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Treatment of pulmonary arterial hypertension by vardenafil-solid dispersion lozenges as a potential alternative drug delivery system
Journal of Drug Delivery Science and Technology 55 (2020) 101444
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Treatment of pulmonary arterial hypertension by vardenafil-solid dispersion lozenges as a potential alternative drug delivery system
T
Amr S. Abu Lilaa,b, Eman Gomaaa, Fakhr Eldin S. Ghazya, Azza A. Hasana,∗ a b
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt Department of Pharmaceutics, College of Pharmacy, Hail University, Hail, 81442, Saudi Arabia
ARTICLE INFO
ABSTRACT
Keywords: Vardenafil Pulmonary arterial hypertension Solid dispersion Lozenges Cyclic guanosine monophosphate
Pulmonary Arterial Hypertension (PAH) is a condition of increased blood pressure within the arteries of the lung. Vardenafil (VDF) is commonly used for alleviating pulmonary blood pressure but unfortunately; VDF has low bioavailability due to poor solubility and extensive first-pass metabolism after ordinary oral administration. Therefore, in this study, solid dispersion (SD) of VDF was prepared to enhance VDF dissolution rate and then incorporated into lozenges as a buccal dosage form for systemic drug delivery avoiding first-pass effect. Vardenafil solid dispersion (VDF-SD) lozenges (L1 formula) was selected as master formula due to its high release rate. Organ bio-distribution of VDF (20 mg/kg) following VDF-SD lozenges (L1) administration in adult albino female rats was investigated, and showed a significant increase in Cmax (≈11.55 times) and AUC0–4 h (≈11.81 times) of VDF in lung compared to oral administration of VDF suspension (p < 0.0001). In addition, cyclic guanosine monophosphate (cGMP) serum level, used as indicator of VDF in-vivo efficacy, was higher following VDF-SD lozenges (L1) administration, compared to that following oral administration of VDF suspension. This study suggests that administration of VDF as lozenges in the mouth cavity to be a potential alternative to oral route with superior therapeutic effect targeting pulmonary arterial hypertension.
1. Introduction Pulmonary arterial hypertension (PAH) is a progressive debilitating disease characterized by an increase in pulmonary vascular resistance; leading to right heart failure and premature death [1]. Vardenafil (VDF) is a potent and highly selective phosphodiesterase type 5 (PDE-5) inhibitor that hase been shown in numerous clinical trials to improve erectile function in men [2]. VDF can also be effective as a therapy for a range of cardiovascular diseases. It is a more selective PDE5 inhibitor than tadalafil or sildenafil and is ten times more potent than sildenafil [3]. The favorable effects of VDF therapy on symptoms, exercise capacity, hemodynamics and clinical outcome in treating naive patients with PAH and the relatively low cost of this medication suggest its usefulness as a first-line treatment in developing countries [4]. Vardenafil is an inhibitor of PDE5, which is an enzyme that acts on cyclic Guanosine Mono Phosphate (cGMP) and breaks it down. This inhibitor binds to the catalytic site of PDE5 with high affinity and specificity, and thus, inhibit PDE-5 enzyme activity, preventing the break bown of cGMP into inactive GMP. Consequently, VDF could result in an increase in serum cGMP levels, which in turn, can lead to smooth muscle relaxation and vasodilation.
∗
Nonetheless, vardenafil suffers from poor oral bioavailability due to extensive first-pass metabolism and low water solubility; VDF is designated as a class II drug according to BCS [5]. Accordingly, there is an urgent need for developing a dosage form that can enhance VDF dissolution and at the same time bypass the first-pass metabolism. Lozenges are palatable solid unit dosage form administered in the oral cavity. They are meant to dissolve in mouth or pharynx for its local effect [6]. They may contain one or more medicaments in a flavored and sweetened base [7]. Recently, lozenges have been challenged for their potential to enhance the systemic effect of some drugs provided that the drug is well absorbed through the buccal linings or when it is swallowed [8]. Ebbert et al. [9] have evaluated the efficacy of 12 weeks of 4 mg nicotine lozenges in a randomized clinical trial for smokeless tobacco use. They confirmed that nicotine lozenges could produce a 25% increase in nicotine concentration area under the curve compared to that achieved with the nicotine gum. In the same context, Menon et al. [10] have also addressed the potential of mouth dissolving lozenges to enhance the bioavailability of curcumin as compared with the conventional hard gelatin capsule in fourteen healthy male subjects. They reported that administration of even lower dose (one fifth the conventional dose) of curcumin lozenges could result in a two-fold
Corresponding author. E-mail address: [email protected] (A.A. Hasan).
https://doi.org/10.1016/j.jddst.2019.101444 Received 11 October 2019; Received in revised form 25 November 2019; Accepted 1 December 2019 Available online 02 December 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 55 (2020) 101444
A.S. Abu Lila, et al.
increase in Cmax as compared to that achieved with a full conventional dose of curcumin-containing hard gelatin capsule. Soft Lozenges [11] are meant for either chewing or even melt to release the drug in mouth. They can be made from PEG 1000 or 1450, chocolate or sugar-acacia base while some soft candy formulations may also contain acacia and silica gel. Acacia is used to provide texture and smoothness and silica gel is used as a suspending agent to avoid settling of materials to the bottom of the mould cavity during the cooling. Including VDF as solid dispersion (SD) in soft lozenges may give further enhancement in dissolution rate in buccal cavity. The best improvement of dissolution rate of VDF was by using tartaric acid SDs. This may be attributed to several mechanisms; such as reduction of particle size, solubilizing effect of the carrier, and absence of crystals aggregation, improved wettability and dispersibility of the drug, drug dissolution in hydrophilic carrier, drug conversion to amorphous state, and combination of the above mentioned factors [12]. In this study, therefore, medicated soft lozenges with VDF or VDFSD were prepared and evaluated for its weight variation, hardness and thickness, erosion time, drug content, friability, surface pH and in-vitro release. Moreover, in-vivo drug distribution study and measurement of cGMP serum level were carried out for in-vivo evaluation of the optimized formula compared to VDF oral suspension.
evaporation was continued until constant weight was obtained. Solid residues were dried in a vacuum oven for 24 h at room temperature, pulverized and sieved using the sieve (size 40 #) [12]. 2.3. Evaluation of medicated lozenges The formulated lozenges were evaluated for the following parameters: 2.3.1. Weight variation Lozenges were randomly tested to ensure their weight uniformity. A number of 20 lozenges were weighed; average weight and % weight variation were calculated. For USP; the maximum difference of only 10% was allowed for each lozenge [8]. 2.3.2. Hardness and thickness The prepared lozenges were evaluated for hardness and thickness (using hardness tester and Vernier Calipers). The extent to which the thickness of each lozenge deviated from the standard value was determined [14].
2.1. Materials
2.3.3. Erosion time The test was carried out for the prepared lozenges in Sӧrensen's phosphate buffer of pH 6.8 using USP Disintegration apparatus. The lozenges pass the test if all six have been disintegrated [15]. In-vitro erosion time was recorded and expressed in seconds [16].
Gum acacia, gum tragacanth, and xanthan gum were kindly supplied by El-Nasr Pharmaceuticals Chemicals Co. (Qalyubia, Egypt). Polyethylene glycol (PEG) 400 and 1450 were purchased from Hoechest Chemikalien (Werk Gendort, Germany). Tartaric acid, Acetone and tricholoroacetic acid (TCA) were obtained from Analytical Department, Faculty of Pharmacy, Zagazig University. VDF was kindly supplied by G.N.P. Co. (6th of October City, Giza, Egypt).
2.3.4. Drug content Lozenges from each batch were selected, weighed individually and crushed in a mortar. The resultant powder was dispersed in 100 ml using pH 6.8 buffer and sonicated for 30 min. The solution in the volumetric flask was filtered, diluted suitably, and analyzed spectrophotometrically at 270 nm using UV spectrophotometer. All the formulations should be within the standard limits [8].
2. Materials and methods
2.3.5. Friability test Friability of the prepared lozenges was tested using (Roche friabilator) [17]. Six tablets from each batch were examined for friability and the equipment was run for 4 min at 25 revolutions/min. The lozenges were taken out, dedusted and reweighed. Friability of the formulations should be not more than 1% [18].
2.2. Formulation of medicated PEG-based soft lozenges by melting and congealing technique Powders were blended together to form uniform mixture. PEG mixture was melted in a beaker; the powder mixture was added to the molten base and blended thoroughly. The mixture was cooled to less than 50 °C, drug was added and mixed well. Silica gel was added to prevent sedimentation of drug. The blend was poured into moulds and allowed to cool. The prepared lozenges were stored under refrigeration [13]. Composition of soft lozenges using different polymers was listed in Table 1. All formulae were prepared by incorporating 20 mg of VDF or its equivalent amount of VRD: Tartaric acid (1:1) Solid dispersions (VDF-SDs), prepared by solvent evaporation method. To prepare VDFSD, accurately weighed quantities of VDF and Tartaric acid were dissolved by adding sufficient quantity of methanol. The mixture was stirred at room temperature and methanol was then removed under vacuum at a maximum temperature of 40 °C. The process of
2.3.6. Surface pH The lozenges were first allowed to swell by keeping them in contact with Sӧrensen's phosphate buffer of pH 6.8 for 2 h. The pH value was recorded by bringing electrode into solution and allows equilibrating for 1min [19]. 2.4. In-vitro studies 2.4.1. In-vitro release studies In-vitro release studies were carried out using USP dissolution test apparatus type II (paddle type) at 50 rpm and 37 ± 0.5 °C using 100 ml of a Sӧrensen's phosphate buffer of pH 6.8 as a dissolution medium. One lozenge was placed in each flask of the dissolution apparatus and 5 ml samples were withdrawn at an interval of 10 min up to 60 min. In order to maintain sink conditions, an equal volume of dissolution medium was replaced. The samples were analyzed by using UV–Visible spectrophotometer at λmax 270 nm and percentage drug released was calculated. This experiment was done in triplicate; the average percentage release and standard deviation were calculated.
Table 1 Composition of VDF soft lozenges. Composition
PEG 1450: PEG 400 (7:3) Silica gel Gum acacia Gum tragacanth Xanthan gum
Formula L1
L2
L3
L4
3 Q.S – – –
2.7 Q.S 0.3 – –
2.7 Q.S – 0.3 –
2.7 Q.S – – 0.3
2.4.2. Kinetics evaluation of the in-vitro release data The data of drug release from the tested formulations were subjected to analysis to determine the order of kinetic release according to the equations previously mentioned.
Weight of blank soft lozenges = 3 g 2
Journal of Drug Delivery Science and Technology 55 (2020) 101444
A.S. Abu Lila, et al.
(AUC
2.5. In-vivo drug distribution studies
test/
AUC
reference)
X (Dose
reference/
Dose
test)
x 100%
Where; AUC is the area under drug concentration-time curve from time zero to the last sampling time.
2.5.1. Animals Adult albino female rats, weighing 200–250 gm each, were used. Rats were obtained from animal center, Faculty of pharmacy, Zagazig University, Egypt and treated according to Ethical Committee of animal handling in Zagazig University “ECAHZU” (approval number:P17-072017). Rats were maintained on sawdust bedding free of any known chemical contaminants in animal facility at 25 ± 5 °C and 50–80% relative humidity. The animals were fasted for 24 h prior to dosage administration with free access to water. All rats received an equivalent amount of 20 mg VDF per Kg body weight [20] according to the following arrangement: Group I: (Control group): Rats didn't receive any drug. Groups II: Rats received a calculated volume of VDF suspension via gastric gavage. Groups III: Rats were anaesthetized by intravenous injection of pentobarbital at a dose of 25 mg/kg. The rats were then placed on a table with the lower jaw supported in a horizontal position and oral soft lozenges (L1) containing the calculated amount of VDF-SD were placed carefully on the rat's tongue. The rats were anaesthetized to ensure the maintenance of the dosage forms in the oral cavity without escaping down to the gastrointestinal tract [21].
2.5.3. Measurement of cGMP level in serum The aim of this experiment was to measure cGMP in serum as an indicator for VDF concentration in the body after administration of L1 (VDF-SD). For the measurement of serum cGMP, rats were randomly divided into three groups each containing 6 rats; Group 1, negative control (non-treated rats), Group 2, rats treated with an oral VDF suspension, Group 3, rats treated with L1 lozenges containing VDF-SD. All treated animals received VDF at a dose of 20 mg/kg. At different time points (0.5, 1, 2, and 4 h) post VDF administration, blood samples were withdrawn from the tail vein of each rat. To obtain serum, the blood was placed at room temperature for 30 min and then centrifuged at 5000 rpm and 4 °C for 15 min. The serum collected from non-treated rats was used as a control. Quantitative measurement of cGMP level was carried out using cGMP Direct Immunoassay Kit (Abcam, MA, USA) according to general principle of ELISA technique and the manufacturer's recommended instructions [24]. The percentage increase of cGMP was calculated using the following formula.
(average cGMP after the tested route
2.5.2. In-vivo organ distribution studies In-vivo drug distribution studies were carried out to compare the tissue distribution of VDF or its equivalent SD from the tested formula with that of the free oral drug in terms of percentage increase in AUC in various organs namely; liver, lungs, kidney and Uterus/vagina [22]. Female rats were randomly divided into 3 groups containing 12 rats each. Three animals from each group were sacrificed at 0.5, 1, 2, and 4 h after administration. The organs were collected immediately, and cleaned with saline solution several times to ensure removal of blood and adhered drug and/or dosage forms. The removed tissues were weighed after removing the adhered fats or any other extra tissues. Exactly 2 ml of Phosphate Buffer Solution (PBS) of pH 7 was added to 0.1 g of tissue, and homogenized using Cole Parmer homogenizer, at 20 000 rpm under ice bath. The diluted homogenates were transferred into respective tubes and stored in deep-freeze (−70 °C) to precipitate protein as shown in Fig. 1 [23]. The concentration of VDF versus time profile was constructed, analyzed and the relative bioavailability of VDF was calculated relative to oral route. Relative bioavailability was calculated using the following equation; Relative bioavailability % =
% increase in cGMP =
average cGMP in Control group) X 100 average cGMP in Control group
2.6. Statistical analysis Statistical analysis was performed using one-way Anova using GraphPad InStat Software (GraphPad Software, CA, USA). The level of significance was set at p < 0.05. 3. Results and discussion 3.1. Evaluation of the prepared lozenges All lozenges prepared with various excipients were visually inspected (Fig. 2). 3.1.1. Evaluation of the prepared PEG-based soft lozenges containing VDF The formulated soft lozenges of VDF L1, L2, L3 and L4 were evaluated for their dug content, weight variation, thickness, diameter, hardness, pH, and friability. The drug content of all formulations was within the specified range of 95–105%. Other parameters were
Fig. 1. Photographic images for steps of tissue preparation.
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3.2. In-vitro release from soft lozenges Polyethylene glycol (PEG1450) has been potentially utilized as a matrix component for soft lozenges prepared with melting and congealing method. It is considered as an inert carrier to improve the solubility of poorly water-soluble drugs [26]. The in-vitro release study for all soft lozenge formulations were demonstrated in Fig. 3. It was found that incorporation of SDs has fastened the release rate than freely incorporated drug, and was 99.8 ± 0.6% for the master formula (L1) in only 30 min and more than 50% of the drug released in the first 10 min (Fig. 3B), compared to 63.3 ± 0.4% when free drug was incorporated in the same formula (Fig. 3A). The release of drug was delayed by presence of gum acacia and tragacanth (L2&L3) to release about all the drug at the end of the first hour, and the least percent released was in case of xanthan gum (L4) which was very close to that of L1 with free drug. Similar results were obtained by Vidyadhara et al. [28]; who formulated Amoxicillin trihydrate soft lozenges and examined the effect of xanthan gum (polymer), sodium carboxymethyl cellulose (polymer) as excipients. They found that the formulations containing PEG 1500 and sodium carboxymethyl cellulose showed the slower release of the drug i.e. up to 45 min.
Fig. 2. Photographic image for the prepared PEG-based soft lozenges.
illustrated in Table 2. Generally, the heating and congealing technique was found to be suitable for moulding the soft lozenge formulations [25]. Soft lozenges were prepared with melting and congealing technique as previously reported and the formula comprising a ratio of 7:3 (PEG1540: PEG400) was the most suitable for use since lozenges can be easily removed from the mould. This was in accordance with Phaemachud and Tuntarawongsa [26], who used the same ratio for preparing soft lozenges of clotrimazole. All PEG-based soft lozenges were transparent, smooth with an elegant appearance except for lozenges formulated using the xanthan gum which was sticky, greasy and difficult to be molded. Hardness of soft lozenges was ranged from 6.9 ± 1.9 to 8 ± 0.3 kg/cm2. Weight loss due to friability test was < 1% in all cases which indicates the tablet's ability to withstand shock during handling. The pH of the formulations was more or less neutral, not irritate the mucosa. These results were in accordance with Modyala et al. [27]; who prepared Itraconazole soft either hand-rolled or PEG-based for treatment of invasive fungal disease and the lozenges were formulated using different excipients like gum acacia and xanthan gum to reach the objective for successful formulation of Itraconazole lozenges.
3.3. Kinetics analysis of release data The drug release mechanism was studied by comparing the respective rate constants of various kinetics equations (zero-order, firstorder and Higuchi models) to find out the mechanism of drug release. Table 3 represent the kinetics data of the release of VDF or its solid dispersion from oral lozenges in pH 6.8, and it was observed that the release of drug obeyed diffusion model. 3.4. Study of VDF distribution in different body organs This study was performed to investigate the concentration of VDF in different body organs in terms of AUC following administration of VDF suspension and the master formula (L1) containing VDF-SD to female rats and to determine the corresponding relative bioavailability. The mean concentration-time profile of VDF in liver, lung, kidney and uterus/vagina after each route of administration was constructed and analyzed by PK-solver program. Cmax, Tmax, elimination rate constant (Ke) and the half-life in each organ were determined. The area under the drug concentration in organ-versus-time curve (AUC) was calculated by the trapezoidal method. Other parameters like AUCMC and its corresponding mean residence time (MRT) of the drug following each route of administration were also inspected by the program. From the obtained results; it was clear that after administration of VDF; it was widely distributed into organs with different concentrations. VDF is a weakly basic compound (pKa values = 4.72 & 6.21), therefore, due to its moderately lipophilic character, it was extensively distributed in different body organs [29]. After administration of oral suspension; VDF was found to be
3.1.2. VDF.SD containing PEG-based soft lozenges PEG-based soft lozenges were inspected after incorporation of VDFSD and all parameters were within the accepted limits as shown in Table 2. It was noticed that incorporation of SD increased the hardness values and made lozenges more rigid against handling and transport.
Table 2 Physical evaluation of the prepared PEG-based soft lozenges containing VDF and VDF-SD. Parameter
Weight variation (g) Thickness (cm) Diameter (cm) Hardness (kg/cm2) pH Friability (%)
Formula L1 (VDF)
L1 (VDF-SD)
L2 (VDF)
L2 (VDF-SD)
L3 (VDF)
L3 (VDF-SD)
L4 (VDF)
L4 (VDF-SD)
2.9 ± 0.8 0.62 ± 0.1 1.52 ± 0.02 7.2 ± 0.2 6.90 0.66 ± 0.01
3.1 ± 0.5 0.61 ± 0.05 1.49 ± 0.01 9.8 ± 0.4 7.30 0.7 ± 0.2
2.95 ± 0.5 0.63 ± 0.05 1.5 ± 0.2 7.8 ± 0.8 6.98 0.4 ± 0.01
3.05 ± 0.3 0.59 ± 0.01 1.48 ± 0.3 9.9 ± 0.8 7.16 0.65 ± 0.05
3.3 ± 0.8 0.61 ± 0.01 1.5 ± 0.5 8 ± 0.3 6.93 0.5 ± 0.02
2.9 ± 0.8 0.6 ± 0.02 1.46 ± 0.3 10.7 ± 0.9 6.97 0.35 ± 0.01
3.1 ± 0.2 0.64 ± 0.03 1.49 ± 0.4 6.9 ± 1.9 6.94 0.6 ± 0.02
2.91 ± 0.5 0.63 ± 0.03 1.47 ± 0.2 8.5 ± 1.9 6.98 0.33 ± 0.03
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A.S. Abu Lila, et al.
Fig. 3. Cumulative % release of A) VDF and B) VDF-SD from soft lozenges.
that; high-fat meal delays the absorption of the drug with a mean delay in Tmax and Cmax [30]. Concerning lozenges; (Group III), results were shown in Table 4. It was clear that; lozenges improved the oral absorption of VDF, and its localization in lung. This was expressed by the higher Cmax (117.7 ± 10.3 μg/ml) and the significantly high AUC0–4h (407.6 ± 13.34 μg h/ml) (Table 5). The improved rate of absorption and hence bioavailability of VDF might be due to the rapid disintegration, fast dissolution of lozenges with no variation in Tmax [31]. Moreover, the rapid transport of VDF across a single epithelial layer of the oral mucosa into the interstitial fluid on the basolateral side of the epithelial cells and then into the venous circulation might be the reason for the high absorption of VDF from such formulations [32]. It is worthy to note that oral cavity formulations had a significant effect (P < 0.05) on VDF bioavailability, as evidenced by increasing the Cmax in lung by 11.5 folds for lozenge, compared to oral route. Moreover, the AUC0–4h of the lozenges (Group III) was 11.8 folds higher than that of oral route with a relative bioavailability value of 1180%. This may be attributed to the fact that the formulation process imparts an increase in the dissolution rate of VDF and thereby its absorption and corresponding bioavailability [33] (Table 5).
Table 3 Kinetics data of the release of the VDF from soft lozenges containing VDF and VDF. SD in pH 6.8. Lozenges
Zero
First
Diffusion
Observed Order
2
Correlation coefficient (r ) L1
VDF VDF-SD VDF VDF-SD VDF VDF-SD VDF VDF-SD
L2 L3 L4
0.758 0.968 0.768 0.937 0.778 0.961 0.871 0.923
−0.959 0.288 −0.838 0.164 −0.824 0.0289 −0.895 −0.966
0.981 0.994 0.929 0.991 0.934 0.997 0.973 0.977
Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion
distributed in the examined organs after being absorbed. Table 4 demonstrated the cumulative existence of VDF in different organs after administration of its oral suspension. Liver was the organ in which VDF was much localized with a mean Tmax of 1 h. The distribution pattern of VDF was in the order of liver > lung > uterus > kidney with a mean Cmax of 71.9 ± 2.033 > 10.199 ± 0.01 > 5.25 ± 0.049 > 3.25 ± 0.057 μg/ml; respectively (Table 5). That distribution may be attributed to the extensive first-pass effect occurred when the drug was orally administered and was responsible for its very low bioavailability. For group II; the oral dosing was in the fasted state as it was known
3.5. Serum level of cGMP It was clear that VDF oral suspension markedly raised cGMP levels in serum by 99% as expected due to its pharmacological activity. cGMP
Table 4 Mean concentration-time profile of VDF in different body organs, following oral administration of VDF suspension and L1 (VDF-SD) Lozenges. Time (h)
Concentration (μg/ml) Lung Oral Susp.
0 0.5 1 2 4
0 5.42 ± 0.052 10.19 ± 0.01 9.99 ± 0.025 9.19 ± 0.037
Uterus Lozenges 104.13 117.70 107.05 106.70
0 ± ± ± ±
13.3 10.3 10.2 15.2
Oral Susp. 4.22 5.25 5.22 5.13
0 ± ± ± ±
0.048 0.049 0.021 0.015
Kidney Lozenges
Oral Susp.
0 ± ± ± ±
0 3.25 ± 0.057 3.12 ± 0.04 2.99 ± 0.041 2.89 ± 0.051
7.37 7.45 7.32 7.12
0.037 0.040 0.030 0.022
5
Liver Lozenges
Oral Susp.
0 ± ± ± ±
0 5.96 ± 0.040 71.96 ± 2.033 71.54 ± 2.035 69.49 ± 1.022
4.58 4.84 4.46 4.42
0.025 0.037 0.024 0.020
Lozenges 2.47 2.56 1.56 1.52
0 ± ± ± ±
0.002 0.002 0.002 0.001
Journal of Drug Delivery Science and Technology 55 (2020) 101444 0.150 ± 0.0017 4.60 ± 0.02 1 2.56 ± 0.002 7.0 ± 0.05 17.115 ± 0.01 120.86 ± 5.0 7.1 ± 0.55
Medicated soft lozenges of VDF were formulated to bypass first pass metabolism and increase bioavailability by absorption through oral mucosa. Incorporation of VDF-SD resulted in an enhancement in drug release compared to free drug in all tested lozenges. The in-vitro release of VDF from the selected soft lozenge master formula L1 (containing VDF-SD) was found to be 99.8% in only 30 min. The oral route for administration of VDF suspension showed extensive body distribution in various organs, in the order of liver > lung > uterus > kidney, and due to the first-pass effect it has a very low bioavailability. On the other hand, the in-vivo biodistribution study demonstrated significant higher Cmax and AUC0–4h (P < 0.05) of VDF in Lung following buccal administration of VDF-SD lozenges compared to oral administration of VDF suspension with relative bioavailability of 1180%, that favors the use of this route for treatment of PAH.
0.012 ± 0.003 57.65 ± 7.49 1 71.90 ± 2.033 233.7 ± 3.97 6014.10 ± 104.83 504444.30 ± 944.8 83.9 ± 6.57 0.026 ± 0.0001 26.10 ± 1.21 1 4.84 ± 0.037 17.0 ± 0.06 183.38 ± 12.8 6966.40 ± 55.8 38.0 ± 4.22 0.014 ± 0.0005 47.34 ± 1.51 1 7.45 ± 0.040 27.4 ± 0.80 513.88 ± 14.45 35228.86 ± 1028.10 68.6 ± 2.18 0.036 ± 0.001 19.45 ± 0.38 1 10.20 ± 0.010 34.50 ± 0.130 292.50 ± 5.94 8345.78 ± 335 28.5 ± 0.55
0.028 ± 0.001 24.50 ± 1.65 1 117.70 ± 10.300 407.6 ± 13.30 4179.47 ± 112.80 149279.29 ± 1150.2 35.7 ± 3.82
0.008 ± 0.0013 89.57 ± 12.16 1 5.25 ± 0.049 19.1 ± 0.25 682.98 ± 88.85 88501.79 ± 947.5 129.6 ± 17.54
4. Conclusion
Ke (h ) T1/2 (h) Tmax (h) Cmax (μg/ml) AUC0–4h (μg.h/ml) AUC0-inf (μg.h/ml) AUMC0-inf (μg.h2/ml) MRT0-inf (h)
Lozenges
level increased to a very high percentage in group III that received lozenges by 304% (Fig. 4.). This came in agreement with the pharmacokinetics of drug, and provided a proof-of-concept that the buccal route could efficiently reduced the possibility of hepatic metabolism of the drug via. bypassing the enterohepatic circulation, and thereby, allowing the direct presentation of the drug into the systemic circulation. From the previous pharmacokinetics parameters it is concluded that VDF-SD Lozenges can be the potential alternative to oral VDF suspension for treatment of PAH. In ordinary VDF oral dosage forms, the distribution pattern of VDF was in the order of liver > lung > uterus > kidney due to extensive first-pass effect. Hence, very low bioavailability. On the other hand, lozenges improved the oral absorption of VDF, and its localization in lung due to bypassing the first pass metabolism and increasing bioavailability by absorption through oral mucosa. So, VDF-SD Lozenges give also superior therapeutic benefits to oral and more convenient dosage form, especially for patients who cannot swallow solid oral dosage forms.
CRediT authorship contribution statement Amr S. Abu Lila: Formal analysis, Writing - review & editing. Eman Gomaa: Formal analysis, Writing - review & editing. Fakhr Eldin S. Ghazy: Supervision. Azza A. Hasan: Formal analysis, Writing - review & editing.
−1
Oral Susp.
Fig. 4. Comparison of serum cGMP level for lozenges against oral Vardenafil & normal controls.
0.024 ± 0.0012 28.50 ± 1.45 0.5 3.25 ± 0.057 11.3 ± 0.35 130.17 ± 7.77 5384.90 ± 117.7 41.3 ± 2.09
Oral Susp. Lozenges Lozenges Oral Susp.
Oral Susp.
Kidney Uterus Lung Parameters
Table 5 Pharmacokinetic parameters of VDF distribution in Lung, Uterus, Kidney and Liver; following administration of Oral suspension and L1 (VDF-SD) Lozenges in female rats.
Liver
Lozenges
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Declaration of competing interest
[15] International Pharmacopoeia, fifth ed., WHO, Switzerland, 2015. [16] K. Sondarva, S. Bhadra, Development of cefixime lozenges for the treatment of throat infection, World J. Pharm. Pharm. Sci. 4 (7) (2015) 645–656. [17] R.D. Chaudhari, T.R. Jitendra, C. Amrish, Formulation and in vitro evaluation of taste masked orodispersible dosage form of levocetrizine dihydrochloride, Ind. J Pharm. Educ. Res. 41 (4) (2007) 319–327. [18] F.R. Sheeba, Formulation and evaluation of nifedipine sublingual tablets, Asian J. Pharmaceut. Clin. Res. 2 (3) (2009) 44–48. [19] S.K. Thombre, S.S. Gaikwad, Design and development of mucoadhesive buccal delivery for pantoprazole with stability enhancement in human saliva, Int. J. Pharm. Pharm. Sci. 5 (2) (2013) 122–127. [20] M.A. Taher, Effect of vardenafil on fertility in male rats, I.J.S.N. 5 (2014) 297–303. [21] R.L. Jyothi, Formulation and in vitro & in vivo evaluation of oral dispersible films of lornoxicam, Experiment 24 (1) (2014) 1649–1655. [22] Z.D. Salman, N.K. Maraie, M.G. Alabbassi, M.M. Ghareeb, In vitro/in vivo evaluation and bioavailability study of amitriptyline hydrochloride from the optimized oral fast dissolving films, UK. J. Pharm. Biosci. 2 (6) (2014) 32–42. [23] S. Shanmugam, Y.H. Kim, J.H. Park, H.T. Im, Y.T. Sohn, K.S. Kim, K. Yong-Il, C.S. Yong, J.O. Kim, H.G. Choi, J.S. Woo, Sildenafil vaginal suppositories: preparation, characterization, in vitro and in vivo evaluation, Drug Dev. Ind. Pharm. 40 (6) (2013) 1–10. [24] T. Itoh, N. Nagaya, T. Fujii, T. Iwase, N. Nakanishi, K. Hamada, K. Kangawa, H. Kimura, A combination of oral sildenafil and beraprost ameliorates pulmonary hypertension in rats, Amer. J. Respir. Crit. Care med. 169 (2004) 34–38. [25] A. Mesnukul, K. Yodkhum, J. Mahadlek, T. Phaechamud, Characterization of indomethacin release from polyethylene glycol tablet fabricated with mold, Indian J. Pharm. Sci. 72 (1) (2010) 92–100. [26] T. Phaemachud, S. Tuntarawongsa, Clotrimazole soft lozenges fabricated with melting and mold technique, Res. J. Pharm. Biol. Chem. Sci. 1 (4) (2010) 579–586. [27] D. Modyala, C. Aparna, P. Srinivas, Formulation, evaluation and characterization of Itraconazole lozenges, IOSR J. Pharm. Biol. Sci. 9 (3) (2014) 86–94. [28] S. Vidyadhara, R.L.C. Sasidhar, B. Deepti, E. Wilwin, B. Sowjanyalakshmi, Formulation and evaluation of Amoxicillin trihydrate lozenges, Dhaka Univ. J. Pharm. Sci. 14 (1) (2015) 61–70. [29] D.A. Smith, B.C. Jones, D.K. Walker, Design of drugs involving the concepts and theories of drug metabolism and pharmacokinetics, Med. Res. Rev. 16 (1996) 66243. [30] A.H. Elshafeey, E.R. Bendas, O.H. Mohamed, Intranasal microemulsion of sildenafil citrate: in vitro evaluation and invivo pharmacokinetic study in rabbits, AAPS. Pharm. Sci. Tech. 10 (2009) 361–367. [31] A. Abdelbary, A.H. Elshafeeyand, G. Zidan, Comparative effects of different cellulosic-based directly compressed orodispersable tablets on oral bioavailability of famotidine, Carbohydr. Polym. 77 (4) (2009) 799–806. [32] A.A. Mahmoud, S. Salah, Fast relief from migraine attacks using fast-disintegrating sublingual zolmitriptan tablets, Drug Dev. Ind. Pharm. 38 (6) (2011) 1–8. [33] R. Zayed, O.K. Amany, M.S.A. El-Shamy, An in vitro and in vivo comparative study of directly compressed solid dispersions and freeze dried sildenafil citrate sublingual tablets for management of pulmonary arterial hypertension, Acta Pharm. 62 (2012) 411–432.
The authors report no conflicts of interest in this work. References [1] N. Galiè, L. Rubin, Pulmonary arterial hypertension. Epidemiology, pathobiology, assessment and therapy, J. Am. Coll. Cardiol. 43 (2004) 51–90. [2] W.Q. Li, A.A. Qureshi, K.C. Robinson, J. Han, Sildenafil use and increased risk of incident melanoma in US men: a prospective cohort study, JAMA Intern Med 174 (6) (2014) 964–970. [3] M.K. Choi, I.S. Song, Characterization of efflux transport of the PDE5 inhibitors, vardenafil and sildenafil, J. Pharm. Pharmacol. 64 (2012) 1074–1083. [4] Z.C. Jing, Z.X. Yu, J.Y. Shen, B.X. Wu, K.F. Xu, X.Y. Zhu, L. Pan, Z.L. Zhang, X.Q. Liu, Y.S. Zhang, X. Jiang, N. Galiè, Vardenafil in pulmonary arterial hypertension: a randomized, double-blind, placebo-controlled study, Am. J. Respir. Crit. Care Med. 183 (12) (2011) 1723–1729. [5] J.K. Park, Y.S. Shin, S.W. Lee, K. Park, W.S. Chung, S.W. Kim, J.S. Hyun, D.G. Moon, S.K. Yang, J.K. Ryu, D.Y. Yang, K.H. Moon, K.S. Min, Effect of levitra on sustenance of erection (EROS): an open-label, prospective, multicenter, single-arm study to investigate erection duration measured by stopwatch with flexible dose vardenafil administered for 8 weeks in subjects with erectile dysfunction, Int. J. Impot. Res. 27 (2015) 95–102. [6] M.S. Umashankar, S.R. Dinesh, R. Rini, K.S. Lakshmi, N. Damodharan, A review: chewable lozenge formulation, Int. Res. J. Pharm. 7 (4) (2016) 9–16. [7] R.W. Mendes, H. Bhargava, Lozenges, in: J. Swarbick (Ed.), Encyclopedia of Pharmaceutical Technology, third ed., Informa Healthcare Inc., North California, USA, 2006, pp. 2231–2235. [8] S. Pundir, A.M. Verma, Formulation development and evaluation of antiemetic lozenges of ondasteron hydrochloride, Int. J. Pharm. Res. Biosci 3 (3) (2014) 365–372. [9] J.O. Ebbert, H. Severson, I.T. Croghan, B.G. Danaher, D.R. Schroeder, A randomized clinical trila of nicotine lozenge for smokeless tobacco use, Nicotine Tob. Res. 11 (12) (2009) 1415–1423. [10] S. Menon, M. Mhatre, M. Rajarshi, J. Thakkar, Bioavailability of curcumin from a novel mouth dissolving lozenge, Int. J. Basic Clin. Pharmacol. 7 (3) (2018) 561–568. [11] S.G. Shinde, V. Kadam, G.R. Kapse, M.D. Jadhav, S.B. Zameeruddin, V.B. Bharkad, A review on lozenges, Indo Amer. J. Pharm. Res. 4 (1) (2014) 566–570. [12] S. Chhater, K. Kumar Praveen, Evaporation method for amorphous solid dispersions: predictive tools for improve the dissolution rate of pioglitazone hydrochloride, Int. J. Pharm., Chem. & Bio. Sci. 3 (2) (2013) 350–359. [13] N. Duryodhan, C. Aparna, P. Srinivas, Formulation and evaluation of medicated lozenges of albendazole for paediatric use, Asian J. Biochem. Pharmaceut. Res. 3 (5) (2015) 202–215. [14] A. Aditya, G. Gudas, M. Bingi, S. Debnath, V. Rajesham, Design and evaluation of controlled release mucoadhesive buccal tablets of lisinopril, Int. J. Curr. Pharm. Res. 2 (2010) 24–27.
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Treatment of pulmonary arterial hypertension by vardenafil-solid dispersion lozenges as a potential alternative drug delivery system
T
Amr S. Abu Lilaa,b, Eman Gomaaa, Fakhr Eldin S. Ghazya, Azza A. Hasana,∗ a b
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt Department of Pharmaceutics, College of Pharmacy, Hail University, Hail, 81442, Saudi Arabia
ARTICLE INFO
ABSTRACT
Keywords: Vardenafil Pulmonary arterial hypertension Solid dispersion Lozenges Cyclic guanosine monophosphate
Pulmonary Arterial Hypertension (PAH) is a condition of increased blood pressure within the arteries of the lung. Vardenafil (VDF) is commonly used for alleviating pulmonary blood pressure but unfortunately; VDF has low bioavailability due to poor solubility and extensive first-pass metabolism after ordinary oral administration. Therefore, in this study, solid dispersion (SD) of VDF was prepared to enhance VDF dissolution rate and then incorporated into lozenges as a buccal dosage form for systemic drug delivery avoiding first-pass effect. Vardenafil solid dispersion (VDF-SD) lozenges (L1 formula) was selected as master formula due to its high release rate. Organ bio-distribution of VDF (20 mg/kg) following VDF-SD lozenges (L1) administration in adult albino female rats was investigated, and showed a significant increase in Cmax (≈11.55 times) and AUC0–4 h (≈11.81 times) of VDF in lung compared to oral administration of VDF suspension (p < 0.0001). In addition, cyclic guanosine monophosphate (cGMP) serum level, used as indicator of VDF in-vivo efficacy, was higher following VDF-SD lozenges (L1) administration, compared to that following oral administration of VDF suspension. This study suggests that administration of VDF as lozenges in the mouth cavity to be a potential alternative to oral route with superior therapeutic effect targeting pulmonary arterial hypertension.
1. Introduction Pulmonary arterial hypertension (PAH) is a progressive debilitating disease characterized by an increase in pulmonary vascular resistance; leading to right heart failure and premature death [1]. Vardenafil (VDF) is a potent and highly selective phosphodiesterase type 5 (PDE-5) inhibitor that hase been shown in numerous clinical trials to improve erectile function in men [2]. VDF can also be effective as a therapy for a range of cardiovascular diseases. It is a more selective PDE5 inhibitor than tadalafil or sildenafil and is ten times more potent than sildenafil [3]. The favorable effects of VDF therapy on symptoms, exercise capacity, hemodynamics and clinical outcome in treating naive patients with PAH and the relatively low cost of this medication suggest its usefulness as a first-line treatment in developing countries [4]. Vardenafil is an inhibitor of PDE5, which is an enzyme that acts on cyclic Guanosine Mono Phosphate (cGMP) and breaks it down. This inhibitor binds to the catalytic site of PDE5 with high affinity and specificity, and thus, inhibit PDE-5 enzyme activity, preventing the break bown of cGMP into inactive GMP. Consequently, VDF could result in an increase in serum cGMP levels, which in turn, can lead to smooth muscle relaxation and vasodilation.
∗
Nonetheless, vardenafil suffers from poor oral bioavailability due to extensive first-pass metabolism and low water solubility; VDF is designated as a class II drug according to BCS [5]. Accordingly, there is an urgent need for developing a dosage form that can enhance VDF dissolution and at the same time bypass the first-pass metabolism. Lozenges are palatable solid unit dosage form administered in the oral cavity. They are meant to dissolve in mouth or pharynx for its local effect [6]. They may contain one or more medicaments in a flavored and sweetened base [7]. Recently, lozenges have been challenged for their potential to enhance the systemic effect of some drugs provided that the drug is well absorbed through the buccal linings or when it is swallowed [8]. Ebbert et al. [9] have evaluated the efficacy of 12 weeks of 4 mg nicotine lozenges in a randomized clinical trial for smokeless tobacco use. They confirmed that nicotine lozenges could produce a 25% increase in nicotine concentration area under the curve compared to that achieved with the nicotine gum. In the same context, Menon et al. [10] have also addressed the potential of mouth dissolving lozenges to enhance the bioavailability of curcumin as compared with the conventional hard gelatin capsule in fourteen healthy male subjects. They reported that administration of even lower dose (one fifth the conventional dose) of curcumin lozenges could result in a two-fold
Corresponding author. E-mail address: [email protected] (A.A. Hasan).
https://doi.org/10.1016/j.jddst.2019.101444 Received 11 October 2019; Received in revised form 25 November 2019; Accepted 1 December 2019 Available online 02 December 2019 1773-2247/ © 2019 Elsevier B.V. All rights reserved.
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increase in Cmax as compared to that achieved with a full conventional dose of curcumin-containing hard gelatin capsule. Soft Lozenges [11] are meant for either chewing or even melt to release the drug in mouth. They can be made from PEG 1000 or 1450, chocolate or sugar-acacia base while some soft candy formulations may also contain acacia and silica gel. Acacia is used to provide texture and smoothness and silica gel is used as a suspending agent to avoid settling of materials to the bottom of the mould cavity during the cooling. Including VDF as solid dispersion (SD) in soft lozenges may give further enhancement in dissolution rate in buccal cavity. The best improvement of dissolution rate of VDF was by using tartaric acid SDs. This may be attributed to several mechanisms; such as reduction of particle size, solubilizing effect of the carrier, and absence of crystals aggregation, improved wettability and dispersibility of the drug, drug dissolution in hydrophilic carrier, drug conversion to amorphous state, and combination of the above mentioned factors [12]. In this study, therefore, medicated soft lozenges with VDF or VDFSD were prepared and evaluated for its weight variation, hardness and thickness, erosion time, drug content, friability, surface pH and in-vitro release. Moreover, in-vivo drug distribution study and measurement of cGMP serum level were carried out for in-vivo evaluation of the optimized formula compared to VDF oral suspension.
evaporation was continued until constant weight was obtained. Solid residues were dried in a vacuum oven for 24 h at room temperature, pulverized and sieved using the sieve (size 40 #) [12]. 2.3. Evaluation of medicated lozenges The formulated lozenges were evaluated for the following parameters: 2.3.1. Weight variation Lozenges were randomly tested to ensure their weight uniformity. A number of 20 lozenges were weighed; average weight and % weight variation were calculated. For USP; the maximum difference of only 10% was allowed for each lozenge [8]. 2.3.2. Hardness and thickness The prepared lozenges were evaluated for hardness and thickness (using hardness tester and Vernier Calipers). The extent to which the thickness of each lozenge deviated from the standard value was determined [14].
2.1. Materials
2.3.3. Erosion time The test was carried out for the prepared lozenges in Sӧrensen's phosphate buffer of pH 6.8 using USP Disintegration apparatus. The lozenges pass the test if all six have been disintegrated [15]. In-vitro erosion time was recorded and expressed in seconds [16].
Gum acacia, gum tragacanth, and xanthan gum were kindly supplied by El-Nasr Pharmaceuticals Chemicals Co. (Qalyubia, Egypt). Polyethylene glycol (PEG) 400 and 1450 were purchased from Hoechest Chemikalien (Werk Gendort, Germany). Tartaric acid, Acetone and tricholoroacetic acid (TCA) were obtained from Analytical Department, Faculty of Pharmacy, Zagazig University. VDF was kindly supplied by G.N.P. Co. (6th of October City, Giza, Egypt).
2.3.4. Drug content Lozenges from each batch were selected, weighed individually and crushed in a mortar. The resultant powder was dispersed in 100 ml using pH 6.8 buffer and sonicated for 30 min. The solution in the volumetric flask was filtered, diluted suitably, and analyzed spectrophotometrically at 270 nm using UV spectrophotometer. All the formulations should be within the standard limits [8].
2. Materials and methods
2.3.5. Friability test Friability of the prepared lozenges was tested using (Roche friabilator) [17]. Six tablets from each batch were examined for friability and the equipment was run for 4 min at 25 revolutions/min. The lozenges were taken out, dedusted and reweighed. Friability of the formulations should be not more than 1% [18].
2.2. Formulation of medicated PEG-based soft lozenges by melting and congealing technique Powders were blended together to form uniform mixture. PEG mixture was melted in a beaker; the powder mixture was added to the molten base and blended thoroughly. The mixture was cooled to less than 50 °C, drug was added and mixed well. Silica gel was added to prevent sedimentation of drug. The blend was poured into moulds and allowed to cool. The prepared lozenges were stored under refrigeration [13]. Composition of soft lozenges using different polymers was listed in Table 1. All formulae were prepared by incorporating 20 mg of VDF or its equivalent amount of VRD: Tartaric acid (1:1) Solid dispersions (VDF-SDs), prepared by solvent evaporation method. To prepare VDFSD, accurately weighed quantities of VDF and Tartaric acid were dissolved by adding sufficient quantity of methanol. The mixture was stirred at room temperature and methanol was then removed under vacuum at a maximum temperature of 40 °C. The process of
2.3.6. Surface pH The lozenges were first allowed to swell by keeping them in contact with Sӧrensen's phosphate buffer of pH 6.8 for 2 h. The pH value was recorded by bringing electrode into solution and allows equilibrating for 1min [19]. 2.4. In-vitro studies 2.4.1. In-vitro release studies In-vitro release studies were carried out using USP dissolution test apparatus type II (paddle type) at 50 rpm and 37 ± 0.5 °C using 100 ml of a Sӧrensen's phosphate buffer of pH 6.8 as a dissolution medium. One lozenge was placed in each flask of the dissolution apparatus and 5 ml samples were withdrawn at an interval of 10 min up to 60 min. In order to maintain sink conditions, an equal volume of dissolution medium was replaced. The samples were analyzed by using UV–Visible spectrophotometer at λmax 270 nm and percentage drug released was calculated. This experiment was done in triplicate; the average percentage release and standard deviation were calculated.
Table 1 Composition of VDF soft lozenges. Composition
PEG 1450: PEG 400 (7:3) Silica gel Gum acacia Gum tragacanth Xanthan gum
Formula L1
L2
L3
L4
3 Q.S – – –
2.7 Q.S 0.3 – –
2.7 Q.S – 0.3 –
2.7 Q.S – – 0.3
2.4.2. Kinetics evaluation of the in-vitro release data The data of drug release from the tested formulations were subjected to analysis to determine the order of kinetic release according to the equations previously mentioned.
Weight of blank soft lozenges = 3 g 2
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(AUC
2.5. In-vivo drug distribution studies
test/
AUC
reference)
X (Dose
reference/
Dose
test)
x 100%
Where; AUC is the area under drug concentration-time curve from time zero to the last sampling time.
2.5.1. Animals Adult albino female rats, weighing 200–250 gm each, were used. Rats were obtained from animal center, Faculty of pharmacy, Zagazig University, Egypt and treated according to Ethical Committee of animal handling in Zagazig University “ECAHZU” (approval number:P17-072017). Rats were maintained on sawdust bedding free of any known chemical contaminants in animal facility at 25 ± 5 °C and 50–80% relative humidity. The animals were fasted for 24 h prior to dosage administration with free access to water. All rats received an equivalent amount of 20 mg VDF per Kg body weight [20] according to the following arrangement: Group I: (Control group): Rats didn't receive any drug. Groups II: Rats received a calculated volume of VDF suspension via gastric gavage. Groups III: Rats were anaesthetized by intravenous injection of pentobarbital at a dose of 25 mg/kg. The rats were then placed on a table with the lower jaw supported in a horizontal position and oral soft lozenges (L1) containing the calculated amount of VDF-SD were placed carefully on the rat's tongue. The rats were anaesthetized to ensure the maintenance of the dosage forms in the oral cavity without escaping down to the gastrointestinal tract [21].
2.5.3. Measurement of cGMP level in serum The aim of this experiment was to measure cGMP in serum as an indicator for VDF concentration in the body after administration of L1 (VDF-SD). For the measurement of serum cGMP, rats were randomly divided into three groups each containing 6 rats; Group 1, negative control (non-treated rats), Group 2, rats treated with an oral VDF suspension, Group 3, rats treated with L1 lozenges containing VDF-SD. All treated animals received VDF at a dose of 20 mg/kg. At different time points (0.5, 1, 2, and 4 h) post VDF administration, blood samples were withdrawn from the tail vein of each rat. To obtain serum, the blood was placed at room temperature for 30 min and then centrifuged at 5000 rpm and 4 °C for 15 min. The serum collected from non-treated rats was used as a control. Quantitative measurement of cGMP level was carried out using cGMP Direct Immunoassay Kit (Abcam, MA, USA) according to general principle of ELISA technique and the manufacturer's recommended instructions [24]. The percentage increase of cGMP was calculated using the following formula.
(average cGMP after the tested route
2.5.2. In-vivo organ distribution studies In-vivo drug distribution studies were carried out to compare the tissue distribution of VDF or its equivalent SD from the tested formula with that of the free oral drug in terms of percentage increase in AUC in various organs namely; liver, lungs, kidney and Uterus/vagina [22]. Female rats were randomly divided into 3 groups containing 12 rats each. Three animals from each group were sacrificed at 0.5, 1, 2, and 4 h after administration. The organs were collected immediately, and cleaned with saline solution several times to ensure removal of blood and adhered drug and/or dosage forms. The removed tissues were weighed after removing the adhered fats or any other extra tissues. Exactly 2 ml of Phosphate Buffer Solution (PBS) of pH 7 was added to 0.1 g of tissue, and homogenized using Cole Parmer homogenizer, at 20 000 rpm under ice bath. The diluted homogenates were transferred into respective tubes and stored in deep-freeze (−70 °C) to precipitate protein as shown in Fig. 1 [23]. The concentration of VDF versus time profile was constructed, analyzed and the relative bioavailability of VDF was calculated relative to oral route. Relative bioavailability was calculated using the following equation; Relative bioavailability % =
% increase in cGMP =
average cGMP in Control group) X 100 average cGMP in Control group
2.6. Statistical analysis Statistical analysis was performed using one-way Anova using GraphPad InStat Software (GraphPad Software, CA, USA). The level of significance was set at p < 0.05. 3. Results and discussion 3.1. Evaluation of the prepared lozenges All lozenges prepared with various excipients were visually inspected (Fig. 2). 3.1.1. Evaluation of the prepared PEG-based soft lozenges containing VDF The formulated soft lozenges of VDF L1, L2, L3 and L4 were evaluated for their dug content, weight variation, thickness, diameter, hardness, pH, and friability. The drug content of all formulations was within the specified range of 95–105%. Other parameters were
Fig. 1. Photographic images for steps of tissue preparation.
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3.2. In-vitro release from soft lozenges Polyethylene glycol (PEG1450) has been potentially utilized as a matrix component for soft lozenges prepared with melting and congealing method. It is considered as an inert carrier to improve the solubility of poorly water-soluble drugs [26]. The in-vitro release study for all soft lozenge formulations were demonstrated in Fig. 3. It was found that incorporation of SDs has fastened the release rate than freely incorporated drug, and was 99.8 ± 0.6% for the master formula (L1) in only 30 min and more than 50% of the drug released in the first 10 min (Fig. 3B), compared to 63.3 ± 0.4% when free drug was incorporated in the same formula (Fig. 3A). The release of drug was delayed by presence of gum acacia and tragacanth (L2&L3) to release about all the drug at the end of the first hour, and the least percent released was in case of xanthan gum (L4) which was very close to that of L1 with free drug. Similar results were obtained by Vidyadhara et al. [28]; who formulated Amoxicillin trihydrate soft lozenges and examined the effect of xanthan gum (polymer), sodium carboxymethyl cellulose (polymer) as excipients. They found that the formulations containing PEG 1500 and sodium carboxymethyl cellulose showed the slower release of the drug i.e. up to 45 min.
Fig. 2. Photographic image for the prepared PEG-based soft lozenges.
illustrated in Table 2. Generally, the heating and congealing technique was found to be suitable for moulding the soft lozenge formulations [25]. Soft lozenges were prepared with melting and congealing technique as previously reported and the formula comprising a ratio of 7:3 (PEG1540: PEG400) was the most suitable for use since lozenges can be easily removed from the mould. This was in accordance with Phaemachud and Tuntarawongsa [26], who used the same ratio for preparing soft lozenges of clotrimazole. All PEG-based soft lozenges were transparent, smooth with an elegant appearance except for lozenges formulated using the xanthan gum which was sticky, greasy and difficult to be molded. Hardness of soft lozenges was ranged from 6.9 ± 1.9 to 8 ± 0.3 kg/cm2. Weight loss due to friability test was < 1% in all cases which indicates the tablet's ability to withstand shock during handling. The pH of the formulations was more or less neutral, not irritate the mucosa. These results were in accordance with Modyala et al. [27]; who prepared Itraconazole soft either hand-rolled or PEG-based for treatment of invasive fungal disease and the lozenges were formulated using different excipients like gum acacia and xanthan gum to reach the objective for successful formulation of Itraconazole lozenges.
3.3. Kinetics analysis of release data The drug release mechanism was studied by comparing the respective rate constants of various kinetics equations (zero-order, firstorder and Higuchi models) to find out the mechanism of drug release. Table 3 represent the kinetics data of the release of VDF or its solid dispersion from oral lozenges in pH 6.8, and it was observed that the release of drug obeyed diffusion model. 3.4. Study of VDF distribution in different body organs This study was performed to investigate the concentration of VDF in different body organs in terms of AUC following administration of VDF suspension and the master formula (L1) containing VDF-SD to female rats and to determine the corresponding relative bioavailability. The mean concentration-time profile of VDF in liver, lung, kidney and uterus/vagina after each route of administration was constructed and analyzed by PK-solver program. Cmax, Tmax, elimination rate constant (Ke) and the half-life in each organ were determined. The area under the drug concentration in organ-versus-time curve (AUC) was calculated by the trapezoidal method. Other parameters like AUCMC and its corresponding mean residence time (MRT) of the drug following each route of administration were also inspected by the program. From the obtained results; it was clear that after administration of VDF; it was widely distributed into organs with different concentrations. VDF is a weakly basic compound (pKa values = 4.72 & 6.21), therefore, due to its moderately lipophilic character, it was extensively distributed in different body organs [29]. After administration of oral suspension; VDF was found to be
3.1.2. VDF.SD containing PEG-based soft lozenges PEG-based soft lozenges were inspected after incorporation of VDFSD and all parameters were within the accepted limits as shown in Table 2. It was noticed that incorporation of SD increased the hardness values and made lozenges more rigid against handling and transport.
Table 2 Physical evaluation of the prepared PEG-based soft lozenges containing VDF and VDF-SD. Parameter
Weight variation (g) Thickness (cm) Diameter (cm) Hardness (kg/cm2) pH Friability (%)
Formula L1 (VDF)
L1 (VDF-SD)
L2 (VDF)
L2 (VDF-SD)
L3 (VDF)
L3 (VDF-SD)
L4 (VDF)
L4 (VDF-SD)
2.9 ± 0.8 0.62 ± 0.1 1.52 ± 0.02 7.2 ± 0.2 6.90 0.66 ± 0.01
3.1 ± 0.5 0.61 ± 0.05 1.49 ± 0.01 9.8 ± 0.4 7.30 0.7 ± 0.2
2.95 ± 0.5 0.63 ± 0.05 1.5 ± 0.2 7.8 ± 0.8 6.98 0.4 ± 0.01
3.05 ± 0.3 0.59 ± 0.01 1.48 ± 0.3 9.9 ± 0.8 7.16 0.65 ± 0.05
3.3 ± 0.8 0.61 ± 0.01 1.5 ± 0.5 8 ± 0.3 6.93 0.5 ± 0.02
2.9 ± 0.8 0.6 ± 0.02 1.46 ± 0.3 10.7 ± 0.9 6.97 0.35 ± 0.01
3.1 ± 0.2 0.64 ± 0.03 1.49 ± 0.4 6.9 ± 1.9 6.94 0.6 ± 0.02
2.91 ± 0.5 0.63 ± 0.03 1.47 ± 0.2 8.5 ± 1.9 6.98 0.33 ± 0.03
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Fig. 3. Cumulative % release of A) VDF and B) VDF-SD from soft lozenges.
that; high-fat meal delays the absorption of the drug with a mean delay in Tmax and Cmax [30]. Concerning lozenges; (Group III), results were shown in Table 4. It was clear that; lozenges improved the oral absorption of VDF, and its localization in lung. This was expressed by the higher Cmax (117.7 ± 10.3 μg/ml) and the significantly high AUC0–4h (407.6 ± 13.34 μg h/ml) (Table 5). The improved rate of absorption and hence bioavailability of VDF might be due to the rapid disintegration, fast dissolution of lozenges with no variation in Tmax [31]. Moreover, the rapid transport of VDF across a single epithelial layer of the oral mucosa into the interstitial fluid on the basolateral side of the epithelial cells and then into the venous circulation might be the reason for the high absorption of VDF from such formulations [32]. It is worthy to note that oral cavity formulations had a significant effect (P < 0.05) on VDF bioavailability, as evidenced by increasing the Cmax in lung by 11.5 folds for lozenge, compared to oral route. Moreover, the AUC0–4h of the lozenges (Group III) was 11.8 folds higher than that of oral route with a relative bioavailability value of 1180%. This may be attributed to the fact that the formulation process imparts an increase in the dissolution rate of VDF and thereby its absorption and corresponding bioavailability [33] (Table 5).
Table 3 Kinetics data of the release of the VDF from soft lozenges containing VDF and VDF. SD in pH 6.8. Lozenges
Zero
First
Diffusion
Observed Order
2
Correlation coefficient (r ) L1
VDF VDF-SD VDF VDF-SD VDF VDF-SD VDF VDF-SD
L2 L3 L4
0.758 0.968 0.768 0.937 0.778 0.961 0.871 0.923
−0.959 0.288 −0.838 0.164 −0.824 0.0289 −0.895 −0.966
0.981 0.994 0.929 0.991 0.934 0.997 0.973 0.977
Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion Diffusion
distributed in the examined organs after being absorbed. Table 4 demonstrated the cumulative existence of VDF in different organs after administration of its oral suspension. Liver was the organ in which VDF was much localized with a mean Tmax of 1 h. The distribution pattern of VDF was in the order of liver > lung > uterus > kidney with a mean Cmax of 71.9 ± 2.033 > 10.199 ± 0.01 > 5.25 ± 0.049 > 3.25 ± 0.057 μg/ml; respectively (Table 5). That distribution may be attributed to the extensive first-pass effect occurred when the drug was orally administered and was responsible for its very low bioavailability. For group II; the oral dosing was in the fasted state as it was known
3.5. Serum level of cGMP It was clear that VDF oral suspension markedly raised cGMP levels in serum by 99% as expected due to its pharmacological activity. cGMP
Table 4 Mean concentration-time profile of VDF in different body organs, following oral administration of VDF suspension and L1 (VDF-SD) Lozenges. Time (h)
Concentration (μg/ml) Lung Oral Susp.
0 0.5 1 2 4
0 5.42 ± 0.052 10.19 ± 0.01 9.99 ± 0.025 9.19 ± 0.037
Uterus Lozenges 104.13 117.70 107.05 106.70
0 ± ± ± ±
13.3 10.3 10.2 15.2
Oral Susp. 4.22 5.25 5.22 5.13
0 ± ± ± ±
0.048 0.049 0.021 0.015
Kidney Lozenges
Oral Susp.
0 ± ± ± ±
0 3.25 ± 0.057 3.12 ± 0.04 2.99 ± 0.041 2.89 ± 0.051
7.37 7.45 7.32 7.12
0.037 0.040 0.030 0.022
5
Liver Lozenges
Oral Susp.
0 ± ± ± ±
0 5.96 ± 0.040 71.96 ± 2.033 71.54 ± 2.035 69.49 ± 1.022
4.58 4.84 4.46 4.42
0.025 0.037 0.024 0.020
Lozenges 2.47 2.56 1.56 1.52
0 ± ± ± ±
0.002 0.002 0.002 0.001
Journal of Drug Delivery Science and Technology 55 (2020) 101444 0.150 ± 0.0017 4.60 ± 0.02 1 2.56 ± 0.002 7.0 ± 0.05 17.115 ± 0.01 120.86 ± 5.0 7.1 ± 0.55
Medicated soft lozenges of VDF were formulated to bypass first pass metabolism and increase bioavailability by absorption through oral mucosa. Incorporation of VDF-SD resulted in an enhancement in drug release compared to free drug in all tested lozenges. The in-vitro release of VDF from the selected soft lozenge master formula L1 (containing VDF-SD) was found to be 99.8% in only 30 min. The oral route for administration of VDF suspension showed extensive body distribution in various organs, in the order of liver > lung > uterus > kidney, and due to the first-pass effect it has a very low bioavailability. On the other hand, the in-vivo biodistribution study demonstrated significant higher Cmax and AUC0–4h (P < 0.05) of VDF in Lung following buccal administration of VDF-SD lozenges compared to oral administration of VDF suspension with relative bioavailability of 1180%, that favors the use of this route for treatment of PAH.
0.012 ± 0.003 57.65 ± 7.49 1 71.90 ± 2.033 233.7 ± 3.97 6014.10 ± 104.83 504444.30 ± 944.8 83.9 ± 6.57 0.026 ± 0.0001 26.10 ± 1.21 1 4.84 ± 0.037 17.0 ± 0.06 183.38 ± 12.8 6966.40 ± 55.8 38.0 ± 4.22 0.014 ± 0.0005 47.34 ± 1.51 1 7.45 ± 0.040 27.4 ± 0.80 513.88 ± 14.45 35228.86 ± 1028.10 68.6 ± 2.18 0.036 ± 0.001 19.45 ± 0.38 1 10.20 ± 0.010 34.50 ± 0.130 292.50 ± 5.94 8345.78 ± 335 28.5 ± 0.55
0.028 ± 0.001 24.50 ± 1.65 1 117.70 ± 10.300 407.6 ± 13.30 4179.47 ± 112.80 149279.29 ± 1150.2 35.7 ± 3.82
0.008 ± 0.0013 89.57 ± 12.16 1 5.25 ± 0.049 19.1 ± 0.25 682.98 ± 88.85 88501.79 ± 947.5 129.6 ± 17.54
4. Conclusion
Ke (h ) T1/2 (h) Tmax (h) Cmax (μg/ml) AUC0–4h (μg.h/ml) AUC0-inf (μg.h/ml) AUMC0-inf (μg.h2/ml) MRT0-inf (h)
Lozenges
level increased to a very high percentage in group III that received lozenges by 304% (Fig. 4.). This came in agreement with the pharmacokinetics of drug, and provided a proof-of-concept that the buccal route could efficiently reduced the possibility of hepatic metabolism of the drug via. bypassing the enterohepatic circulation, and thereby, allowing the direct presentation of the drug into the systemic circulation. From the previous pharmacokinetics parameters it is concluded that VDF-SD Lozenges can be the potential alternative to oral VDF suspension for treatment of PAH. In ordinary VDF oral dosage forms, the distribution pattern of VDF was in the order of liver > lung > uterus > kidney due to extensive first-pass effect. Hence, very low bioavailability. On the other hand, lozenges improved the oral absorption of VDF, and its localization in lung due to bypassing the first pass metabolism and increasing bioavailability by absorption through oral mucosa. So, VDF-SD Lozenges give also superior therapeutic benefits to oral and more convenient dosage form, especially for patients who cannot swallow solid oral dosage forms.
CRediT authorship contribution statement Amr S. Abu Lila: Formal analysis, Writing - review & editing. Eman Gomaa: Formal analysis, Writing - review & editing. Fakhr Eldin S. Ghazy: Supervision. Azza A. Hasan: Formal analysis, Writing - review & editing.
−1
Oral Susp.
Fig. 4. Comparison of serum cGMP level for lozenges against oral Vardenafil & normal controls.
0.024 ± 0.0012 28.50 ± 1.45 0.5 3.25 ± 0.057 11.3 ± 0.35 130.17 ± 7.77 5384.90 ± 117.7 41.3 ± 2.09
Oral Susp. Lozenges Lozenges Oral Susp.
Oral Susp.
Kidney Uterus Lung Parameters
Table 5 Pharmacokinetic parameters of VDF distribution in Lung, Uterus, Kidney and Liver; following administration of Oral suspension and L1 (VDF-SD) Lozenges in female rats.
Liver
Lozenges
A.S. Abu Lila, et al.
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Journal of Drug Delivery Science and Technology 55 (2020) 101444
A.S. Abu Lila, et al.
Declaration of competing interest
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