Pharmacokinetics and tissue distribution studies of orally administered nanoparticles encapsulated ethionamide used as potential drug delivery system in management of multi-drug resistant tuberculosis

Figures & data

Table 1.  Accuracy and precision data of ethionamide in spiked mice plasma.

	LOQ	LQC	MQC	HQC
Concentration added (µg/ml)	0.1	0.3	25	45
Within-run
Concentration found (µg/ml)	0.08 ± 0.01	0.26 ± 0.03	20.43 ± 2.96	39.59 ± 3.93
Accuracy (%) (mean found/added)	85.00	87.00	81.72	87.98
Precision (%RSD)	11.76	11.49	14.49	9.93
Between-run
Concentration found (µg/ml)	0.08 ± 0.01	0.24 ± 0.02	21.31 ± 1.83	40.63 ± 1.48
Accuracy (%) (mean found/added)	83.00	81.00	85.24	90.29
Precision (%RSD)	19.28	8.23	5.77	3.64

LOQ, Limit of quantitation; LQC, Lowest quality control; MQC, Medium quality control; HQC, Highest quality control; * RSD%, percentage relative standard deviation; Values are reported as mean ± SD; Number of replicate measurements (n) = 5/concentration.

Table 2.  Recovery of ethionamide in spiked mice plasma.

Concentration(µg/ml)	Unextracted (spiked after extraction) (µg/ml)	Extracted (spiked before extraction) (µg/ml)	Recovery(%)
0.3	0.24 ± 0.04	0.21 ± 0.04	87.5
25	19.48 ± 0.80	19.43 ± 0.79	99.7
45	40.49 ± 0.88	40.09 ± 1.14	99.0

Values are expressed as mean ± SD; Number of replicate measurements (n) = 5/concentration.

Table 3.  Stability of ethionamide in spiked mice plasma.

Concentration (µg/ml)	Day 1 (µg/ml)	Day 6 (µg/ml)
0.3	0.24 ± 0.02 (8.33)	0.21 ± 0.005 (2.38)^NS
25	21.31 ± 1.83 (8.59)	19.44 ± 1.16 (5.97)^NS
45	40.65 ± 1.48 (3.64)	38.31 ± 1.78 (4.65)^NS

Values in parentheses indicate %CV (Percentage coefficient of variation); Values are reported as mean ± SD; Number of replicate measurements, n = 3/concentration; NS, not significant.

Figure 1.  Calibration curve of ethionamide in mice plasma.

Table 4.  Characterization of formulation before and after 90 days of storage at 4–8°C.

Parameters	Initial	Final
Particle size (nm)	280 ± 21	300 ± 10
PDI	0.2 ± 0.02	0.2 ± 0.03
Zeta potential (mV)	−12.8 ± 2.9	−13.2 ± 1.5
Entrapment efficiency (% w/w)	34.9 ± 3.6	32.6 ± 2.9
Volume of reconstitution per ml	5 mg	5 mg

Values reported are mean ± SD.

Table 5.  Pharmacokinetic parameters of free ethionamide (ETH) and ethionamide-loaded nanoparticles (ETH NPs) upon oral administration in the mice.

Drugs	Cmax (µg/ml)	tmax (h)	AUC0–∞ (µg/ml) × h	AUMC0–∞ (µg/ml) × h^2	MRT (h)	ke (h^−1)
ETH (130 mg/kg)	20.71 ± 1.8	0.25	21.89 ± 3.2	27.90 ± 2.9	1.27 ± 0.2	−0.732 ± 0.08
ETH NPs (equivalent to 130 mg/kg of ETH)	29.22 ± 1.0 *	1	657.45 ± 52.9*	23,743.62 ±1549.5*	36.11 ± 3.9*	−0.044 ±0.001*
ETH NPs (equivalent to 260 mg/kg of ETH)	43.90 ± 2.6*^,$	1	1250.58 ± 135.7^*,$	43,846.82 ±1915.7*^,$	35.06 ± 6.9*	−0.048 ±.001*

C_max, peak plasma concentration; tmax, peak time; AUC0–∞, area under the plasma concentration–time curve; AUMC0–∞, area under moment curve; MRT, mean residence time; ke, elimination constant; * p < 0.05 vs ETH (130 mg/kg), $ p < 0.05 vs ETH NPs. (equivalent to130 mg/kg of ethionamide)

Figure 2.  In-vivo plasma concentration–time profile of orally-administered free ethionamide (ETH) and ethionamide-loaded nanoparticles (ETH NPs) equivalent to 130 mg/kg and 260 mg/kg of ethionamide in mice (n = 3–4 mice at each time point). Insert show an expanded view of 0–24 h plasma profile.

Table 6.  Tissues drug levels following single oral administration of ethionamide-loaded PLGA nanoparticles (ETH NPs) at two doses in mice.

Drugs	Organs	72 h		96 h		120 h		144 h		168 h
		µg/ml	µg/g	µg/ml	µg/g	µg/ml	µg/g	µg/ml	µg/g	µg/ml	µg/g
ETH NPs (equivalent to 130 mg/kg)	Lung	2.3 ± 0.2	11.5 ± 1.0	1.2 ± 0.03	5.0 ± 0.1	1.2 ± 0.05	5.0 ± 0.2	ND	ND	ND	ND
	Liver	2.9 ± 0.3	14.5 ± 1.5	1.7 ± 0.1	8.5 ± 0.5	1.9 ± 0.1	9.5 ± 0.5	0.3 ± 0.03	1.5 ± 0.1	ND	ND
	Spleen	2.1 ± 0.3	10.5 ± 1.5	1.7 ± 0.2	8.5 ± 1.0	1.4 ± 0.08	7.0 ± 0.4	0.2 ± 0.04	1.0 ± 0.2	ND	ND
ETH NPs(equivalent to 260 mg/kg )	Lung	3.1 ± 0.5	15.5 ± 2.5	1.6 ± 0.1	8.0 ± 0.5	1.5 ± 0.1	7.5 ± 0.5	0.2 ± 0.05	1.0 ± 0.2	ND	ND
	Liver	3.9 ± 0.5	19.5 ± 2.5	2.9 ± 0.3	14.5 ± 1.5	2.0 ± 0.1	10.0 ± 0.5	0.5 ± 0.09	2.5 ± 0.4	0.3 ± 0.05	1.5 ± 0.2
	Spleen	3.8 ± 0.3	19.0 ± 1.5	1.8 ± 0.2	9.0 ± 1.0	2.6 ± 0.1	13.0 ± 0.5	0.3 ± 0.06	1.5 ± 0.3	0.1 ± 0.02	0.5 ± 0.01

All values are mean ± SD of 3–4 animals; ND, not detected. Concentration of ethionamide is expressed as µg/ml in the 20 % w/v tissue homogenates and µg/g of the tissue.

Table 7.  Calculated values for AUC above MIC, C_max above MIC, and time above MIC for free ethionamide (ETH) and ethionamide-loaded nanoparticles (ETH NPs) at a MIC₉₀ (0.6 µg/ml).

Drugs	Parameters	Values
ETH (130 mg/kg)	AUC0–24/MIC	36.11 ± 5.33
	Cmax/MIC	34.51 ± 3.00
	Time > MIC (h)	2
ETH NPs (equivalent to130 mg/kg of ETH)	AUC0–24/MIC	465.50 ± 20.17*
	Cmax/MIC	48.7 ± 1.66*
	Time > MIC (h)	120 *
ETH NPs (equivalent to 260 mg/kg of ETH)	AUC0–24/MIC	902.61 ± 121.98*^,$
	Cmax/MIC	73.16 ± 4.33*^,$
	Time > MIC (h)	120*

AUC_0–24, area under the curve at 24 h; t, time; MIC, minimum inhibitory concentration; C_max, maximum concentration in blood; * p < 0.05 vs ETH (130 mg/kg), $ p < 0.05 vs ETH NPs (equivalent to 130 mg/kg of ethionamide).