Mitigation of Rheumatic Arthritis in a Rat Model via Transdermal Delivery of Dapoxetine HCl Amalgamated as a Nanoplatform: In vitro and in vivo Assessment

Figures & data

Table 1 The Independent Variables, Their Respective Levels, and the Summarize Statistics Model of Box–Behnken Design Used for Optimization of DH Trans- Ethosomes

Variable		Level Used
		Low (−1)			Medium (0)		High (+1)
Independent variables (Factors)
A = PC concentration % (w/w)		1			2		3
B = Ethanol concentration % (w/w)		10			25		40
C = SDC concentration % (w/w)		0.1			0.15		0.2
Dependent variables	R2	Adjusted R2	Predicted R2	Constraints	p value	F value	Adequate precision
Y1: EE%	0.9970	0.9932	0.9828	Maximize	0.0001	259.92	56.83
Y2: Vesicle size (nm)	0.9909	0.9792	0.9248	Minimize	0.0001	84.61	33.035
Y3: zeta potential (mV)	0.9975	0.9943	0.9774	Maximize	0.0001	312.88	62.675
Y4: Q8h (%)	0.9922	0.9903	0.9846	Maximize	0.0001	548.21	81.3748
Y5: Q24 (µg/cm2)	0.970	0.980	0.9979	Maximize	0.0001	1.412	1322.74

Abbreviations: PC, phosphatidyl choline; SDC, sodium deoxycholate; EE%, entrapment efficiency percent; Q8h, cumulative release after 8 h; Q24, cumulative amount permeated/unit area in 24 h.

Table 2 Observed Response in Box–Behnken Design for Optimization of DH Loaded TENVs

Formulation	Independent Variables				Dependent Variables				PDI
	A: PC Concentration (%w/w)	B: Ethanol Concentration (%w/w)	C: SDC Concentration (% w/w)	Y1: EE%	Y2: Vesicle Size (nm)	Y3: Zeta Potential (mV)	Y4: Q8h (%)	Y5: Q24 (µg/cm2)
F1	1	10	0.15	39.80± 1.75	168.50± 11.03	-19.20± 3.20	76.70± 13.57	238.40± 6.96	0.32
F2	2	25	0.15	54.60± 4.13	195.10± 19.69	-32.20± 5.56	67.10± 10.00	334.70± 24.10	0.47
F3	3	25	0.1	75.50± 3.72	285.20± 24.01	-42.30± 6.25	53.10± 3.50	345.83± 21.79	0.27
F4	2	40	0.1	61.50± 4.61	220.40± 20.05	-36.40± 4.30	63.20± 12.25	269.50± 25.00	0.50
F5	2	25	0.15	55.70± 3.39	199.20± 13.26	-32.10± 3.60	67.70± 4.55	334.23± 23.91	0.24
F6	1	40	0.15	35.50± 2.06	165.40± 20.44	-26.90± 4.80	81.30± 11.85	259.13± 27.01	0.42
F7	1	25	0.1	42.40± 2.79	179.60± 18.96	-24.60±5.36	71.40± 7.17	174.38± 15.12	0.39
F8	3	10	0.15	69.50± 3.80	273.40± 19.33	-38.30± 1.00	57.40± 4.19	409.50± 21.50	0.48
F9	2	25	0.15	54.20± 1.06	193.60± 22.84	-32.20± 8.42	68.60± 3.74	334.10± 22.10	0.39
F10	2	10	0.2	53.10± 2.14	190.40± 17.86	-31.10±8.94	73.60± 6.88	398.60± 23.01	0.33
F11	3	40	0.15	66.90± 5.95	262.10± 22.59	-43.80± 9.41	60.70± 4.06	429.83± 27.01	0.39
F12	2	25	0.15	53.70± 3.42	192.70± 25.29	-32.10±7.60	68.20± 3.23	334.87± 20.12	0.36
F13	2	40	0.2	48.60± 3.59	187.90± 8.77	-37.00±6.95	78.90± 9.63	418.43± 26.87	0.22
F14	1	25	0.2	32.90± 0.55	142.60± 10.45	-26.00± 2.76	85.90± 14.45	322.06± 23.98	0.17
F15	3	25	0.2	59.70± 2.25	218.50± 14.00	-43.50±7.61	65.10± 4.70	493.74±30.08	0.38
F16	2	25	0.15	56.30± 3.36	206.10± 9.63	-33.20± 3.39	68.60± 5.00	334.53± 19.28	0.44
F17	2	10	0.1	62.20± 4.14	245.90± 23.41	-27.20± 2.11	59.10± 3.62	251.28 ±21.72	0.32
Fop	2.68	32.66	0.2
Observed values				52.62	216.53	-38.14	61.69	467.87
Predicted values				56.55	208.48	-41.75	69.90	471.29

Note: Data are mean values (n = 3) ± SD.

Abbreviations: PC, phosphatidyl choline; SDC, sodium deoxycholate; EE%, entrapment efficiency percent; Q8h, cumulative release after 8 h (%); Q24, cumulative amount permeated/unit area in 24 h; PDI, polydispersity index; Fop, optimal TENV.

Figure 1 Response surface plot for the effect of PC concentration (A), ethanol concentration (B) at the middle levels of the 3rd (SDC concentration) on (A) EE%, (B) vesicle size, (C) zeta potential, (D) Q8h, (e) Q24 and (F) desirability of the developed TENVs dispersions.

Table 3 Ex vivo Permeation Parameters of DH-TENVs Formulation versus DH Solution

Formulation No.	Lag Time (min)	Jss (µg/cm2.h)	Kp (cm/h)	EI
F1	75.32 ± 0.33	9.93± 0.71	0.0060± 0.0012	1.88
F2	38.65 ± 4.70	13.95± 1.00	0.0108± 0.0018	3.26
F3	29.34 ± 7.72	14.41± 0.96	0.0115± 0.0008	3.57
F4	57.33 ± 8.39	11.23± 1.04	0.0083± 0.0017	2.62
F5	38.38 ± 4.80	13.93± 1.00	0.0098± 0.0015	3.06
F6	69.77 ± 1.12	10.80± 1.13	0.0079± 0.0012	2.48
F7	82.42 ± 11.31	7.27± 0.63	0.0057± 0.0014	1.82
F8	23.14 ± 4.93	17.06± 0.90	0.0138± 0.0018	4.17
F9	43.72 ± 1.13	13.92± 0.92	0.0097± 0.0017	3.03
F10	26.86 ± 3.67	16.61± 0.96	0.0127± 0.0018	3.98
F11	25.44 ± 0.94	17.91± 0.91	0.0182± 0.0030	5.72
F12	38.82 ± 8.88	13.95± 0.84	0.0110± 0.0015	3.44
F13	28.85 ± 6.08	17.43± 1.12	0.0145± 0.0021	4.52
F14	49.37 ± 5.14	13.42± 1.00	0.0094± 0.0013	2.93
F15	15.31 ± 3.80	20.57± 1.25	0.0242± 0.0023	7.54
F16	50.47 ± 0.44	13.94± 0.80	0.0099± 0.0013	3.08
F17	65.42 ± 0.12	10.47± 0.90	0.0069± 0.0012	2.17
DH solution	91.10 ± 20.67	5.65± 0.71	0.0034± 0.0009	–

Note: Data are mean values (n = 3) ± SD.

Abbreviations: Jss, drug flux; Kp, permeability coefficient; EI, enhancement index.

Figure 2 Transmission electron micrograph of the optimized DH-TENV formulation.

Figure 3 DSC thermograms of DH, Phosphatidyl choline, SDC and the optimized TENVs.

Figure 4 Effect of storage at 4°C for 3 months on entrapment efficiency (EE) and vesicle size (VS) of the optimized DH-TENV formulation.

Figure 5 Light photomicrographs showing histopathological sections of (A) Normal untreated rat skin and (B) Rat skin treated with DH- TENV gel (X200 H&E stain).

Table 4 Pharmacokinetic Parameters of DH in Rat Plasma After Administration of an Oral Solution, TENVs Loaded Gel, and Control Gel

Pharmacokinetic Parameter	Oral DH Solution	TENVs Loaded DH gel	Control DH Gel
Cmax (ng/mL)	197.61± 16.43	120.72± 9.50^a	32.27± 2.71^a,b
tmax (h)	1.50± 0.06	4.00± 0.55^a	3.00± 0.34^a,b
t1/2 (h)	5.46± 2.87	7.45± 1.32	9.24± 1.86 ^a
MRT (h)	5.74± 1.56	11.97± 1.71^a	13.56± 2.04^a
AUC0–24 (ng.h/mL)	701.05± 25.81	1544.64± 130.82^a	353.82± 31.84^a,b
AUC0–∞ (ng.h/mL)	733.65± 51.08	1773.35± 171.05^a	426.33± 26.55^a,b
Frel (%)	–	242.99± 32.18	58.25± 4.17^b

Notes: Values are means ± SD, with the number of animals =6 for each group. ^ap < 0.05 versus oral DH solution. ^bp < 0.05 versus TENVs loaded DH gel. Using one-way ANOVA.

Table 5 Biochemical Analysis in Different Studied Groups

Groups	Parameter
	Anti-CCP (ng/mL)	COMP (pg/mL)	IL-6 (Pg/mL)
Normal control	1.88 ± 0.12	32.20 ± 1.66	18.91 ± 1.71
DH - TENV gel	1.91 ± 0.04	27.43 ± 1.41	14.23 ± 0.68
RA group	9.27 ± 0.52^a, b	117.7 ± 3.48^a, b	80.33 ± 5.19^a, b
MTX+ RA	4.00 ± 0.03^a, b, c	51.63 ± 4.96^a, b, c	37.27 ± 1.25^a, b, c
DH -TENV gel+ RA	2.83 ± 0.12^c, d	48.50 ± 1.22^b, c	30.43 ± 3.05^b, c
DH - gel+ RA	4.46 ± 0.13^a, b, c, d, e	72.33 ± 2.61^a, b, c, d, e	54.97 ± 2.07^a, b, c, d, e
Oral DH+ RA	4.64 ± 0.15^a, b, c, d, e	70.30 ± 5.73^a, b, c, d, e	77.03 ± 1.15^a, b, c, d, e

Notes: Each value represents the mean of 8 experimental rat ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer post- multiple comparisons test. ^aSignificantly different from normal control group at p < 0.05. ^bSignificantly different from DH TENV gel group at P < 0.05. ^cSignificantly different from RA group at P < 0.05. ^dSignificantly different from MTX+ RA group at P < 0.05. ^eSignificantly different from RA+ DH TENV gel group at P < 0.05.

Abbreviations: ANOVA, analysis of variance; RA, Rheumatoid Arthritis; MTX, Methotrexate; DH, dapoxetine HCl; TENV, transethosome nanovesicle; anti-CCP, anti-cyclic cirtullinated peptide antibody; COMP, cartilage oligomeric matrix protein; IL-6, interleukin 6.

Figure 6 Effect of transdermal application of DH- TENV gel on RANKL relative expression as compared to arthritic group using Western blot analysis. Each value represents the mean of 8 experimental rat ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer post- multiple comparisons test. ^aSignificantly different from normal control group at p < 0.05. ^bSignificantly different from DH -TENV gel group at P < 0.05. ^cSignificantly different from RA group at P < 0.05. ^dSignificantly different from MTX+ RA group at P < 0.05. ^eSignificantly different from DH-TENV gel+ RA group at P < 0.05. ^fSignificantly different from oral DH+ RAgroup at P < 0.05.

Abbreviations: ANOVA, analysis of variance; RA, Rheumatoid Arthritis; MTX, Methotrexate; DH, dapoxetine HCl; TENV, transethosome nanovesicle; RANKL, Receptor activator of nuclear factor kappa-Β ligand; B-Actin, Beta-Actin.

Figure 7 Radiographic analysis of rat hind paw from different groups. (A) Normal control group and (B) DH - TENV gel group showed normal tissue with no signs of inflammation, bone enlargement or bone damage. (C) Complete Freund’s adjuvant (CFA) arthritic group showed observable excess soft tissue volume, joint space, sub-chondral erosion, periostitis, osteolysis, subluxation, degenerative joint changes with signs of inflammation at the metatarsal-phalangeal joint and the regions in-between the bones of the phalanges and the metatarsals. (D) Rheumatoid arthritis group treated with methotrexate showed almost normal soft tissue with disappearance of inflammatory signs and no bone enlargement was observed. (E) Rats treated with DH - gel, (F) DH - TENV gel and (G) Oral DH exhibited significant and comparable inhibition of soft tissues swelling that surrounded the bones of the foot and bone enlargement.

Figure 8 Photomicrographs of joint sections, stained with routine H&E stain (×100) and obtained from different groups. (A) Sections from normal control rats showing articulating ends of two bones that covered by articular surface (black arrow) and normal synovial membrane (blue arrow) lining the inner surface of capsule with normal meniscus (orange arrow). (B) Sections from DH - TENV gel group illustrated normal articular surface (black arrow) with normal synovial membrane (blue arrow) and normal meniscus (orange arrow). (C) RA- arthritic rats showing bone in the focal manner in the articular surface (black arrow). Also resorption of meniscus is appeared, as well as the resorbed area is reached with collaged and fibroblast cell together with a few chondroblast (blue arrow). (D) RA group that treated with MIX showed slight smooth articular surface (black arrow) with thickened articular cartilage with proliferating chondrocytes (blue arrow) and synovial membrane showing congestion (arrow head) and edema (orange arrow). (E) Articular surface of rheumatoid arthritis group that treated with DH - TENV gel showed smooth articular surface (black arrow). The proliferating chondrocytes showing hypercellularity, cloning and slightly ordered distribution (blue arrow) and nearly normal synovial membrane (orange arrow). (F) The joint capsule and most of the synovial membrane of RA- treated gel group showed slight normal articular surface (black arrow) and synovial membrane showed focal inflammatory cell aggregation, moderate edema, capillary are increased in number, thickness and hypercellularity (blue arrow). (G) Rheumatoid arthritis group that treated with oral DH showed rough cerrated articular surface (black arrow), smooth intact synovial membrane (blue arrow) and the associated connective tissue showing multiple capillaries (red arrow) and moderate edema (purple arrow). Photomicrographs sections of spleen that obtained from different groups and stained with routine H&E stain (×100) were are followed: (H) Normal control rats showing Normal white pulb containing follicular artery (black arrow) and normal red pulb containing numerous blood sinusoids (blue arrow). (I) Sections from DH- TENV gel group illustrated normal white pulb (black arrow) with dilated in sinusoid in red pulb (blue arrow). (J) RA- arthritic rats showing marked atrophy of white pulp (black arrow). (K) RA group that treated with MIX showed hyperplagia in the white pulb (blue circle), congestion and dilation in sinusoid of the red pulp (black arrow). (L) Sections from DH - TENV gel group illustrated hyperplasia in white pulb with multiple macrophages indicated immunoreaction (black arrow). (M) spleen sections of RA- treated gel group showed Hyperplasia in white pulb (black arrow) and dilation in sinusoid in the red pulb (blue arrow). (N) Rheumatoid arthritis group that treated with oral DH showed marked activation in the macrophage in the white pulb (black arrow) and nearly normal red pulb (blue arrow). Liver photomicrographs sections obtained from different groups and stained with routine H&E stain (×200) were are followed: (O) Normal control rats showing normal liver with normal central vein (blue arrow) with normal hepatic cords (black arrow). (P) Sections from DH - TENV gel group illustrated nearly normal liver with normal central vein (blue arrow) and portal area (orange arrow). (Q) RA- arthritic rats showing slight congestion (orange arrow) and edema (black arrow) together with sever vascular degeneration in the hepatocyte (blue arrow). (R) RA group that treated with MIX showed dilated central vein (blue arrow) and dilation in hepatic sinusoid (black arrow) with some with vascular degeneration in the hepatocyte (arrow head). (S) Sections from DH - TENV gel group illustrated nearly normal liver except slight congestion in central (black arrow) and portal vein (blue arrow). (T) liver sections of RA- treated gel group showed slight congestion in both central vein and portal vein (blue arrow) and hepatic cord and hepatocyte appear normally (black arrow). (U) Rheumatoid arthritis group that treated with DH oral solution showed marked congestion of central veins (blue arrow) with dilated engorged sinusoid (arrow head) and focal coagulative necrosis of sore hepatocyte (black arrow).