, entrapment efficiency; ddC, dideoxy-cytosine.

Table 2 Linearity, accuracy, and precision data of tenofovir analysis

Linearity data of tenofovir calibration sets
Standard curve	Range (µg/mL)	Calibrators	Slope	y-Intercept	R^2 value
Validation 1	1.0–10	6	0.0566	−0.0691	0.9995
Validation 2	1.0–10	6	0.0579	−0.0948	0.9996
Validation 3	1.0–10	6	0.0603	−0.0166	0.9995
Accuracy and precision: drug substance (n = 6)
Validation parameters	Validation solutions	1 µg/mL	2 µg/mL	6 µg/mL	10 µg/mL
Accuracy (%)	Set 1	94.6	96.8	101.2	99.4
	Set 2	95.5	98.6	100.0	99.5
	Set 3	98.0	99.1	100.7	99.8
Precision (%RSD)	Set 1	2.58	1.51	0.625	1.15
	Set 2	0.84	1.17	0.334	0.49
	Set 3	0.98	1.61	0.72	1.70

Abbreviation: RSD, relative standard deviation.

Table 3 Robustness data (mean ± relative standard deviation) expressed as nominal, low, and high values for flow rate, injection volume, column temperature, organic-phase fraction, and pH value of mobile-phase variations

Parameters	Change	Retention time	Capacity factor (k’)	Symmetry	Area	Resolution	Plates (half width), m
Nominal condition		4.055 ± 0.092	0.674 ± 0.225	0.668 ± 0.744	218.63 ± 0.152	15.748 ± 0.497	31,804 ± 1.122
Flow rate, mL/min	(Low) 0.975	4.149 ± 0.158	0.713 ± 0.414	0.671 ± 0293	224.4 ± 0.293	15.838 ± 0.092	32,472 ± 0.091
	(High) 1.025	3.966 ± 0.065	0.637 ± 0.209	0.666 ± 0.551	213.58 ± 0.069	15.652 ± 0.269	31,360 ± 1.226
Injection volume, µL	(Low) 7.5	4.314 ± 0.085	0.780 ± 0.170	0.710 ± 0.928	161.113 ± 0.203	15.352 ± 0.419	35,811 ± 1.468
	(High) 12.5	4.326 ±0.14	0.785 ± 0.253	0.716 ± 0.293	272.5 ± 0.231	15.166 ± 0.318	34,952 ± 1.673
Temperature, °C	(Low) 24	3.897 ± 0.462	0.612 ± 0.419	0.680 ± 0.365	201.6 ± 0.275	15.173 ± 0.397	31,526 ± 0.306
	(High) 28	3.865 ± 0.076	0.595 ± 0.203	0.688 ± 0.704	202.733 ± 0.138	15.108 ± 0.591	32,442 ± 1.395
Organic phase, %	(Low) 4.9	4.082 ± 0.164	0.685 ± 0.430	0.660 ± 0.606	224.9 ± 0.275	15.807 ± 0.336	30,072 ± 1.328
	(High) 5.1	3.89 ± 0.068	0.605 ± 0.171	0.655 ± 0.648	217.7 ± 0.369	15.493 ± 0.362	29,410 ± 0.134
pH	(Low) 7.3	3.799 ± 0.060	0.568 ± 0.133	0.632 ± 0.527	234.0 ± 0.307	14.139 ± 0.396	27,362 ± 1.247
	(High) 7.5	3.475 ± 0.148	0.434 ± 0.553	0.671 ± 0.523	233.8 ± 0.349	16.391 ± 0.484	28,403 ± 1.974

Table 4 Entrapment efficiency and apparent permeability (Caco-2 transwells, n = 4) data for tenofovir liposomal formulations

Formulation #	Entrapment efficiency (%)	Apparent permeability (cm/s)
Tenofovir solution A	–	3.71E−07 ± 1.21E−07
Formulation A	39.8 ± 8.09	4.18E−06 ± 1.92E−07
Formulation B	68.1 ± 2.55	1.19E−06 ± 1.51E−07

Notes: Formulations were prepared using 50 mg cholesterol, either 7.5% (formulation A) or 15% (formulation B) stearylamine as a positive charge imparting agent and an amount of phospholipon 100H to make a total lipid pool of 150 mg. Tenofovir solution A was prepared in correspondence to the EF value of Formulation A in isotonic phosphate buffer.

Abbreviation: EF, entrapment efficiency.