Intranasal brain-targeted clonazepam polymeric micelles for immediate control of status epilepticus: in vitro optimization, ex vivo determination of cytotoxicity, in vivo biodistribution and pharmacodynamics studies

Figures & data

Table 1. Variables in 3³ central composite face-centered design (CCFD) for CZ^a polymeric micelles.

	Levels
Factors	−1	0	1
X1: P123 conc.	50%	75%	100%
X2: Pluronics:drug ratio^b	20:1	30:1	40:1
X3: Hydration volume	5	7.5	10

^aWeight of CZ = 10 mg.

^b Mixture of Pluronics® (P123 and L121) according to X₁ levels.

Table 2. Factors’ levels for the 3³ central composite face-centered design (CCFD) used to prepare CZ polymeric micelles.

	Factors’ levels
Formula code	X1	X2	X3
Factorial points
PM1	50	20	5
PM2	100	20	5
PM3	50	40	5
PM4	100	40	5
PM5	50	20	10
PM6	100	20	10
PM7	50	40	10
PM8	100	40	10
Axial points
PM9	50	30	7.5
PM10	100	30	7.5
PM11	75	20	7.5
PM12	75	40	7.5
PM13	75	30	5
PM14	75	30	10
Center points
PM15	75	30	7.5
PM16	75	30	7.5
PM17	75	30	7.5

X₁: P123 conc., X₂: Pluronic®: drug ratio, and X₃: hydration volume.

Table 3. The measured responses of the central composite face-centered design (CCFD) of CZ polymeric micelles (mean ± SD, n = 3).

Formula code	Y1 = EE (%)	PS (nm)	Y2 = PDI	ZP (mV)	Y3 = Q8h (%)	Y4 = t50% (h)
PM1	25.40 ± 2.39	113.25 ± 1.13	0.20 ± 0.02	−20.90 ± 3.15	96.82 ± 3.80	3.14 ± 0.18
PM2	13.21 ± 3.28	87.34 ± 2.31	0.34 ± 0.01	−7.12 ± 2.21	67.12 ± 1.75	5.58 ± 0.27
PM3	66.87 ± 1.36	83.77 ± 2.18	0.28 ± 0.05	−23.30 ± 2.08	80.90 ± 1.98	4.24 ± 0.06
PM4	31.01 ± 4.03	95.40 ± 0.19	0.41 ± 0.03	−15.70 ± 1.28	64.53 ± 4.06	5.75 ± 0.80
PM5	35.87 ± 1.82	88.67 ± 1.14	0.20 ± 0.01	−20.55 ± 2.56	74.28 ± 3.13	3.78 ± 0.20
PM6	13.46 ± 1.92	132.70 ± 4.23	0.19 ± 0.07	−18.25 ± 3.15	70.97 ± 0.37	3.72 ± 0.03
PM7	85.62 ± 0.63	124.15 ± 5.56	0.19 ± 0.05	−24.60 ± 4.25	97.21 ± 2.04	2.21 ± 0.22
PM8	45.75 ± 0.27	95.75 ± 4.56	0.30 ± 0.02	−19.85 ± 1.22	86.54 ± 1.70	3.71 ± 1.09
PM9	51.42 ± 4.25	102.40 ± 2.22	0.26 ± 0.01	−18.95 ± 0.98	86.09 ± 1.34	3.70 ± 0.99
PM10	16.55 ± 1.84	125 ± 5.18	0.45 ± 0.07	−15.70 ± 1.28	70.90 ± 0.28	6.35 ± 0.08
PM11	12.70 ± 2.56	106.07 ± 3.47	0.23 ± 0.05	−17.95 ± 0.67	72.90 ± 0.93	4.58 ± 0.04
PM12	43.99 ± 3.47	91.24 ± 4.5	0.26 ± 0.01	−15.45 ± 1.15	89.85 ± 0.35	2.81 ± 0.90
PM13	18.19 ± 1.02	93.74 ± 5.13	0.31 ± 0.08	−17.80 ± 3.55	89.34 ± 2.06	2.85 ± 0.09
PM14	25.1 ± 2.73	102.69 ± 5.33	0.19 ± 0.04	−19.25 ± 1.98	68.11 ± 0.46	5.10 ± 1.37
PM15	22.2 ± 1.15	112.70 ± 2.23	0.25 ± 0.03	−28.00 ± 1.08	73.13 ± 0.86	4.28 ± 0.18
PM16	18.8 ± 2.34	119.90 ± 1.23	0.36 ± 0.02	−25.80 ± 2.66	74.99 ± 1.39	4.81 ± 0.44
PM17	24.2 ± 2.18	116.30 ± 4.56	0.28 ± 0.02	−26.90 ± 2.50	73.69 ± 0.92	4.50 ± 0.41

Figure 1. In vitro CZ release profiles from investigated polymeric micelle and the drug solution in ethanol:water (1:1) at 37 ± 0.5 °C, mean ± SD, n = 3.

Table 4. Fitting CZ release to zero, first, and Higuchi diffusion models.

	Correlation coefficient
Formula code	Zero order	First order	Higuchi diffusion	Best fitting model
PM1	0.832	0.733	0.941	Higuchi diffusion
PM2	0.885	0.792	0.968
PM3	0.723	0.584	0.862
PM4	0.904	0.771	0.977
PM5	0.946	0.820	0.994
PM6	0.907	0.773	0.981
PM7	0.799	0.634	0.920
PM8	0.684	0.560	0.831
PM9	0.796	0.650	0.903
PM10	0.879	0.753	0.961
PM11	0.873	0.711	0.956
PM12	0.823	0.684	0.929
PM13	0.790	0.657	0.914
PM14	0.872	0.757	0.960
PM15	0.913	0.758	0.981
PM16	0.928	0.636	0.937
PM17	0.926	0.789	0.988

Figure 2. DSC thermograms of CZ, Pluronic® P123, Pluronic® L121, and the optimized polymeric micelle (PM7).

Figure 3. TEM photomicrographs of mixed Pluronic® L121/P123 polymeric micelles (PM7).

Figure 4. Photomicrographs of the anterior segments of sheep nasal mucosa treated with pH 6.4 PBS (negative control, a), isopropyl alcohol (positive control, b), and CZ-loaded polymeric micelles (c) (100×).

Figure 5. Photomicrographs of the posterior segments of sheep nasal mucosa treated with pH 6.4 PBS (negative control, a), isopropyl alcohol (positive control, b), and CZ-loaded polymeric micelles (c) (100×).

Figure 6. Variation of the radiochemical yield of ^99mTc-clonazepam as a function of clonazepam amount (a), Na₂S₂O₄ amount (b), pH (c), reaction temperature, (d) and time (e).

Figure 7. CZ concentration in brain at different time intervals following administration of intranasal ^99mTc-CZ solution, intranasal ^99mTc-PM7 and intravenous ^99mTc-PM7, mean ± SD, n = 3, in male Swiss albino mice.

Figure 8. CZ concentration in blood at different time intervals following administration of intranasal ^99mTc-CZ solution, intranasal ^99mTc-PM7 and intravenous ^99mTc-PM7, mean ± SD, n = 3, in male Swiss albino mice.

Table 5. Brain/blood distribution of CZ administration as intranasal ^99mTc solution, intranasal ^99mTc-PM7, and intravenous ^99mTc-PM7 in male Swiss albino mice (mean ± SD, n = 3).

		Time
Formulation/route of administration	Organ or tissue	0.25	0.5	1	2	4	8
^99mTc-CZ solution/intranasal	Brain	0.24 ± 0.01	0.16 ± 0.01	0.12 ± 0.01	0.02 ± 0.00	0.02 ± 0.00	0.00 ± 0.00
	Blood	1.03 ± 0.27	1.37 ± 0.43	0.95 ± 0.09	0.83 ± 0.08	0.67 ± 0.15	0.44 ± 0.15
	Brain/blood	0.24 ± 0.05	0.13 ± 0.05	0.13 ± 0.03	0.02 ± 0.00	0.02 ± 0.00	0.00 ± 0.00
^99mTc-PM7/intranasal	Brain	4.28 ± 0.69	1.35 ± 0.24	0.43 ± 0.11	0.16 ± 0.03	0.13 ± 0.00	0.02 ± 0.00
	Blood	0.85 ± 0.20	0.63 ± 0.12	0.32 ± 0.05	0.10 ± 0.02	0.06 ± 0.02	0.01 ± 0.01
	Brain/blood	5.10 ± 0.45	2.14 ± 0.18	1.31 ± 0.13	1.72 ± 0.37	2.04 ± 0.15	1.5 ± 0.45
^99mTc-PM7/intravenous	Brain	0.24 ± 0.05	0.29 ± 0.08	0.14 ± 0.01	0.10 ± 0.02	0.08 ± 0.01	0.01 ± 0.02
	Blood	9.62 ± 1.30	8.72 ± 1.29	7.48 ± 0.97	6.42 ± 0.86	5.25 ± 1.33	2.76 ± 0.85
	Brain/blood	0.02 ± 0.00	0.03 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.00 ± 0.00

Table 6. Pharmacokinetics parameters for CZ administration as intranasal ^99mTc solution, intranasal ^99mTc-PM7, and intravenous ^99mTc-PM7 in male Swiss albino mice (mean ± SD, n = 3).

		Time
Formulation/route of administration	Organ or tissue	Cmax (%ID/g)	Tmax (h)	AUC0–∞ (h%ID/g)	AUC0–8 (h%ID/g)	Relative bioavailability^a
^99mTc CZ solution/intranasal	Brain	0.24 ± 0.01	0.25	0.32 ± 0.01	0.31 ± 0.01
	Blood	1.37 ± 0.43	0.5	10.18 ± 4.05	5.65 ± 1.20
^99mTc PM7/intranasal	Brain	4.28 ± 0.69	0.25	2.68 ± 0.51	2.62 ± 0.47	812.96 ± 154.6
	Blood	0.85 ± 0.20	0.25	1.12 ± 0.22	1.08 ± 0.22	11.83 ± 2.88
^99mTc PM7/intravenous	Brain	0.29 ± 0.08	0.5	0.80 ± 0.33	0.71 ± 0.19
	Blood	9.62 ± 1.30	0.25	61.15 ± 17.10	42.23 ± 8.48

^aRelative bioavailability (RBA) in comparison to I.N CZ solution.

Table 7. The DTE%, DTI%, and DTP% of intranasal ^99mTc-CZ solution and intranasal ^99mTc-PM7 polymeric micelles relative to the intravenous ^99mTc-PM7 in male Swiss albino mice (mean ± SD, n = 3).

Formulation/route of administration	DTE%	DTI	DTP%
^99mTc-CZ solution/intranasal	5.78 ± 1.01	3.46 ± 0.81	70.07 ± 7.04
^99mTc-PM7/intranasal	242.39 ± 10.30	144.25 ± 10.22	99.30 ± 0.04

DTE: drug targeting efficiency; DTI: drug targeting index; DTP: nose-to-brain direct transport percentage.

Table 8. Time (s) for the development of seizures in male Swiss albino mice (mean ± SD, n = 5).

Formula administered	Administration after 15 min	Administration after 30 min	Administration after 45 min
Control group	59.33 ± 3.6	56 ± 6.02	59.66 ± 2.08
CZS(i.n)	98 ± 10.5	84.33 ± 10.53	71.33 ± 9.07
CZS(i.v)	62 ± 6.55	160.6 ± 7.02	139 ± 8.18
PM7(i.n)	424.33 ± 31.5	332.33 ± 41.10	314.66 ± 24.58

EE: entrapment efficiency; PS: particle size; PDI: polydispersity index; ZP: zeta potential Q8h: amount released after 8 h and t50%: amount for the release of 50% of the drug.