|Year : 2013 | Volume
| Issue : 3 | Page : 191-201
Development and evaluation of fixed dose bi therapy sublingual tablets for treatment stress hypertension and anxiety
Mohamed A El-Nabarawi1, Saadia A Tayel1, Nadia A Soliman2, Hadel A Abo Enin2
1 Department of Pharmaceutics and Industerial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
2 Department of Pharmaceutics, National Organization of Drug Control and Research (NODCAR), Giza, Egypt
|Date of Submission||24-Sep-2012|
|Date of Decision||07-Dec-2012|
|Date of Acceptance||28-Mar-2013|
|Date of Web Publication||23-Aug-2013|
Hadel A Abo Enin
Department of Pharmaceutics, National Organization of Drug Control and Research (NODCAR), Giza
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: A stress induced rise in the blood pressure. Some believe that patients with hypertension are characterized by a generalized state of increased anxiety. Aim: The purpose of this study is to prepare a fixed dose bi therapy using bisoprolol hemifumarate (BH) as antihypertensive drug and buspirone hydrochloride (BuHCl) as anxiolytic drug, which can be used to treat both diseases concomitantly. Using sublingual tablets is hopeful to improve the BuHCl poor oral bioavailability and to facilitate administration to patients experiencing problems with swallowing. Materials and Methods: A total of 5mg BH and 10mg BuHCl were selected based on compatibility study. A 3×22 full factorial design was adopted for the optimization of the tablets prepared by direct compression method. The effects of the filler type, the binder molecular weight, and the binder type were studied. The prepared formulae were evaluated according to their physical characters as hardness, friability, disintegration time (new modified method and in vivo disintegration time) and wetting properties. In vitro drugs dissolute, permeation through the buccal mucosa and the effect of storage were analyzed by a new valid high pressure liquid chromatography (HPLC) method. Bioavailability study of the selected formula study was carried out and followed by the clinical. Results: The optimized tablet formulation showed accepted average weight, hardness, wetting time, friability, content uniformity, disintegration time (less than 3 min). Maximum drug release could be achieved with in 10 min. In addition enhancing drug permeation through the buccal mucosa and, the maximum concentration of the drug that reached the blood was in the first 10 min which means a rapid onset of action and improved the extent of both drug's absorption. Conclusion: The results revealed that sublingual (F6) tablets containing both drugs would maintain rapid onset of action, and increase bioavailability. BuHCl with BH can be attributed to the marked decline in DBP and SBP. That led to a reduction in the MAP.
Keywords: Bisoprolol hemifumarate, buspirone hudrochloride, sublingual tablets; transmucosal study, bioavailability study
|How to cite this article:|
El-Nabarawi MA, Tayel SA, Soliman NA, Abo Enin HA. Development and evaluation of fixed dose bi therapy sublingual tablets for treatment stress hypertension and anxiety. J Pharm Bioall Sci 2013;5:191-201
|How to cite this URL:|
El-Nabarawi MA, Tayel SA, Soliman NA, Abo Enin HA. Development and evaluation of fixed dose bi therapy sublingual tablets for treatment stress hypertension and anxiety. J Pharm Bioall Sci [serial online] 2013 [cited 2019 May 27];5:191-201. Available from: http://www.jpbsonline.org/text.asp?2013/5/3/191/116803
Stress of modern life and work can be the cause of many diseases, for example; anxiety, depression, hypertension, and an increased risk of cancer and coronary heart disease.
Some believe that patients with the hypertension are characterized by a generalized state of increased anxiety, which has been reported in those with a family history of the high blood pressure (BP). ,
Hence, the need for combination therapy of anxiolytic drug and antihypertensive drug is common, especially for the hypertensive patients who suffering from daytime alertness.
FDC had many advantages as convenience, cost savings and an increase the patient compliance.  This study is concerned in the formulation and evaluation of bisoprolol hemifumarate (BH) and buspirone HCl (Bu) combination therapy in sublingual tablets as a combination of Beta blocker (BH) and antianxiety (Bu) which both induced a significant decrease in BP. They reduced the mean arterial pressure (MAP) through decreasing the BP and reducing the cardiovascular responses to the 5-HT1A receptor agonists. 
It was useful for long-term treatment of anxiety; another alternative is the use of adrenergic beta-blocking drugs.  Furthermore, by investigating the antihypertensive effect of 5-HT1A agonist, it was found that the chronic administration of bu, produced a significant reduction in BP.  The use of certain beta-adrenoceptor antagonists, such as propranolol and metoprolol, might improve the antiepileptic potential of benzodiazepines.  In addition, Beta-adrenoceptor blockers may be useful as adjunctive medication in the treatment of depression. As they show affinities to 5-HT1A. 
BH is a selective beta-1 receptor blocker and is an alternative first-line choice in the treatment of heart failure and congestive heart failure. It almost completely absorbed from the gastrointestinal tract with small first-pass effect (<10 %)
in man, the plasma elimination half-life is 10-12 hours, resulting in the duration of action of 24 hours. The high absorption rate and small first-pass effect result in an absolute bioavailability of 88%. Concomitant food intake does not delay the absorption. 
The molecular weight of bisoprolol is 325.443 g/mol.
BH leads to 46% reduction in sudden death after 1 year. BH administered once daily appears to be an effective and safe as an antianginal agent. It acts essentially through the reduction of myocardial oxygen consumption. 
Bu is structurally unrelated to the benzodiazepines but possesses comparable anxiolytic activity, especially in patients in whom daytime alertness is particularly important.  Because Bu lacks hypnotic, anticonvulsant and muscle relaxant properties, it has been termed ''anxioselective''. It seems unlikely to generate the problems of dependence, abuse and rebound. Bu appears to be efficacious and well tolerated in the treatment of generalized anxiety disorder in elderly patients.  It rapidly absorbed from the gastrointestinal tract reaching peak plasma concentration within 40 to 90 minutes after an oral dose. The systemic bioavailability is low and extensive first-pass effect ( > 90 %). Concomitant food intake delays the absorption. Half-life of Bu ranged from 2h to 3h. The molecular weight of buspiron is 385.50314 g/mol. 
The sublingual route for drug administration has been associated with numerous advantages over oral administration as avoidance of both hepatic and intra-alimentary canal metabolism; convenient taking and high bioavailability furthermore, the sublingual route of administration is useful when an immediate response to drug action is desired furthermore, the inconvenience associated with the administering standard dosage forms applies to people who are ill in bed and to those actively working patients who are busy or traveling and who have no access to water. 
In this context, a sublingual tablet containing bi therapy of BH and Bu was developed and evaluated for various pharmaceutics properties including dissolution and stability testing to be used as a promising dosage form easy for handling of elderly patients suffering from stress hypertension and anxiety.
| Materials and Methods|| |
BH was purchased from Merck (Barcelona, Spain), and BuHCl was purchased from Bristol Mayer Squibb (New York, USA). Avicel PH 101, Avicel PH 102, polyethylene glycol 4000, polyethylene glycol 6000, Ac-Di-Sol, magnesium stearate and aspartame purchased from Fluka; Germany. Mannitol (El -Nasr Pharmaceuticals, Egypt). Anhydrous lactose, polyvinyl pyrrolidone K30 and polyvinyl pyrrolidone K90 (Sigma; USA).
Drug-drug and drug - excepients interaction
Differential scanning calorimetric (DSC) study (Differential Scanning Calorimeter, Shimadzu DSC-50. Japan) and Infrared spectroscopy (IR) (IR spectrometer, Shimadzu IR-470. Japan) were used to characterize (BH), (Bu), mixtures of both BH and Bu (M), and the mixture of (M) with each excipients (1:1 w/w). 
Factorial design for tablet formulation
×2 2 factorial design was used for tablet preparation. Two factors were used at two levels as follows; (1) Binders (low particle size and high particle size). (2) Diluents (mannitol and lactose) in order to formulate 4 formulas.
The design was repeated 3 times for each binder as; Avicel (PH 101 and PH 102), PEG (PEG 4000 and PEG 6000) and PVP (K30 and K90) in order to formulate twelve formulae.
| Tablet preparation|| |
The tablet formulae were directly compressed after sieving using single punch tablet machine with 7 mm flat-faced punch [The composition as presented in [Table 1]  (Single punch tablet machine, Shang Hai Hua's Mao industrial and commercial Co .China). The compaction force was maintained during compression at 20 KN to maintain the tablets porosity. Ingredients of each formula were mixed using the geometric dilution method. The produced mixture passed through a 200 μm sieve and retained on a 100μm sieve in order to maintain the particle size of all components within the range of 75-106 μm. The produced mixture was finally mixed with magnesium stearate for 2 minutes.
|Table 1: The composition of different BH and BuHCl mixture sublingual tablets |
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| Physical evaluation|| |
Tablets' hardness and tablets' friability were measured according to the United States pharmacopeia  using a tablet hardness tester (Hardness Tester, D.R. Schleunger, 6D tablet tester, Germany) and a friability tester (Friabilator, Van Ceuard, PNC, Germany) respectively. The average hardness and the friability percent were calculated.
| Tablets wetting time (WT) and tablets wetting absorption ratio (WAR)|| |
Tablets WT was measured by a modified procedure from that reported by Bi et al. 1999.  The procedure was modified as follows: Five circular tissue paper of 10 cm diameter were placed in a petri dish containing an eosin dye solution in water (10 ml of 0.05% w/v). A tablet was placed carefully on the surface of the tissue paper. The time required for the dye solution to appear on the upper surface of tablet. A WT was recorded using a stopwatch. The water absorption ratio was calculated using the following equation: WAR = (Wb-Wa) / Wa. The test was repeated three timed.
Where Wa and Wb are the weights before and after water absorption, respectively. 
| Disintegration time (DT)|| |
Measurement of DT by a modified apparatus
Instead of the conventional disintegration apparatus described in United States Pharmacopoeia USP, a modified Shukla et al, 2009  method was used, 500 ml of phosphate buffer pH 6.8 maintained at 37 ± 0.5°C and stirred with a paddle at 50 rpm. The tablets were placed in a sinker. (Dimensions of the sinker: 3 cm in height and 3.5 cm in width, mesh #10).
In-vivo oral disintegration time
test was performed on six healthy adult volunteers. Before the test, the volunteers were asked to rinse their mouth with distilled water. The tablet was placed under the subject's tongue, and immediately a chronometer was started until complete disintegration. The chronometer was stopped, and the time recorded.  Permission to carry out this work was obtained from the Institutional Ethics Committee (National Organization of Drug Control and Research [NODCAR],Giza,Egypt).
HPLC method for the determination of (BH) in the presence of (Bu)
The stock solutions of BH and Bu were prepared in a phosphate buffer pH 6.8 at a free base concentration of 1mg/ml (A) respectively. Secondary standard solution for BH and Bu mixture (M) 10μg/ml was prepared as a stock solution. Working standard solutions of (M), 1, 2, 5 μg/ml, 500, 200, 100, 50, 20, 10 ng/ml were prepared from secondary standard solution by dilution with phosphate buffer pH 6.8 respectively.
HPLC procedure was proposed using a 30mm × 4mm (Luna eclips® XBB C18 analytical column) with the aid of a guard column. The mobile phase consisted of acetonitrile and 0.01M phosphate buffer (pH 5.5) at a ratio (30:70) v/v. The absorbance of the prepared solutions was measured at λ max 229 nm. Triplicate injections were made for each sample.
The assay method was done and validated with respect to intra- and inter-day accuracy and precision as per ICH guidelines for 3 days.
In vitro dissolution study
Tablet formulas were introduced individually into each vessel containing 500 ml of phosphate buffer solution pH 6.8, 37 ± 0.5°C and at a speed of 50 rpm. (USP Apparatus Π) (Dissolution tester, Pharma test, PTZ, Germany). Aliquots of 5 ml were withdrawn from the dissolution medium at 5, 10, 15, 20, 30, 45 and 60 minutes. The drug content was determined by HPLC method. Each experiment was carried out in triplicate. The test was adopted after several trial on the modified procedures adopted by Aburahma et al,*for evaluation of sublingual tablets containing vinpocetine. 
In vitro permeation through the buccal mucosa
A test performed using the Franz-type diffusion cell. The buccal mucosa was obtained from a local slaughterhouse (goat buccal mucosa) and used within 3 hours of slaughter. The tissue was stored in a phosphate buffer at 4°C upon collection.
The medium was equilibrated at 37°C ± 0.5°C and stirred 25rpm over a 3-hr. An amount of the drug released from these formulas was determined by the HPLC method.
The percent drug permeated to time was constructed for each experiment, and the slopes of these graphs were used to calculate the permeability coefficients (PC: cm / min) as follows:
PC = slope × Vd / S
Where Vd: is the volume of the donor solution.
S: is the surface area of the tissue (1.77 cm 2 ). 
| Accelerated stability testing|| |
The selected formulae were stored at 60°C and 75% relative humidity (maintained using a saturated solution of NaCl) for 3 weeks.  The stored tablets were examined visually for any changes in color and/or appearance every week. Physical evaluation of the tablets, including dissolution was repeated at the end of the storage period as the same procedures adopted for the fresh tablets.
Dissolution profiles of fresh and stored tablets were compared according to the model independent mathematical approach of Moore and Flanner, 1996.  The similarity factor (ƒ2) was calculated according to the following equation:
Where n is the number of sampling points. Rt, and Tt, are the mean percent dissolved of the reference (fresh) and the test (stored) at time t respectively. ƒ2 represents a logarithmic transformation of the sum of squared error of differences between the reference and test products over all time points.
| Bioavailability of BH and Bu in the selected formulae|| |
A simple crossover design was applied on three phases, using male Albino rabbits (Albino rabbits weighing 2.0-3.0 kg. They were obtained from veterinary service (NODCAR) Egypt.). They were randomly divided into three groups, each containing six rabbits. A group received an oral dose of the market products (Concor® and Buspare® ), in a dose 1 mg/Kg/ day  and 2 mg/Kg/ day  of the body weight in each phase respectively. The other two groups received F6 and F12 sublingually [The composition was as in [Table 1]] respectively after suitable washout period. The optimizing buccal tablet formula were inserted sublingually and positioned in such a way that, the tablet surfaces came into contact with the ventral tongue and the floor of the mouth of anesthetized rabbits for 3 minutes after shifting the tongue. The rabbits were anesthetized with pentobarbital (25 mg/kg). 
All animals were handled in agreement with the ethical principles in animal experimentation adopted by the Ethics Committees Accreditation of Laboratory Animal Experimentation Care (AAALAC) with protocol no. 25/2002.
A volume of blood samples (2.0 ml) was drawn from the terminal veins of the ears at the following time points: 0 (prior to dosing), 5, 10, 15, 20, 30, 60, 90, 120 and 240 minutes and collected in heparinized tubes. Plasma samples were separated and were deproteinized by acetonitrile then analyzed using a HPLC (agillent 1100). A modified Braza et al., 2002,  and a modified Pehourcq,  were used and validated for the determination of BH and Bu respectively. Triplicate injections were made for each sample.
The pharmacokinetic parameters were calculated and statistically compared; Cmax(μg/ml), Tmax(Hours), Auc (0-4) (μg.h/ml), Auc (0-∞) (μg.h/ml) and Relative Bioavailability.
The pharmacokinetic data were computed using Kinetica 2000 Version 3.0 (Inna Phase Corporation, USA).
| Clinical study|| |
Thirty patients with primary hypertension (age: 50.9 ± 2.38 years, weight: 83.9 ± 2.81 kg, BP: 165.8 ± 3.90/102.5 ± 1.86 mm Hg, heart rate: 63.6 ± 2.98 min−1) were included in this study. The study protocol was approved by the Human Ethics Committee of NODCAR. The procedures followed in this study were in accordance with institutional guidelines. Written informed consent was obtained from all the enrolled patients. Patient compliance was documented on the patient's case report form. Safety was assessed throughout the study and included monitoring of adverse events, concomitant medications, routine laboratory safety tests (creatinine clearance, Hematology, serum chemistry, and urinalysis values), and physical examination findings.
The test was performed to compare the pharmacodynamic effect of sublingual tablet (F6) to commercial oral tablet (5mgConcor ® and 10mgBuspare® ) by studying the change of diastolic blood pressure (DBP) and systolic blood pressure (SBP) and recorded them. The sublingual tablet (F6) and commercial oral tablet (5mgConcor ® and 10mgBuspare® ) were given once daily. The non- blind, three treatments, three periods and randomized crossover study were followed. Under this design, a third of the subjects were given orally 5mg Concor® , the other third was given orally 10mg Buspare® and the last group were given F6 buccally (The composition in [Table 1]). BP was measured for 6 hours. The response to the formula was expressed by the area under the curve of the mean arterial pressure (MAP-AUC). The difference of the BP peak induced by both drugs expressed as the change in both and SBP. The data collections at different time intervals as 10, 20, 30, 60, 90, 120,180,240 and 360 minutes recorded with Mercurial sphygmomanometer. The MAP was calculated according to the following equation followed by statistical analysis.
MAP ~ DBP + 1/3(SBP-DBP)
| Statistical analysis|| |
All tests were conducted in triplicates. The results were expressed as the mean ± SD followed by paired t test. One-way Analysis of Variance (ANOVA) was applied to assess the significance of the effect of storage on the physical properties of the tested formulae and the fresh formulae (In All experiments).Two-way ANOVA was applied to assess the significance of the effect of formulation and subject factors on the pharmacokinetic parameters, and the pharmacodynamic parameters of the tested formulae and the oral commercial formulae Concor® and Buspar® tablets. Tukey's HSD (Honestly Significant Difference) test and Guassian's test for multiple comparisons were then performed to determine the source of difference respectively using SPSS® software version 7.5 (SPSS Inc., Chicago, IL). Differences are considered to be significant at P< 0.05.
| Result and Discussion|| |
Drug-drug and drug - excipient's interaction
DSC thermo grams and IR spectrum of M and M-excipient mixtures revealed that; there was not any significant change in the onset (thew point), endothermic peak or the characteristic bands of each drug as in [Figure 1]. The insignificant change in the endothermic peak (DSC) or the characteristic bands of each drug (IR) revealed to the absence of possible chemical or physical interaction between both drugs
|Figure 1: Physical evaluation of the both drugs possible compatibility: (a) differential scanning calorimetric thermogram of buspirone hydrochloride (BuHCl), bisoprolol hrmifmarate (BH) and M*(b) infra red spectroscopy spectrum of (i) BH, (ii) BuHCl, (iii) mixture of both drugs (M*). M*: mixture of BuHCl and BH (1:1)|
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All formula showed hardness values ranging from 2 to 5, which are within the pharmacopoeia acceptable ranges of 2-8Kg (USP, 2009) except F8 [Table 2]. Taking the binder factor in consideration, significant difference occurred when changing the binder of smaller particle size with that of larger particle size. The value of hardness is increased (P-value=0.158, SE=0.156). The analysis of interaction revealed that; mannitol had the lowest hardness value with low particle size binder (P-value=0.138, SE=0.062) (Data not shown).
|Table 2: Physical evaluation of the stored and the fresh sublingual tablets |
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The hardness results suggest that the particle size influences of the binder use were more than the type of binder used (p < 0.05) and that agreement with Bolourchian et al., 2009. 
tablets accepted friability percent, i.e., less than 1%. All the factors studied had a significant effect on the tablets' friability (p < 0.05). Studying the interaction found that Diluents*PEG interaction only had a significant effect on the tablets' friability (p > 0.05) (P-value=0.369, SE = 0.100) (Data not shown). All tablets have accepted friability because it was reported that the binder concentration at 10% resulted in a decrease in the fracture of the tablets but caused compression problems like stickiness to the punches of compression machine.  However, the addition of 1% (w/w) magnesium stearate as a lubricant improves tablets' compression and reduces its friction with compression machine and has the least effect on the tablet characteristics such as the disintegration time, the hardness and the friability of the tablets. 
Tablets WT and WAR
Tablets prepared to use a different formula showed different WT and WAR as in [Table 2]. A ranking of the waiting time and the wetting ratio indicated that F 7 has the tallest WT and the lowest wetting ratio due to its highest hardness value and vice versa in F6 and F12. The reduced tablet porosity retards water penetration and delays or even inhibits the role of the super disintegrant.  In addition, tablets when moistened, the super disintegrant expands and swells to cause rupture and complete the disintegration of the tablet.  Hence, there was an indirect correlation between DT and WT. The decrease in the wetting time was indicating the enhancement of the tablet disintegration. The high absorption ratio indicating the large amount of water absorbed, which enhances disintegration of all tablets.
Measurement of DT by a modified apparatus
USP disintegration apparatus is not intended to measure accurately the DT of sublingual tablets because agitation is more vigorous. To overcome this problem, a modified dissolution test was employed.
In this test, the sinker (wire basket) was positioned in the vessel with the help of a hook in a way that the basket had only 10.5 ml of it.
A ranking of the disintegration times indicates that: F7 and F10 have the highest DT as there was a direct correlation between hardness value and disintegration time. The greatest compact force leads to lower the tablets' porosity. Luginbόhl and Leuenberger  confirmed that the water uptake must be the first step in the process of disintegration, which can be limited by the rate and extent of liquid absorption of the system.
Avicel type and diluents' types had the significant effect on the DT (P < 0.05). Not only there was no interaction between the effect of avicel type and the diluents used, but also it had no significant effect on the tablets' disintegration (Data not shown) (P-value=0.0159, SE=1.844).
Only two formula namely F6 and F12 have disintegration times lesser than 1min. Avicel PH 102 has the smallest particle size in addition Avicel is a water insoluble binder. The high water solubility of other binders leads to increase in the viscosity of water, which brake or be a barrier formed against its penetration into the tablet resulting in delaying the DT. 
Except all formulae have the same type and concentration of the disintegrant (5% Ac Di Sol) but each formula has different DT. This was explained by the fact that the breakup of the tablets was not governed by the disintegration, but it depends on the rate at which the binder dissolved, since the latter was distributed across the particle surface. 
F6 < F12 due to Avicel with Lactose as a diluent gives higher DT than with mannitol, These results are supported by the observation of some investigators who found an additive effect on the DT between lactose as fillers when it was combined with Avicel. 
F1 has low DT. The uneven surface of PVP with a folded structure increases the area subjected to the disintegration media. 
In-vivo oral disintegration time
Six formula namely F6, F12, F9, F3, F1 and F11 showed disintegration times less than 3 min, 61, 71, 124, 125, 144 and 178 seconds respectively (European Pharmacopoeia, 2002, adopted the term orodis-persible tablet as a tablet to be placed in the mouth and disintegrated in less than 3 min). 
From the previous study tablets formula F1, F3, F6, F9, F11 and F12, have the best physical characters of all previous evaluation tests and hence, they were used for the following evaluation tests.
HPLC method for the determination of (BH) in the presence of (Bu)
HLPC separation method explains; there was no interference between both drugs. Statistical evaluation found that the recovery% = 96.399% and 96.78% for BH and Bu respectively with a mean recovery 99.395% ± 0.47. The high recovery% value (95% -102%) indicated reduced the interference between both drugs.
The relative standard deviation was lesser than 2% in both cases. The LLOQ of the assay was calculated to be 0.05μg/ml. The assay showed acceptable precision and accuracy as precision ranged from 0.055 to 7.425 (C.V. %) and the accuracy ranged from 5.865 to 6.45 (relative error %). At all the results indicated that; the suitability of the HPLC method for the determination of BH and Bu respectively in unknown mixture samples. It also identified that the absence of possible chemical interaction between both drugs [Figure 2].
|Figure 2: HPLC method for the determination of (bisoprolol hemifumarate [BH]) in the presence of buspirone (chemical compatibility test): (a) HPLC separation of bisoprolol hemifumarate (BH) (b) HPLC separation of buspirone hydrochloride (BuHCl) (c) HPLC separation of BuHCl and BH mixture|
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In vitro dissolution study
The best selected tablets prepared to use different formula were compared in this respect with the release of the market product (5mg Concor® tablets and 10 mg Buspare® ). The values were calculated as the percentage of BH and Bu dissolute according to the predetermined drug content (Data not shown).
Ranking of dissolution rate of sublingual tablets for Bu was F6 > F3 > in 15 minutes, F11 in 20 minutes, F1 in 25 minutes. F12 showed the quickest dissolution rate at 10 minutes. Altough for BH was F9 > F6 > F12 > F1 in 15 minutes, F11 in 20 minutes, and only F 3 showed the quickest dissolution rate at 10 minutes.
Tablets containing Avicel and PVP showed the enhanced dissolution rate and this attribute to their rapid disintegration property (F1, F3, F6, F9 and F12). Tablets containing Avicel PH 102 (F6 and F12) showed a fast dissolution rate than that containing Avicel PH 101 (F3 and F9); as they have a smaller particle size and because Avicel PH-101 belongs to the cellulose I polymorphic form which formed stronger tablets. 
PVP enhances the dissolution rate as it has the uneven surface with a folded structure, which increase the area subjected to the media.  In addition, the presence of PVP K30 in F1 can probably be increased the wettability of drugs as, PVP K30 causes decreased the interfacial tension between the drug and the dissolution media. 
All formula gave dissolution rate >70% after the dissolution during first 10 min. So, for assessment and comparison, the dissolution test was repeated for 10 minutes and aliquots were withdrawn from the release medium every 1 min and analyzed as mentioned before [Figure 3]. The dissolution rate of the drug during the first 10 minutes (DR-10) and the extent of dissolution after 10 minutes (DP10) were calculated.
|Figure 3: Graduate dissolution profile of the sublingual tablets from the best selected formulae (a) buspirone hydrochloride (b) bisoprolo hemifumarate.Standard deviation was emitted to increase the clarity of the figure|
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In addition, the rate and extent of drug dissolution, defined;
Where the integral is the area under the dissolution curve up to dissolution time t and y100 is the area of the rectangle described by 100 % dissolution at the same time  (data not shown).
There was a significant increase in both the DE% and the (DR-10) in all formula than that of the market products for both drugs. To be more precise, the highest percentage of drug dissolved after 3 minutes was from F6, F3, F12, and F1. Hence F1, F3, F6 and F12 were selected for storage study and in vitro permeation through the buccal mucosa.
In vitro permeation through the buccal mucosa
As illustrated in [Figure 4], it was noted that, F1, F3, F6 and F12 had diffusion percent 99.6%, 103.5%, 98.56% and 98.80 % after 120 minutes for BH and for Bu 98.96%, 101.02%, 99.56% and 98.78% respectively. The PC for BH was 11.64, 9.77, 9.11, 9.17 cm/min and for Bu were 9.93, 8.14, 8.56 and 8.44 cm/min to formula F1, F3, F6 and F12 respectively.
|Figure 4: Diffusion profile of bisoprolol hemifumarate (BH) and buspirone hydrochloride (BuHCl) from different sublingual formulae in compared to the oral commercial one (Concor® and Buspare®) in phosphate buffer pH=6.8 using Franz diffusion cell. (a) BuHCl (B) BH. Standard deviation was emitted to increase the clarity of the figure|
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In vitro, permeation study may offer a means of enhancing drug permeation through the buccal mucosa. Statistical analyses of the given results show no significant difference between either of each formula in both drugs even in diffused percent or in the PC.
Kinetic analysis of in vitro release and the in-vitro permeation data of BH and BuHCl from the prepared formula
The Kinetic analysis of the in-vitro release date of BH and BuHCl from different formulae was studied according to the determination coefficient (R2). It was found that different formulae had first order and 0.5 > n > 1; this indicates Non Fickian or Anomalous, However; the Kinetic analysis of the in-vitro permeation data of BH and Bu from different formulae was diffusion order and n<0.5, this indicates case, I or simple Fickian diffusion (Data not shown).
Hence, Drug release depends on two simultaneous rates' processes, water migration into the matrix and the drug diffusion through continuously swelling strands.
| Accelerated stability testing|| |
None of the stored formulae (F1, F3, F6 and F12) showed any change in color or appearance throughout the storage period. On the other hand, some tablets of F12 showed faint discoloration by the 2nd week. The characteristics of the stored tablets are in the [Table 2]. The discoloration and the brown spots at the end of the storage period might be due to the presence of a high percentage of lactose in this formula. Reducing sugars, including lactose reported to undergo a non-enzymatic browning reaction with amines generally known as the Maillard reaction. 
According to Moore and Flanner for dissolution profiles to be considered similar, value of similarity factor (ƒ2) should be as close as possible to 100 (range from 50 - 100, corresponds to 10% and 0% differences, respectively). The computed (ƒ2) values were 51.96%, 56.222%, 75.277% and 69.05% for F1, F3, F6 and F12, respectively, indicating that the dissolution profiles of fresh and stored tablets could be considered similar, [Figure 5].
|Figure 5: Comparison between the dissolution profile of the fresh and the stored formulae F6 and F12. (a) buspirone hydrochloride(b) bisoprolol hemifumarate|
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The higher (ƒ2) value in case of F6 and F12 indicated the higher the similarity between the dissolution profiles of fresh and stored tablets of these formulae. Except F12 showed marked decrease in the mechanical strength compared to F6. Statistical analysis approved that; all results (hardness, friability and disintegration time) had no significant difference. Hence, these formulas were physically stable after storage.
Based on all results of storage at 60°C and 75% R.H. for 3 weeks, and other physical characters F6 and F12 were the best selected formulae.
Bioavailability of BH and Bu in the selected formulae
The sublingual absorption of BH and Bu from different formulae compared to oral formulae is illustrated in [Figure 6].
|Figure 6: Mean plasma bisoprolol hemifumarate(BH) and buspirone hydrochloride (BuHCl) concentration following sublingual dose of the tested formulae (5 mg BH and 10 mg BuHCl and the market product (5 mg Concor® and 10 mg Buspare®) respectively. (a) BH (b) BuHCl|
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All formulae have Cmax higher than the Cmax of standard tablet (10mg Buspare® ), as It was reported that; Bu is rapidly absorbed and undergoes extensive first-pass metabolism (90% hepatic metabolism). Following oral administration, 10 mg oral dose, lower peak plasma levels of unchanged drug, of 1 to 6 ng/ml was observed at 40 to 90 minutes. 
The presence of two peaks in the curve of tablet formulae 6 and 12 can be attributed to the presence of two absorption phases, namely; sublingual absorption that is characterized by a rapid onset of absorption and sharp decline, followed by intestinal absorption of the swallowed portion of the dose, which is characterized by a delayed onset. It was reported that the maximum effect of BH and Bu after oral administration occurred within 1.7-3h and 1-1.5 h respectively.
The results of the statistical analysis revealed that the formulation had a significant effect on all the tested parameters at P < 0.05 (F5, 17 n = 11.424, 6.469 and 43.890 for Cpmax, Tmax and AUC (0.∞) of BH and F5, 17 n = 194.95, 2.002 and 967.92 for Cpmax, Tmax and AUC (0.∞) of Bu respectively). Based on these results, it was evident that the formulation exhibited the most significant effect on AUC (o-∞). On the other hand, there was no significant difference between the subjects (rabbits) for all the tested parameters.
Multiple comparisons using the Tukey's HSD test revealed that Cpmax of the 3 formula extremely differed significantly from each other with the highest value observed for F6 followed by F12 and finally, the market product.
The relative bioavailability was 156.185%, 421.86% and 123.398%, 270.426% for BH and Bu of F6 and F12 respectively in compared to 5mg Concor® and 10mg Buspare® .
Based on all results, F6 showed the highest Cmax and relative bioavailability and the lowest Tmax value when compared to any other formula, even with the oral commercial tablet (5mg concor® and 10mg Buspare® ). In addition, the maximum concentration of the drug that reached the blood was in the first 10 min which means a rapid onset of action and improved the extent of both drug's absorption.
| Clinical study|| |
The physical examination of Patients did not show moderate dizziness considered possibly related to treatment and no trends or clinically relevant changes were noted. Substantially during the study, Patients were required to have a normal creatinine clearance >25 mL/min 1.73 m 2 . Their hematology, serum chemistry, and urinalysis values were normal indicate the absence of possible toxicity appeared from that combination.
The rate and extent of DBP and SBP reduction were represented as the (MAP- AUC). The mean values for the area under the mean reduction of MAP-time curve AUC (0-6) were 79.79 ± 4.62, 11.39 ± 1.92 and 131.95 ± 11.06 after the oral administration of the market product 5 mg Concor® , 10 mg Buspare® and the sublingual administration of F6 to the thirty subjects, respectively.
The sublingual tablets have SBP and DBP lesser than that of oral tablets, and the effect began from the first 10 min when compared that to oral dose Tmax (DBP=2h , 4.25 h, 0.5 h and SBP=1.5h and 0.33 h for commercial formulae and F6 respectively) as in [Figure 7].
|Figure 7: Mean reduction of mean arterial pressure following sublingual dose of the tested formula F6 (5 mg BH and 10 mg BuHCl), the market product (5 mg Concor®) and the market product (10 mg Buspare®) respectively.|
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The results of the statistical analysis revealed that the formulation had a significant effect on the tested parameter MAP. (Using AUC as an expression of the rate and the extent at P < 0.05 (P-value = 0.351 and 0.212 for 5mg Concor-F6 and 10 mg Buspare-F6 respectively). Therefore, it was evident that, the prepared formula surpassed the market product concerning the both SBP and DBP.
The presence of Bu with BH in F6 can be attributed to the marked decline in DBP and SBP. That led to a reduction in the MAP. The main mode of action of BH is inhibition of chronic beta-1 stimulation-induced myocardial dysfunction. That led to decrease in SBP  and improve diastolic function in the left ventricle, which led to decrease in DBP.  Bu as reported by Shingala and Balaraman, 2005,  has an antihypertensive effect through aortic effect and vascular activity (DBP) Furthermore, it was reported that Bu reduced the SBP. 
| Conclusion|| |
The suggested sublingual tablet (F6), which contains 5mg BH and 10mg BuHCl as a combination therapy showed higher bioavailability than commercial oral tablets. In addition, it considered a promising dosage form easy for handling of elderly patients suffering from stress hypertension and angina pectoris and improved the bioavailability of BuHCl. The significant findings presented here encourage further studies.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]