Oral administration of amphotericin B nanoparticles: antifungal activity, bioavailability and toxicity in rats

Figures & data

Table 1. The characteristics of the selected formulations.

Parameter Formulation	Polymer	Drug content, mg/batch	Mean particle size, nm	Drug encapsulation efficacy, %	Drug release within 24 h, %
F1	PLGA-PEG	20	23.8 ± 4.8	48.3 ± 4.2	61.2 ± 3.2
F2	PLGA-PEG	40	25.3 ± 2.7	56.5 ± 3.9	59.4 ± 5.7
F3	PLA	40	539.9 ± 51.1	27.2 ± 3.2	42.8 ± 2.6

Table 2. The composition, dose and route of administration of the selected formulations.

Animal group	Route of administration	Formulation	Carrier/polymer	Drug amount, mg	GA % IN	GA % OUT	Dose mg/kg
I	PO	Fungizone®	Sodium deoxycholate	50	–	–	10
II	iv	F1	PLGA–PEG	20	–	–	1
III	PO	F1		20	–	–	10
IV	PO	F1-GA-out-1		20	–	1	10
V	PO	F1-GA-out-2		20	–	2	10
VI	PO	F1-GA-in-2		20	2	–	10
VII	PO	F2-GA-in-2		40	2	–	20
VIII	PO	F2-GA-out-2		40	–	2	20
IX	PO	F3	PLA	40	–	–	20

Figure 1. Mean plasma AMB concentration–time profiles after intravenous administration of 1.0 mg/kg of F1-iv and oral administrations of 10 mg/kg of F1-PO and Fungizone® to rats (n = 6).

Table 3. Pharmacokinetic parameters of AMB after iv and oral administrations of AMB-loaded PLGA-PEG formulations and fungizone in rats (n = 6).

Tested parameters	F1-iv	F1-PO	Fungizone®-PO
Dose, mg/kg	1	10	10
Cmax, ng/mL	–	480.2 ± 38.7	298.2 ± 28.3
AUC0–24h (μg.h/l)	–	6100 ± 514.9	3720.8 ± 617.5
AUC (μg.h/l)	84211.2 ± 33935	12325.1 ± 1511.3	9832.4 ± 1657.5
k (h^−1)	0.0208 ± 0.0084	0.0298 ± 0.0060	0.0019 ± 0.0015
Cl (ml/h/kg)	13.8 ± 6.1
t1/2 (h)	38.1 ± 14.8	24.0 ± 4.4	35.3 ± 2.6
V (ml/kg)	666.1 ± 109.7
F	–	0.0146	0.0117
Frel Fungizone	–	1.3

Figure 2. Mean plasma AMB concentration–time profiles following single oral administration of 10.0 mg/kg of AMB-loaded PLGA-PEG formulations in rats (n = 6).

Table 4. Pharmacokinetic parameters of AMB (mean ± SD) after a 10 mg/kg single oral administration of AMB as Fungizone® and AMB-loaded PLGA-PEG NP formulations in rats (n = 6).

Parameters	F1-PO	F1-GA-out-1	F1-GA-out-2	F1-GA-in-2	F2-GA-in-2	F2-GA-out-2	Fungizone®-PO
AUC0–24h (μg.h/l)	6100 ± 514.9	18 818.7 ± 2851	21 803.2 ± 1346	20 216.2 ± 1593	38 808.6 ± 4033	37 364.6 ± 2883	3720.8 ± 617.5
AUC (μg.h/l)	12325.1 ± 1511	53 287.0 ± 24 997	60 478.8 ± 7437	88 307.9 ± 36092	12 0689.8 ± 30 628	10 8366.9 ± 19 736	9836.3 ± 1654
Cmax (ng/ml)	480.2 ± 38.7	1115.0 ± 172	1426.3 ± 133	1239.4 ± 75	2387.2 ± 294	2147.2 ± 176	298.2 ± 28
ka,/h	0.994 ± 0.196	0.72 ± 0.35	1.244 ± 0.513	1.684 ± 1.374	1.68 ± 1.15	1.35 ± 0.38	0.904 ± 0.0.72
t1/2 (h)	24.0 ± 4.4	34.6 ± 15.1	36.0 ± 5.5	42.7 ± 4.2	41.3 ± 11.5	37.1 ± 6.5	35.3 ± 2.6
Mean F	0.015	0.063	0.072	0.105	0.072	0.064	0.0117
Mean Frel, F1	–	4.3	4.9	7.2	4.9	4.4	0.78
Mean Frel, Fungizone	1.3	5.4	6.1	9.0	6.1	5.5	–

Figure 3. BUN, mg/dL, (mean ± SD) after single and multiple iv doses (5 mg/kg) of AMB and AMB loaded to PLGA-PEG NP to rats (n = 3).

Figure 4. Plasma Cr, mg/dL, (mean ± SD) after single and multiple iv doses (5 mg/kg) of AMB and AMB loaded to PLGA-PEG NP to rats (n = 3).

Figure 5. Typical kidney tissue alterations verified in rats treated with AMB or its equivalent dose as 1.0 mg/kg of body weight as iv administration of different Amb-PLGAPEG copolymer. 1) normal kidney tissue; 2,3,4) Fungizone® f1 and f2, respectively, varying degree of nephrotoxicity necrosis related to iv administration of iv doses (5 mg/kg).

Figure 6. In vitro mean RBCs hemolysis following incubation of RBC with Fungizone® and different AMB-NPs formulations at concentrations of 20, 50 and 100 μg/ml (n = 3).

Table 5. Checkboard assay of AMB against C. albicans (n = 6).

Tested form	MIC-0 (μg/ml) after 24 h	MIC-0 (μg/ml) after 48 h
AMB	0.5 ± 0.01	1.0 ± 0.01
Fungizone®	0.5 ± 0.01	1.0 ± 0.01
F1	0.125 ± 0.01	0.125 ± 0.01
F2	0.125 ± 0.01	0.125 ± 0.01

Table 6. Relative bioavailability of AMB in rats, after PO administration of various AMB-loaded NPs in different formulations, in comparison to Fungizone®.

Reference	Formulation/Polymer	Dose of fungizone, mg/kg	AMB Dose in tested formulation, mg/kg	Frel to fungizone %
Current study	Copolymer (PLGA-PEG)	10	10	798
(Yang et al., 2012)	Cubosomes	10	10	285
		10	20	702
(Italia et al., 2009)	PLGA	10	10	693
(Sachs-Barrable et al., 2008)	Peceol/AMB	50	50	2197
		50	5	8506

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