- Email: [email protected]
in Vivo Evaluation
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Yujian Zhang1, 2#, Nianqin Yang3#, Jianwei Lv2*, Haibo Song2, Xuehui Duan2, Jing Leng2, Juanjie Bo2, Dongming Liu2, Yiran Huang2 1
Department of Urology, Fourth Affiliated Hospital of Nantong University (First People’s Hospital of Yancheng), Yancheng, China 2 Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China 3 Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China # These authors contributed equally to this work. *Correspondence: [email protected]
Phentolamine (PT) is a non-selective a-adrenoceptor blocking agent. Oral and intracavernosal PT therapy are effective treatment options for erectile dysfunction. In the present study, a local therapy system of PT preparation was developed, by which its penetration enhancers, safety, stability and in vivo were investigated. The effect of permeation enhancers on in vitro mucosa permeability was assessed using sodium dodecyl sulphate (SDS), azone, propylene glycol (PG), Pharmasolve, Labrasol, Transcutol P, oleic acid, peppermint oil (lipophilicity), peppermint oil (hydrophilic) and their concentrations for absorption enhancement were optimized. The rabbit model was used to evaluate the safety and efficacy. peppermint oil appeared to be the most effective one for enhancing the mucosa permeation of PT than the other enhancers. These enhancements by peppermint oil were statistically significant (p < 0.05) compared to control. The order of permeation enhancement was PO (hydrophilic) > PO (lipophilicity) > azone > Transcutol P ≈ Labrasol > SDS ≈ PG ≈ Pharmasolve ≈ oleic acid. Histological investigations were performed on buccal mucosa and indicated no major morphological changes. The enhancing effect of peppermint oil on the mucosa absorption of PT was evaluated from the mucoadhesive gels in rabbits. Phentolamine local therapy would produce an ideal therapeutic effect on ED. Key words: Phentolamine – Permeation enhancers – Mucoadhesive gels – Mucosa delivery.
Male impotency or erectile dysfunction (ED) is a problem affecting millions of men across the world. Up to thirty million men are affected by male impotency in China alone. The problem is especially common in aged men. There are several treatments available for this condition. Although many treatment options such as intracavernosal vasoactive drugs, medical devices [1-3], surgery and psychotherapy have been used over the years for the treatment of ED, the introduction of an effective oral agent, sildenafil (Viagra, Pfizer, New York, NY, USA), has revolutionized the treatment of this condition [4, 5]. Efforts to develop minimally invasive local therapies for ED continue because they can conceivably circumvent many of the systemic side effects caused by oral medications. In addition, local therapies may benefit those patients who are unresponsive to current oral phosphodiesterase type 5 (PDE5) inhibitors or those who have contraindications to the use of oral therapies, such as in men receiving oral nitrates and men with severe cardiovascular disease. Intracavernosal and transurethral alprostadil therapies are effective with high success rates, but also with high rates of discontinuation because of discomfort, injection pain, or local fibrosis [6, 7]. Topical vasoactive drug delivery is a simple, safe, reversible, and relatively noninvasive alternative modality for the treatment of ED [8]. Phentolamine (PT) is a non-selective a-adrenoceptor blocking agent. The relaxation of vascular smooth muscle and increase of the blood flow can be triggered by the combination of PT and A-adrenoceptor within the cavernous tissue. Oral and intracavernosal PT therapy are effective treatment options for ED [9]. Transdermal delivery of PT alone for ED treatment is rarely reported by laboratory and clinical studies [10, 11]. In the present study, a local therapies system of PT preparation was developed, and its penetration enhancers, safety and stability in vitro were investigated. The effects of PT on conscious rabbits model was compared to that of sildenafil (Viagra) in order to confirm whether the use of PT local therapy would produce an ideal therapeutic effect for ED.
I. MATERIALS AND METHODS 1. Reagents and chemicals
PT was gifted by the Department of Pharmaceutics, Shanghai Xudong Haipu Pharmaceuticals Ltd. (China). Permeation enhancers (PE): sodium dodecyl sulphate (SDS), azone, propylene glycol (PG), Pharmasolve, Labrasol, Transcutol P, oleic acid, peppermint oil (lipophilicity), peppermint oil (hydrophilic) were purchased from Shanghai Chemical Reagents Research Institute (China). All other chemicals were of commercially analytical grade.
2. Tissue preparation
Guinea pig buccal tissue models were prepared for permeation studies of selecting the enhancers. Firstly, Guinea pig buccal tissue was chosen as its non keratinized morphology is quite similar to human buccal epithelium [12]. Buccal tissue was removed after sacrificing the animal and was stored in phosphate buffer (pH 6.8) then immediately transported to the experimental setup. The buccal mucosal membranes were separated by removing the underlying connective tissues using surgical scissors making sure that the basal membrane was still present. The tissue was rinsed and then stored in ice-cold phosphate buffer until mounted in the Franz diffusion cell (within 1 h upon removal) [13]. Slice thickness ranged from 2.2 to 2.5 mm and was mounted between donor and receiver chambers of the diffusion cells for permeation studies.
3. In vitro diffusion of permeation studies
The isolated buccal mucosa (Guinea pig buccal tissue) was mounted in Franz diffusion cells with a diffusion area of 1.2 cm2 and a compartment volume of 17 mL. After an equilibrium period with phosphate buffer on both sides, the acceptor side was filled with phosphate buffer and donor side with PT gel (2.5 mg/mL) in phosphate buffer alone or with the permeation enhancers. The solution in the receptor compartments was continuously stirred at 600 rpm using a magnetic stirring bead on electric stirrer. The diffusion studies were 22
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
carried out at 32 ± 1 °C. Samples of 0.2 mL were taken from the acceptor side at 1, 3, 5, 7, 9 and 12 h, replaced with the same amount of fresh buffer solution, and subsequently assayed using HPLC.
8. Rabbit irritation and efficacy test
The rabbit irritation and efficacy test was conducted in accordance with the requirements of current, internationally recognized good laboratory practice standards. In the beginning of the treatment, the white New Zealand male rabbits were 12-15 weeks old and weighed between 2.4 and 3.6 kg. The animals were kept singly in cages in a room in which the temperature and the relative humidity were maintained within the range of 16-20 ℃ and 40-70 %, respectively. A 12-h light/dark cycle was maintained. In the irritation test, the external genitalia of rabbits were daubed with PT gel once daily with 0.1 mL for 10 consecutive days. On day 11, the rabbits were sacrificed and the genitalia was carefully separated from their bodies. The genitalia was fixed with 10 % formalin, sliced on a microtome, stained with haematoxylin-eosin and observed using a light microscope (Axiovert 200MAT, Carl Zeiss, Germany). The external genitalia of rabbits daubed with saline were used as a control. For each group five rabbits were used. In the efficacy test [13, 16, 17], 24 rabbits were used in this experiment and randomly divided into 4 groups. The grouping of the rabbits is summarized in Table II. From the previous data, the minimal dose of different administration was confirmed. Group A were given PT solution by intravenous. Group B were given sildenafil suspension by PO (proper Viagra tablets with 0.5 % HPMC). Group C and D were given PE gel by the transdermal in the presence of PE or without it. Group E was given normal saline as the negative control. After given, all subjects were observed two leading indicators. One was the onset time of the drug and the other was the maximum effect of the different formulation. The onset time was defined as the time when rabbits penises were erected obviously. The maximum effect was defined as the maximum length of the penis.
4. Determination of drug content
The amount of PT in each sample was determined by HPLC (LC10A, Shimadzu Co. Ltd., Kyoto, Japan). Chromatographic separation was achieved using a Dikma Diamonsil TM C18 column (10 μm, 150 mm × 3.9 mm) at 30 ℃. The mobile phase was a mixture of methanol-water-acetic acid-triethylamine (10:10:1:0.1, v/v) at a flow rate of 1.0ml/min. The UV absorbance of the effluent was monitored (SPD-10A, Shimadzu) at a wavelength of 278 nm.
5. Treatment of permeation data
The cumulative amount of permeated drug (from mucosal side to serosal side) was plotted versus time and the flux was calculated by the steady state part of the curve. The permeability coefficient (Kp) was calculated using the Equation 1 described by Senel et al. [14]: Eq. 1
Kp = (dQ/dt)/(ΔC × A)
where dQ/dt is the average slope from steady state portion of the curve, ΔC the concentration difference across the mucosa, A the area of diffusion. The efficacy of the different enhancers was determined by comparing the permeation rate of PE in the presence and absence of enhancers. It was defined as the enhancement factor (EF) which was calculated using the following Equation 2 [15]: EF = phentolamine permeation rate at steady state in the presence of enhancer/phentolamine permeation rate at steady state in the absence of enhancer
Eq. 2
Table II - The grouping of the rabbits.
One-way analysis of variance (ANOVA) was used for statistical evaluation.
6. Preparation for PT gel
Firstly, the chosen enhancers (the better one in the in vitro study) was sprinkled on chlorhexidine acetate solution and totally swelled. PT powder, wetted with glycerol and grinded, was added to 0.5 % HPMC with sodium bisulfate solution. After stirring for 30 min, phosphoric acid was added to adjust the pH of the system to around 4-6 and a brown clearing gel was formed (total volume 10 mL). A basic formulation with respect to the content of different ingredients and their function was listed in Table I.
PT glycerol chosen enhancers HPMC sodium bisulfite chlorhexidine acetate phosphoric acid Distilled water
10 % 5% Better one 0.5 % 0.5 % 0.01 % Appropriate amount Up to 100 %
Active agent humectant penetration enhancer gel base oxidation inhibitor antiseptic pH adjustor Dispersion medium
Dose
Route of administration
A B C D E
PT solution Sildenafil tablet PT spray with PE PT spray without PE Normal saline
0.6 mg/kg 15 mg/kg 0.25 mg/kg 0.25 mg/kg --
i.v. p.o. Transdermal Transdermal Transdermal
II. RESULTS AND DISCUSSION 1. Selection of permeation enhancers
The representative chromatograms of blank (A); a pure drug sample (B) and sample (C) are shown in Figure 1. The analytical peaks of PT were resolved with good symmetry, the retention time of PT was 12.2 min, no endogenous sources of interference were observed at the retention time of the analyte. The permeation profiles of PT in presence of various enhancers are reported in Figures 2 to 6, while their permeability coefficients (Kp) and the enhancement factors (EF) are reported in Table III. In case of control, 50.2 ± 8.1 % PT was found to permeate through the buccal mucosa in 12 h with permeability coefficient of 0.45 (± 0.21) × 10-4 cm/s. In order to improve mucosa permeation and hence absorption of PT, various permeation enhancers such as SDS, azone, PG, Pharmasolve, Labrasol, Transcutol P, oleic acid, PO (lipophilicity), PO (hydrophilic) were used. EF as a ratio of cumulative PT permeated with PE to the control. From the data of SDS, PG, Pharmasolve, oleic acid, no significant improvement was noticed (p > 0.05) in permeability coefficient and EF, respectively. As a result, the data were not shown in this paper.
Table I - Basic formulation of PT gel. Function
Test design
The results were expressed as mean ± SD. Student’s t-test was used to study the statistical difference and a value of P < 0.05 was considered statistically significant.
A sufficient quantity of PT gel was stored in desiccator containing saturated solution of sodium chloride, which gave a relative humidity of 75 ± 5 %. The desiccator was placed in a hot air oven maintained at 25 ± 2 ℃, and samples were withdrawn at 0, 30, 60, 90, and 180 days. The drug content remaining and physical stability of formulation were measured at predetermined time interval.
Content
Group name
9. Statistical analysis
7. Stability studies
Ingredient
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
23
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
Cumulative PT permeated (%)
100
Figure 1 - HPLC chromatograph of PT. A: blank; B: a pure drug sample (0.1 mg/mL); C: sample (t = 7 h).
Control PO1(2.5%)
90
PO1(1%) PO1(5%)
80 70 60 50 40 30 20 10 0 0
90
Control Transcutol P(2.5%)
80
Transcutol P(1%) Transcutol P(5%)
60 50
100
40
90
30
80
20 10 0 1
3
5
7
9
12
Time (h)
Figure 2 - Permeation of PT in presence of Azone across buccal mucosa.
Cumulative PT permeated (%)
100
5
7
9
12
Control PO2(2.5%)
PO2(1%) PO2(5%)
70 60 50 40 30 20 10 0
90
Control Labrasol(2.5%)
80
0
Labrasol(1%) Labrasol(5%)
1
3
5
7
9
12
Time (h)
70
Figure 6 - Permeation of PT in presence of peppermint oil (hydrophilic) across buccal mucosa.
60 50
Table III - Permeability of PT across buccal mucosa using different penetration enhancers.
40 30 20 10
PE name
Conc. (% g/g)
Kp (10-4 cm/s)
Cumulative PT permeated
EF
Control
-
0.45
50.2
1.00
Azone
1 2.5 5
3.07 3.15 3.17
65.5* 69.3* 67.5*
1.30 1.38 1.34
Labrasol
1 2.5 5
1.45 2.11 2.15
59.4* 58.2* 56.1
1.18 1.16 1.12
Transcutol P
1 2.5 5
0.71 0.81 0.77
50.8 59.4* 54.3
1.01 1.18 1.08
PO (lipophilicity)
1 2.5 5
5.26 5.62 7.06
68.7** 76.4** 85.2**
1.37 1.52 1.70
PO (hydrophilic)
1 2.5 5
6.04 6.53 6.68
72.1** 82.4** 88.4**
1.44 1.64 1.76
0 0
1
3
5
7
9
12
Time (h)
Figure 3 - Permeation of PT in presence of Labrasol across buccal mucosa. 100
Cumulative PT permeated (%)
3
Figure 5 - Permeation of PT in presence of peppermint oil (lipophilicity) across buccal mucosa.
70
0
1
Time (h)
Cumulative PT permeated (%)
Cumulative PT permeated (%)
100
90
Control Transcutol P(2.5%)
80
Transcutol P(1%) Transcutol P(5%)
70 60 50 40 30 20 10 0 0
1
3
5
7
9
12
*p < 0.05 vs control group. **p < 0.01 vs control group.
Time (h)
Figure 4 - Permeation of PT in presence of Transcutol P across buccal mucosa.
24
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
In case of azone, Transcutol P, Labrasol, significant improvement (p < 0.05) in permeation was discerned of the individual concentration. Azone at 1, 2.5, 5 % showed 65.5~69.3 % of PT permeation (p < 0.05). Only Labrasol at 2.5, 5 %; Transcutol P at 2.5 % showed the significant difference and the amount of the permeation was not dose dependent. When using PO (both lipophilicity and hydrophilic), highly significant improvement was noticed (p < 0.01) in permeability coefficient and EF as the values were 68.7~85.2 % and 72.1~88.4 %, respectively. As the concentration of PO was increased and an increase in permeation was observed. The order of permeation enhancement was PO (hydrophilic) > PO (lipophilicity) > azone > Transcutol P ≈ Labrasol > SDS ≈ PG ≈ Pharmasolve ≈ oleic acid. Permeability of mucosa to PT alone was rather low, whereas permeability increased most significantly (p < 0.01) in the presence of PO (hydrophilic). The value of Kp augmented 15.7-fold in presence of PO (hydrophilic) as compared to the control. Hence, PO (hydrophilic) was selected for further studies.
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
Figure 7 - Stability studies of PT gel.
2. Stability studies
Figure 7 depicts results of stability study of PT gel. No microscopical physical changes were observed during storage. Drug content and pH values of the formulations carried out at 6 months showed no significant difference. Hence, it was concluded that the PE gel exhibited good stability.
3. Rabbit irritation
The successful use of transdermal delivery systems is not only limited to their efficacy, but equally important, is their safety. Hence, it was important to investigate the safety of the optimized formulation. Figure 8 shows light photomicrograph taken from anterior cross section of rabbits penis following 10-day exposure to the PT gel. Examination showed buccal mucosa depicts the outermost layer of stratified squamous epithelium, below which can be seen the basement membrane, lamina propria, and submucosa. The boundary between the buccal epithelium and the connective tissue is delineated by the basement membrane. Signs of irritation such as vascular congestion and subepithelial edema were not observed. Moreover, none of severe signs such as appearance of epithelial necrosis, sloughing of epithelial cells and hemorrhage was detected in any of the rabbits. The morphological study revealed the safety of the tested formulation which was deposited for 10 days to the mucosa of rabbits.
Figure 8 - Micrographs of multiple dosing. (A) Negative control (saline); (B) PT gel with peppermint oil (hydrophilic).
4. Efficacy test
Currently, there are usually two animal models for evaluating the treatment of ED. One was rat model and the other was rabbit model. The former mainly stimulates the sponge nerves and induces genital congestion, then the changes of the sponge pressure could reflect the efficacy of the drug. However, this method has many drawbacks such as the animal need to be anesthetized over the whole study and the preparation of process is tedious. Therefore in this paper, Rabbits model was chosen, because its physical morphology is similar to human’s [13, 16]. Table IV and Figure 9 show the pharmacodynamic result of different formulations. Sildenafil tablet was the most common formulation in the commercial market. From the data of group A and B, both PT and Sildenafil could increase the penis of rabbits about 10 mm length. The onset time was 6.5 and 32.4 min, respectively. From the data of group D, the onset time need 1 h and the length of penis was only 9.1 mm. When the PT gel was used with proper PE, PT mucosa delivery showed excellent performance. The onset time and the length of penis of group C were similar with the data of Group B. So it could be suggested that local therapies for ED might be expected. The reason is that they can conceivably circumvent many of the systemic side effects related to oral medications.
Figure 9 - Pharmacodynamic result of different formulations. Table IV - Pharmacodynamic result of different formulations. Group name A B C D E
Test design PT solution Sildenafil tablet PT spray with PE PT spray without PE Normal saline
Onset time (min)
Max length (mm)
6.5 ± 1.8 32.4 ± 3.3 30.8 ± 4.1** 65.6 ± 7.6 30.7 ± 5.2
9.6 ± 0.7 10.5 ± 0.8 10.3 ± 0.6 9.1 ± 0.6 1.2 ± 0.1
**p < 0.01 vs group A.
* In the present study, a local therapy system of PT preparation was developed, and its penetration enhancers, safety, stability and in vivo were investigated. Peppermint oil appeared to be the most effective one for enhancing the mucosa permeation of PT than the other enhancers. Histological investigations were performed on buccal mucosa and 25
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
indicated no major morphological changes. Pharmacodynamic result showed PT local therapy would produce an ideal therapeutic effect on ED.
11. 12.
REFERENCES 1. 2. 3.
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Pinsky M.R., Chawla A., Hellstrom W.J. - Intracavernosal therapy and vacuum devices to treat erectile dysfunction. - Arch. Esp. Urol., 63, 717-725, 2010. Dinsmore W.W., Wyllie M.G. - Vasoactive intestinal polypeptide/ phentolamine for intracavernosal injection in erectile dysfunction. - BJU Int., 102, 933-937, 2008. Shah P.J., Dinsmore W., Oakes R.A., Hackett G. - Injection therapy for the treatment of erectile dysfunction: a comparison between alprostadil and a combination of vasoactive intestinal polypeptide and phentolamine mesilate. - Curr. Med. Res. Opin., 23, 2577-2583, 2007. Levine L.A. - Diagnosis and treatment of erectile dysfunction. - Am. J. Med., 109, 3-30S, 2000. Carson C.C. - Oral and injectable medications for the treatment of erectile dysfunction. - Curr Urol. Rep., 1, 307-312, 2000. Yap R.L., McVary K.T. - Topical agents and erectile dysfunction: is there a place? - Curr Urol. Rep., 3, 471-476, 2002. Porst H. - The rationale for prostaglandin E1 in erectile failure: a survey of worldwide experience. - J. Urol., 155, 802-815, 1996. Goldstein I., Payton T.R., Scheichter P.J. - A double-blind, placebo-controlled, efficacy and safety study of topical gel formulation of 1 % alprostadil (Topiglan) for the in-office treatment of erectile dysfunction. - Urology, 57, 301-305, 2001. Mikami E., Ohno T., Matsumoto H. - Simultaneous identification/ determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition. - Forensic Sci. Int., 130, 140-146, 2002. Zorgniotti A.W. - Experience with buccal phentolamine mesylate for impotence. - Int. J. Impotence Res., 6, 37-41, 1994.
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Gwinup G. - Oral phentolamine in non-specific erectile insufficiency. - Ann. Intern. Med., 109, 162-163, 1988. Deneer V.H., Drese G.B., Roemele P.E., Verhoef J.C., Lie-AHuen L., Kingma J.H., Brouwers J.R., Junginger H.E. - Buccal transport of flecainide and sotalol: effect of a bile salt and ionization state. - Int. J. Pharm., 241, 127-134, 2002. Attia M.A., Gibaly I.E., Shaltout S.E., Fetih G.N. - Transbuccal permeation, anti-inflammatory activity and clinical efficacy of piroxicam formulated in different gels. - Int. J. Pharm., 276, 11-28, 2004. Senel S., Hoogstraate A.J., Spies F., et al. - Enhancement of in vitro permeability of porcine buccal mucosa by bile salts: kinetic and histological studies. - J. Control Rel., 2, 45-56, 1994. Shin S.C., Cho C.W., Yang K.H. - Development of lidocaine gels for enhanced local anesthetic action. - Int. J. Pharm., 287, 73-78, 2004. Wang R., Howard G.E., Hoang A., Yuan J.H., Lin H.C., Dai Y.T. - Prospective and long-term evaluation of erect penile length obtained with inflatable penile prosthesis to that induced by intracavernosal injection. - Asian J. Androl., 11, 411-415, 2009. Lu W., Jiang W., Chen J., Yin M., Wang Z., Jiang X. - Modulation of brain delivery and copulation by intranasal apomorphine hydrochloride. - Int. J. Pharm., 12, 196-205, 2008.
DISCLOSURE The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
MANUSCRIPT Received 10 June 2013, accepted for publication 9 August 2013.
26
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Yujian Zhang1, 2#, Nianqin Yang3#, Jianwei Lv2*, Haibo Song2, Xuehui Duan2, Jing Leng2, Juanjie Bo2, Dongming Liu2, Yiran Huang2 1
Department of Urology, Fourth Affiliated Hospital of Nantong University (First People’s Hospital of Yancheng), Yancheng, China 2 Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China 3 Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China # These authors contributed equally to this work. *Correspondence: [email protected]
Phentolamine (PT) is a non-selective a-adrenoceptor blocking agent. Oral and intracavernosal PT therapy are effective treatment options for erectile dysfunction. In the present study, a local therapy system of PT preparation was developed, by which its penetration enhancers, safety, stability and in vivo were investigated. The effect of permeation enhancers on in vitro mucosa permeability was assessed using sodium dodecyl sulphate (SDS), azone, propylene glycol (PG), Pharmasolve, Labrasol, Transcutol P, oleic acid, peppermint oil (lipophilicity), peppermint oil (hydrophilic) and their concentrations for absorption enhancement were optimized. The rabbit model was used to evaluate the safety and efficacy. peppermint oil appeared to be the most effective one for enhancing the mucosa permeation of PT than the other enhancers. These enhancements by peppermint oil were statistically significant (p < 0.05) compared to control. The order of permeation enhancement was PO (hydrophilic) > PO (lipophilicity) > azone > Transcutol P ≈ Labrasol > SDS ≈ PG ≈ Pharmasolve ≈ oleic acid. Histological investigations were performed on buccal mucosa and indicated no major morphological changes. The enhancing effect of peppermint oil on the mucosa absorption of PT was evaluated from the mucoadhesive gels in rabbits. Phentolamine local therapy would produce an ideal therapeutic effect on ED. Key words: Phentolamine – Permeation enhancers – Mucoadhesive gels – Mucosa delivery.
Male impotency or erectile dysfunction (ED) is a problem affecting millions of men across the world. Up to thirty million men are affected by male impotency in China alone. The problem is especially common in aged men. There are several treatments available for this condition. Although many treatment options such as intracavernosal vasoactive drugs, medical devices [1-3], surgery and psychotherapy have been used over the years for the treatment of ED, the introduction of an effective oral agent, sildenafil (Viagra, Pfizer, New York, NY, USA), has revolutionized the treatment of this condition [4, 5]. Efforts to develop minimally invasive local therapies for ED continue because they can conceivably circumvent many of the systemic side effects caused by oral medications. In addition, local therapies may benefit those patients who are unresponsive to current oral phosphodiesterase type 5 (PDE5) inhibitors or those who have contraindications to the use of oral therapies, such as in men receiving oral nitrates and men with severe cardiovascular disease. Intracavernosal and transurethral alprostadil therapies are effective with high success rates, but also with high rates of discontinuation because of discomfort, injection pain, or local fibrosis [6, 7]. Topical vasoactive drug delivery is a simple, safe, reversible, and relatively noninvasive alternative modality for the treatment of ED [8]. Phentolamine (PT) is a non-selective a-adrenoceptor blocking agent. The relaxation of vascular smooth muscle and increase of the blood flow can be triggered by the combination of PT and A-adrenoceptor within the cavernous tissue. Oral and intracavernosal PT therapy are effective treatment options for ED [9]. Transdermal delivery of PT alone for ED treatment is rarely reported by laboratory and clinical studies [10, 11]. In the present study, a local therapies system of PT preparation was developed, and its penetration enhancers, safety and stability in vitro were investigated. The effects of PT on conscious rabbits model was compared to that of sildenafil (Viagra) in order to confirm whether the use of PT local therapy would produce an ideal therapeutic effect for ED.
I. MATERIALS AND METHODS 1. Reagents and chemicals
PT was gifted by the Department of Pharmaceutics, Shanghai Xudong Haipu Pharmaceuticals Ltd. (China). Permeation enhancers (PE): sodium dodecyl sulphate (SDS), azone, propylene glycol (PG), Pharmasolve, Labrasol, Transcutol P, oleic acid, peppermint oil (lipophilicity), peppermint oil (hydrophilic) were purchased from Shanghai Chemical Reagents Research Institute (China). All other chemicals were of commercially analytical grade.
2. Tissue preparation
Guinea pig buccal tissue models were prepared for permeation studies of selecting the enhancers. Firstly, Guinea pig buccal tissue was chosen as its non keratinized morphology is quite similar to human buccal epithelium [12]. Buccal tissue was removed after sacrificing the animal and was stored in phosphate buffer (pH 6.8) then immediately transported to the experimental setup. The buccal mucosal membranes were separated by removing the underlying connective tissues using surgical scissors making sure that the basal membrane was still present. The tissue was rinsed and then stored in ice-cold phosphate buffer until mounted in the Franz diffusion cell (within 1 h upon removal) [13]. Slice thickness ranged from 2.2 to 2.5 mm and was mounted between donor and receiver chambers of the diffusion cells for permeation studies.
3. In vitro diffusion of permeation studies
The isolated buccal mucosa (Guinea pig buccal tissue) was mounted in Franz diffusion cells with a diffusion area of 1.2 cm2 and a compartment volume of 17 mL. After an equilibrium period with phosphate buffer on both sides, the acceptor side was filled with phosphate buffer and donor side with PT gel (2.5 mg/mL) in phosphate buffer alone or with the permeation enhancers. The solution in the receptor compartments was continuously stirred at 600 rpm using a magnetic stirring bead on electric stirrer. The diffusion studies were 22
A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
carried out at 32 ± 1 °C. Samples of 0.2 mL were taken from the acceptor side at 1, 3, 5, 7, 9 and 12 h, replaced with the same amount of fresh buffer solution, and subsequently assayed using HPLC.
8. Rabbit irritation and efficacy test
The rabbit irritation and efficacy test was conducted in accordance with the requirements of current, internationally recognized good laboratory practice standards. In the beginning of the treatment, the white New Zealand male rabbits were 12-15 weeks old and weighed between 2.4 and 3.6 kg. The animals were kept singly in cages in a room in which the temperature and the relative humidity were maintained within the range of 16-20 ℃ and 40-70 %, respectively. A 12-h light/dark cycle was maintained. In the irritation test, the external genitalia of rabbits were daubed with PT gel once daily with 0.1 mL for 10 consecutive days. On day 11, the rabbits were sacrificed and the genitalia was carefully separated from their bodies. The genitalia was fixed with 10 % formalin, sliced on a microtome, stained with haematoxylin-eosin and observed using a light microscope (Axiovert 200MAT, Carl Zeiss, Germany). The external genitalia of rabbits daubed with saline were used as a control. For each group five rabbits were used. In the efficacy test [13, 16, 17], 24 rabbits were used in this experiment and randomly divided into 4 groups. The grouping of the rabbits is summarized in Table II. From the previous data, the minimal dose of different administration was confirmed. Group A were given PT solution by intravenous. Group B were given sildenafil suspension by PO (proper Viagra tablets with 0.5 % HPMC). Group C and D were given PE gel by the transdermal in the presence of PE or without it. Group E was given normal saline as the negative control. After given, all subjects were observed two leading indicators. One was the onset time of the drug and the other was the maximum effect of the different formulation. The onset time was defined as the time when rabbits penises were erected obviously. The maximum effect was defined as the maximum length of the penis.
4. Determination of drug content
The amount of PT in each sample was determined by HPLC (LC10A, Shimadzu Co. Ltd., Kyoto, Japan). Chromatographic separation was achieved using a Dikma Diamonsil TM C18 column (10 μm, 150 mm × 3.9 mm) at 30 ℃. The mobile phase was a mixture of methanol-water-acetic acid-triethylamine (10:10:1:0.1, v/v) at a flow rate of 1.0ml/min. The UV absorbance of the effluent was monitored (SPD-10A, Shimadzu) at a wavelength of 278 nm.
5. Treatment of permeation data
The cumulative amount of permeated drug (from mucosal side to serosal side) was plotted versus time and the flux was calculated by the steady state part of the curve. The permeability coefficient (Kp) was calculated using the Equation 1 described by Senel et al. [14]: Eq. 1
Kp = (dQ/dt)/(ΔC × A)
where dQ/dt is the average slope from steady state portion of the curve, ΔC the concentration difference across the mucosa, A the area of diffusion. The efficacy of the different enhancers was determined by comparing the permeation rate of PE in the presence and absence of enhancers. It was defined as the enhancement factor (EF) which was calculated using the following Equation 2 [15]: EF = phentolamine permeation rate at steady state in the presence of enhancer/phentolamine permeation rate at steady state in the absence of enhancer
Eq. 2
Table II - The grouping of the rabbits.
One-way analysis of variance (ANOVA) was used for statistical evaluation.
6. Preparation for PT gel
Firstly, the chosen enhancers (the better one in the in vitro study) was sprinkled on chlorhexidine acetate solution and totally swelled. PT powder, wetted with glycerol and grinded, was added to 0.5 % HPMC with sodium bisulfate solution. After stirring for 30 min, phosphoric acid was added to adjust the pH of the system to around 4-6 and a brown clearing gel was formed (total volume 10 mL). A basic formulation with respect to the content of different ingredients and their function was listed in Table I.
PT glycerol chosen enhancers HPMC sodium bisulfite chlorhexidine acetate phosphoric acid Distilled water
10 % 5% Better one 0.5 % 0.5 % 0.01 % Appropriate amount Up to 100 %
Active agent humectant penetration enhancer gel base oxidation inhibitor antiseptic pH adjustor Dispersion medium
Dose
Route of administration
A B C D E
PT solution Sildenafil tablet PT spray with PE PT spray without PE Normal saline
0.6 mg/kg 15 mg/kg 0.25 mg/kg 0.25 mg/kg --
i.v. p.o. Transdermal Transdermal Transdermal
II. RESULTS AND DISCUSSION 1. Selection of permeation enhancers
The representative chromatograms of blank (A); a pure drug sample (B) and sample (C) are shown in Figure 1. The analytical peaks of PT were resolved with good symmetry, the retention time of PT was 12.2 min, no endogenous sources of interference were observed at the retention time of the analyte. The permeation profiles of PT in presence of various enhancers are reported in Figures 2 to 6, while their permeability coefficients (Kp) and the enhancement factors (EF) are reported in Table III. In case of control, 50.2 ± 8.1 % PT was found to permeate through the buccal mucosa in 12 h with permeability coefficient of 0.45 (± 0.21) × 10-4 cm/s. In order to improve mucosa permeation and hence absorption of PT, various permeation enhancers such as SDS, azone, PG, Pharmasolve, Labrasol, Transcutol P, oleic acid, PO (lipophilicity), PO (hydrophilic) were used. EF as a ratio of cumulative PT permeated with PE to the control. From the data of SDS, PG, Pharmasolve, oleic acid, no significant improvement was noticed (p > 0.05) in permeability coefficient and EF, respectively. As a result, the data were not shown in this paper.
Table I - Basic formulation of PT gel. Function
Test design
The results were expressed as mean ± SD. Student’s t-test was used to study the statistical difference and a value of P < 0.05 was considered statistically significant.
A sufficient quantity of PT gel was stored in desiccator containing saturated solution of sodium chloride, which gave a relative humidity of 75 ± 5 %. The desiccator was placed in a hot air oven maintained at 25 ± 2 ℃, and samples were withdrawn at 0, 30, 60, 90, and 180 days. The drug content remaining and physical stability of formulation were measured at predetermined time interval.
Content
Group name
9. Statistical analysis
7. Stability studies
Ingredient
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
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A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
Cumulative PT permeated (%)
100
Figure 1 - HPLC chromatograph of PT. A: blank; B: a pure drug sample (0.1 mg/mL); C: sample (t = 7 h).
Control PO1(2.5%)
90
PO1(1%) PO1(5%)
80 70 60 50 40 30 20 10 0 0
90
Control Transcutol P(2.5%)
80
Transcutol P(1%) Transcutol P(5%)
60 50
100
40
90
30
80
20 10 0 1
3
5
7
9
12
Time (h)
Figure 2 - Permeation of PT in presence of Azone across buccal mucosa.
Cumulative PT permeated (%)
100
5
7
9
12
Control PO2(2.5%)
PO2(1%) PO2(5%)
70 60 50 40 30 20 10 0
90
Control Labrasol(2.5%)
80
0
Labrasol(1%) Labrasol(5%)
1
3
5
7
9
12
Time (h)
70
Figure 6 - Permeation of PT in presence of peppermint oil (hydrophilic) across buccal mucosa.
60 50
Table III - Permeability of PT across buccal mucosa using different penetration enhancers.
40 30 20 10
PE name
Conc. (% g/g)
Kp (10-4 cm/s)
Cumulative PT permeated
EF
Control
-
0.45
50.2
1.00
Azone
1 2.5 5
3.07 3.15 3.17
65.5* 69.3* 67.5*
1.30 1.38 1.34
Labrasol
1 2.5 5
1.45 2.11 2.15
59.4* 58.2* 56.1
1.18 1.16 1.12
Transcutol P
1 2.5 5
0.71 0.81 0.77
50.8 59.4* 54.3
1.01 1.18 1.08
PO (lipophilicity)
1 2.5 5
5.26 5.62 7.06
68.7** 76.4** 85.2**
1.37 1.52 1.70
PO (hydrophilic)
1 2.5 5
6.04 6.53 6.68
72.1** 82.4** 88.4**
1.44 1.64 1.76
0 0
1
3
5
7
9
12
Time (h)
Figure 3 - Permeation of PT in presence of Labrasol across buccal mucosa. 100
Cumulative PT permeated (%)
3
Figure 5 - Permeation of PT in presence of peppermint oil (lipophilicity) across buccal mucosa.
70
0
1
Time (h)
Cumulative PT permeated (%)
Cumulative PT permeated (%)
100
90
Control Transcutol P(2.5%)
80
Transcutol P(1%) Transcutol P(5%)
70 60 50 40 30 20 10 0 0
1
3
5
7
9
12
*p < 0.05 vs control group. **p < 0.01 vs control group.
Time (h)
Figure 4 - Permeation of PT in presence of Transcutol P across buccal mucosa.
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A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
In case of azone, Transcutol P, Labrasol, significant improvement (p < 0.05) in permeation was discerned of the individual concentration. Azone at 1, 2.5, 5 % showed 65.5~69.3 % of PT permeation (p < 0.05). Only Labrasol at 2.5, 5 %; Transcutol P at 2.5 % showed the significant difference and the amount of the permeation was not dose dependent. When using PO (both lipophilicity and hydrophilic), highly significant improvement was noticed (p < 0.01) in permeability coefficient and EF as the values were 68.7~85.2 % and 72.1~88.4 %, respectively. As the concentration of PO was increased and an increase in permeation was observed. The order of permeation enhancement was PO (hydrophilic) > PO (lipophilicity) > azone > Transcutol P ≈ Labrasol > SDS ≈ PG ≈ Pharmasolve ≈ oleic acid. Permeability of mucosa to PT alone was rather low, whereas permeability increased most significantly (p < 0.01) in the presence of PO (hydrophilic). The value of Kp augmented 15.7-fold in presence of PO (hydrophilic) as compared to the control. Hence, PO (hydrophilic) was selected for further studies.
J. DRUG DEL. SCI. TECH., 24 (1) 22-26 2014
Figure 7 - Stability studies of PT gel.
2. Stability studies
Figure 7 depicts results of stability study of PT gel. No microscopical physical changes were observed during storage. Drug content and pH values of the formulations carried out at 6 months showed no significant difference. Hence, it was concluded that the PE gel exhibited good stability.
3. Rabbit irritation
The successful use of transdermal delivery systems is not only limited to their efficacy, but equally important, is their safety. Hence, it was important to investigate the safety of the optimized formulation. Figure 8 shows light photomicrograph taken from anterior cross section of rabbits penis following 10-day exposure to the PT gel. Examination showed buccal mucosa depicts the outermost layer of stratified squamous epithelium, below which can be seen the basement membrane, lamina propria, and submucosa. The boundary between the buccal epithelium and the connective tissue is delineated by the basement membrane. Signs of irritation such as vascular congestion and subepithelial edema were not observed. Moreover, none of severe signs such as appearance of epithelial necrosis, sloughing of epithelial cells and hemorrhage was detected in any of the rabbits. The morphological study revealed the safety of the tested formulation which was deposited for 10 days to the mucosa of rabbits.
Figure 8 - Micrographs of multiple dosing. (A) Negative control (saline); (B) PT gel with peppermint oil (hydrophilic).
4. Efficacy test
Currently, there are usually two animal models for evaluating the treatment of ED. One was rat model and the other was rabbit model. The former mainly stimulates the sponge nerves and induces genital congestion, then the changes of the sponge pressure could reflect the efficacy of the drug. However, this method has many drawbacks such as the animal need to be anesthetized over the whole study and the preparation of process is tedious. Therefore in this paper, Rabbits model was chosen, because its physical morphology is similar to human’s [13, 16]. Table IV and Figure 9 show the pharmacodynamic result of different formulations. Sildenafil tablet was the most common formulation in the commercial market. From the data of group A and B, both PT and Sildenafil could increase the penis of rabbits about 10 mm length. The onset time was 6.5 and 32.4 min, respectively. From the data of group D, the onset time need 1 h and the length of penis was only 9.1 mm. When the PT gel was used with proper PE, PT mucosa delivery showed excellent performance. The onset time and the length of penis of group C were similar with the data of Group B. So it could be suggested that local therapies for ED might be expected. The reason is that they can conceivably circumvent many of the systemic side effects related to oral medications.
Figure 9 - Pharmacodynamic result of different formulations. Table IV - Pharmacodynamic result of different formulations. Group name A B C D E
Test design PT solution Sildenafil tablet PT spray with PE PT spray without PE Normal saline
Onset time (min)
Max length (mm)
6.5 ± 1.8 32.4 ± 3.3 30.8 ± 4.1** 65.6 ± 7.6 30.7 ± 5.2
9.6 ± 0.7 10.5 ± 0.8 10.3 ± 0.6 9.1 ± 0.6 1.2 ± 0.1
**p < 0.01 vs group A.
* In the present study, a local therapy system of PT preparation was developed, and its penetration enhancers, safety, stability and in vivo were investigated. Peppermint oil appeared to be the most effective one for enhancing the mucosa permeation of PT than the other enhancers. Histological investigations were performed on buccal mucosa and 25
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A preclinical study of novel phentolamine formulation and in vitro/in vivo evaluation Y. Zhang, N. Yang, J. Lv, H. Song, X. Duan, J. Leng, J. Bo, D. Liu, Y. Huang
indicated no major morphological changes. Pharmacodynamic result showed PT local therapy would produce an ideal therapeutic effect on ED.
11. 12.
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DISCLOSURE The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
MANUSCRIPT Received 10 June 2013, accepted for publication 9 August 2013.
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