Community and hospital pharmacists are always face the challenge to prepare oral liquid extemporaneous formulations to fit the needs of a specific patient population when commercial forms or the required strength are unavailable. Accordingly, the primary aim of this research was to prepare a stable patient-friendly oral liquid extemporaneous formulation of bisoprolol with a 0.5 mg/mL as a target concentration. Furthermore, an accelerated stability studies were carried out to study the physical and chemical stability of bisoprolol and to evaluate the adequate shelf-life of the optimized extemporaneous suspension. The research was extended to study the bioavailability of the optimized formulation in humans.
Preparation of bisoprolol extemporaneous suspensions
Several trials were conducted in order to obtain the most optimized formulation that met the desired quality requirements in terms of organoleptic properties, dissolution behavior and stability, as depicted in Table 3. All formulations were prepared without the utilization of potentially unsafe excipients, and with a well tolerable pH value. Formulation trials were carried out using various suspending agents to achieve homogeneity of redispersed suspensions. Xanthan gum and sodium alginate are natural polymers whereas, CMC and Carbopolare synthetic polymers. Xanthan gum is safe up to 15 g per day [38].
Sodium benzoate was involved in a small amount (0.05 %w/v) in all formulations as a preservative to prevent microbial growth [34]. The U.S. FDA allows up to a 0.1% w/w of sodium benzoate in foods and beverages [39]. The WHO has set the acceptable daily intake level for sodium benzoate to 0–5 mg/kg [40,41]. Glycerol is usually utilized in the preparation of liquid dosage forms as a solvent, thickening agent, lubricant, demulcent, sweetener, and humectant [42]. It is virtually non-toxic. Glycerol containing-pharmaceutical products are classified as “Generally Recognized as Safe” (GRAS) [42,43].
Extemporaneous preparations should be aesthetically appealing and efficacious, as well as fulfilling the quality requirements [44-46]. The poor palatability of extemporaneous formulations may have a negative impact on the patient compliance, and consequently the overall clinical outcomes. Sweetener in combination with flavoring agent were employed for improving patient adherence and providing safer and more palatable options for younger and elderly patients with dysphagia. Sorbitol is widely used as a diabetic-friendly sweetener [47]. The maximum daily intake of sorbitol in adults and neonates should be less than 20 g/day and 2 g/kg/week, respectively to avoid gastrointestinal (GI) side effects [47,48]. Red fruit (strawberry/raspberry) flavor is the most acceptable flavor in children [49]. Strawberry-flavored medications had the best tasting in comparison to the cherry-, bubble gum-, banana-, grape-, and peppermint-flavored formulations in children aged 3–12 years [50,51]. Accordingly, strawberry flavor was used in the preparation of bisoprolol-containing extemporaneous suspensions.
The selected extemporaneous formulation (F2) can be prepared easily in different types of pharmacy settings with a simple, natural, available, sugar free excipients. It provides several advantages such as high safety, due to lack of possible harmful preservatives as parabens [52]. It does not cause carcinogenicity problems due to the absence of sucrose [53]. Besides, it is appropriate additionally for diabetic patients. Furthermore, the absence of GI side effects was also demonstrated.
Chromatography
A stability indicating method was developed and validated to determine the selectivity of the method in stability and plasma samples in presence of endogenous materials and/or degradation products. The UPLC method had a suitable level of precision, accuracy and linearity. The calibration curves were linear with a correlation coefficient R2 > 0.999 (n=6) throughout the course of the assay (10-100 μg/mL) and (1-100 ng/mL) for stability and human plasma samples, respectively. The UPLC method was accurate and precise. Within-day percent coefficient of variation (%CV) ranged from 0.52 % to 4.45% and 0.244 % to 11.79%, while the between-day %CV ranged from 0.89 % to 4.31 % and 0.712% to 9.411 % for stability and human plasma samples, respectively. Within-day accuracy ranged from 98.99% to 104.8 % and 97.3 % to 104.5 %, while the between-day accuracy ranged from 97.74% to 105.1% and 96.22 % to 103.8% for stability and human plasma samples, respectively. The retention time of bisoprolol and IS was 5.0 ± 0.25 min and 2.5 ± 0.1 minutes respectively. No peaks for any endogenous materials and/or degradation appeared at the same retention time for bisoprolol and IS indicating the selectivity of the assay method.
In vitro evaluation of extemporaneous suspensions
Appearance and pH measurement
All compounded extemporaneous formulations formulated from crushed Concor® tablets (5 mg bisoprolol fumarate) had a pink, milky color. However, the formulation (F2-unprocessed) prepared from pure bisoprolol was a clear liquid. Strawberry flavor along with sorbitol as a sweetener imparted pleasant organoleptic properties to the formulations. The resulting extemporaneous formulations were subjected to various assessments such as; pH, viscosity, sedimentation ratio and resuspendability measurements. Observations of these measurements were listed in Table 3. The pH values of all suspensions (F1-F8) were within the acceptable range (5-8) [54]; ranged from5.2 to 6.4 whereas, the pH of the formulation (F2-unprocessed) was 6.8 (Table 3). The best pH values were noted in xanthan gum- containing extemporaneous formulations (F1, F2, F3, and F2-unprocessed) without the addition of any pH stabilizer. Xanthan gum is safe for oral utilization and supported by the regulatory authorities [38].
Viscosity measurement
The results showed that the viscosity of bisoprolol extemporaneous formulations was increased in the following order: F2 > F2-unprocessed > F7 > F5 >F4 > F8 > F1> F3 > F6 (Table 3). Accordingly, xanthan gum- containing formulations (F2 & F2-unprocessed) possessed the most reasonable viscosity.
Sedimentation volume and resuspendability
The results indicated that the sedimentation ratios of xanthan gum-containing suspensions (F1, F2, F5) was 0.975, 1, 0.98, respectively. F1 (0.1% w/v) and F5 (0.3% w/v) were easily resuspended however, no sedimentation was occurred in case of F2 (0.5% w/v) during the test period (Table 3). Xanthan gum is often utilized as a flocculating agent to increase the homogeneity of suspension particles with no need to other adjuvant [21,26]. The obtained results related to the loose and fluffy network of flocs that formed in the xanthan gum-containing suspension which can extend throughout the extra vehicle [55]. The increase in xanthan gum concentration in F2 (0.5% w/v) revealed a great flocculation and homogeneity of suspension compared to F1 and F5.
On the other hand, Carbopol- containing suspensions (F6 & F7) considered pharmaceutically unacceptable. F6 (0.1% w/v Carbopol) had a sedimentation ratio of 0.9 and was resuspended with difficulty compared to other formulations. In case of F7 (0.5% w/v Carbopol), it was noticed that the increase in Carbopol concentration led to a reduction of the sedimentation ratio (F= 0.65) however, the suspension was resuspended easily by shaking (Table 3).
The other formulations which contain CMC (F3-F5) and sodium alginate (F8) had a sedimentation ratio equal to 0.975, 0.97, 0.98, and 0.925, respectively. Besides, they were easily resuspended by shaking. This difference in the sedimentation ratios might be attributed to the type of the suspending agent used, since CMC and sodium alginate might form homogenous network in all concentrations used [21,56].
The overall appeal and uniformity of the suspension relies upon the sedimentation ratio and the ease of redispersion. Sedimentation ratio nearest to satisfies both the above rules. Accordingly, F2 suspension containing 0.5% w/v xanthan gum was the best one that offered homogeneity without any sedimentation (F = 1) with ease of pouring compared to other formulations which were less viscous and homogenous than F2.
Dissolution studies
The dissolution pattern of bisoprolol from R and different extemporaneous suspensions are illustrated in Fig. 1. Models utilized to describe the release kinetics of bisoprolol were; zero-order, first-order [57], Higuchi kinetics [58], the cubic root law [59], Weibull distribution [60], and the logistic model [61]. The derived parameters of model- dependent and - independent in vitro characterization and other dissolution parameters are presented in Table 4.
The R tablet liberated 82.2% of its potency in the first 5 min (Q5) with complete bisoprolol dissolution after 60 min (Q60) (Table 4). This was concurred with the USP and FDA dissolution specification for Concor® tablets (R) where, not less than 80% of bisoprolol should be dissolved within 20 min in water [62, 63]. Weibull distribution and logistic model can describe the type of dissolution profile and dissolution time as well [29]. The shape parameter (β) describes the shape of the dissolution profile whereas, the time interval necessary to dissolve 63.2% of the drug is represented by the time parameter (Td) [60]. Values for β=1 indicates curve exponential, β>1 shows a sigmoid curve, and β <1 specifies to a parabolic curve with steeper initial slope that is consistent with the exponential [29,60,64,65]. The dissolution data of R product was best fitted the logistic model, while the second best fit was Weibull distribution. The β parameter of R product was > 1 (1.325); indicating a complex dissolution mechanism with a more pronounced sigmoid curve (S-shaped curve) with upward curvature followed by a turning point(Table 4). Also the first-order and Hixson-Crowell models fit gave the statistical parameters as being approximately the same as those of Weibull distribution for the R product (Table 4).
The effect of various types of suspending agents on the dissolution rate of bisoprolol was studied. All extemporaneous suspensions were obeyed logistic model and Weibull distribution; emphasizing the S-shaped dissolution profiles of all extemporaneous suspensions (Table 4). However, the preferred model described drug release kinetics of the prepared suspensions was Weibull model. The β -Values calculated for all bisoprolol extemporaneous suspensions were < 1, which indicated parabolic curves with initial inflection (Table 4). The parabolic curves are common in suspensions that liberate the drug quickly in early times of dissolution and then remain at a constant dissolution [65, 66]. The Td values varied significantly (p˂0.05) from one formulation to another with the recorded values being ranked as F5 > F7 > F8 > F4 > F3 > F6 > F2 > F1(Table 4). However, the time necessary to dissolve 63.2% of bisoprolol from extemporaneous formulations F1 and F2 was shorter than the other suspensions (Table 4). The third best fit model was the first-order model in case of all extemporaneous suspensions, except F5, F7, and F8 followed Higuchi kinetics (Table 4).
Xanthan gum was used as a suspending agent in concentration of 0.1% and 0.5% to prepare F1 and F2 suspensions, respectively. F1 and F2 achieved the best dissolution behavior among other extemporaneous suspensions (Fig. 1). The Q5, Q60, DE, MDT values of F1 were 80.8%, 101.5%, 91.6%, and 4.86 min respectively (Table 4). A similar trend (P > 0.5) was noticed for the dissolution behavior of F2 (f2 =74.5%) (Fig. 1 and Table 4) where, the latter liberated 76.06% of bisoprolol after 5 min with 100% of the drug dissolved after 60 min. Moreover, a similar DE and MDT values (P > 0.5) of F2 (92.8% and 4.58 min) were observed in comparison to R and F1 (Table 4). F2 showed a high DE value (92.8%) concurred with the rapid dissolution rate observed. The dissolution rate of bisoprolol was not changed (P > 0.5) by increasing xanthan gum concentration (Table 4). Suspensions (F1 & F2) liberated bisoprolol at a rate similar to (P > 0.05) R product. This was further indicated from the values of the similarity factor which reflected the sameness of dissolution profiles of F1 & F2 and R. The values of f2 were57.4% and 62.1% for F1 and F2, respectively; confirming their similarity to R product. Xanthan gum is an effective flocculating agent at a relatively low concentration and has excellent suspending and wetting properties [21,67]. Additionally, the rheological stability of xanthan gum toward pH changes encountered during transit through the GI tract providing reasons for its use [21].
According to the logistic model, suspensions (F1 & F2) had the best results of the β (shape factor) parameter where the values were 2.304 and 2.627, respectively compared to R (3.338); indicating that the dissolution profiles of F1 & F2 were similar to the profile of R. This was coincided with the previous results (Table 4). To sum up, F1 and F2 gave the best dissolution behavior among other extemporaneous suspensions however, F2 had a higher viscosity (217 mPa s) compared to F1 (153 mPa s) (Table 3). Furthermore, F2 maintained its flocculated state and poured easily.
F2-unprocessed formulation was prepared using the pure powder of bisoprolol instead of crushed Concor® tablets. F2-unprocessed formulation was a clear viscous solution with a viscosity of 205 mPa s (Table 3). F2-unprocessed formulation liberated 86.7% of its potency in the first 5 min with complete bisoprolol dissolution after 60 min (Fig. 1d and Table 4). The DE and MDT values of F2-unprocessed formulation were 93.4% and 4.25 min, respectively; indicating the sameness between F2-unprocessed and F2 which was further confirmed with f2 results (f2 = 59.76%) (Fig. 1d and Table 4).
CMC was used as a suspending agent in concentration of 0.1% and 0.5% to prepare F3-F5, respectively. Referring to the results of CMC-containing extemporaneous suspensions, the increase in the concentration of CMC resulted in a slower drug release. Extemporaneous suspensions with 0.1% CMC (F3) showed a comparable (P > 0.05) Q5 (84.7%) and DE (94.6 %) to that of R, but the MDT (6.08 min) was increased significantly (P < 0.05) (Fig. 1 and Table 4). The similarity in the overall dissolution of F3 to R was further confirmed by the similarity factor test (f2 = 52.67%). Moreover, dissolution profile of F3 (0.1% CMC) was similar to that of F1 (0.1% xanthan gum) (f2 = 71.1%) where its aqueous dispersion has nearly the same viscosity (152 mPa s) and produced sediment layer that was easily redispersed by shaking (Table 3). In case of F4, the values of Q5, DE, and MDT were significantly changed compared to both R and F3. The increase in CMC from 0.1% (F3) to 0.5% (F4) resulted in a significant decrease in Q5 (67.5 %), and the overall DE (87.5 %). The increase in CMC concentration to 0.5% (w/v) (F4) resulted in the increase in viscosity of the F4 (157 mPa s) compared to F3 (152 mPa s) (Table 3). This was evidenced by the similarity factor test (f2 = 49.09%) and the significant increase in the MDT (8.47 min) (Fig. 1 and Table 4).
In case of F5, the addition of xanthan gum (0.3%) to CMC (0.1%) liberated bisoprolol at significantly lower rate compared to F3 which contains the same amount of CMC (0.1%). The combination of xanthan gum and CMC led to a slightly more viscous suspension (159 mPa s) which significantly (P < 0.5) reduced the dissolution rate of bisoprolol (Table 3). The Q5, DE, and MDT values of F5 were 63.2 %, 85.6 %, and 10.43 min, respectively; indicating the difference between F3 and F5 (f2 = 40.88%) (Fig. 1 and Table 4).
Another pattern was shown with Carbapol-containing suspensions (F6 & F7) (Fig. 1 and Table 4). In case of F6, a low concentration of Carbapol(0.1%) did not significantly change the dissolution parameters of bisoprolol (P > 0.05), compared to R product (f2 = 53.59%) (Fig. 1 and Table 4). However, the increase in Carbapol to 0.5% (w/v) (F7) retarded the dissolution rate of bisoprolol as indicated from the calculated dissolution parameters (Fig. 1 and Table 4). This was concurred with the viscosity results where, F7 was more viscous suspension (164 mPa s) than F6 (145 mPa s). The Q5, Q60, DE, and MDT values were 67.0 %, 92.0 %. 80.4 %, and 7.56 min, respectively (Fig. 1 and Table 4); indicating the difference between F6 and F7 (f2 = 44.42%).
Sodium alginate-containing extemporaneous suspension (F8) liberated 67.5% of bisoprolol in the first 5 min and > 98% after 60 min with the calculated DE and MDT being 85.7% and 7.69 min, respectively. This was further indicated from the similarity factor test (f2 = 49.07%) which reflected the difference of the dissolution profile of bisoprolol of F8 compared to R (Fig. 1 and Table 4). This could be attributed to the hydrated polymer surrounding bisoprolol particles which resulted in the formation of higher viscosity regions that encountered high resistance to the drug release [56].
Accordingly, from the dissolution, viscosity, and sedimentation results, the most satisfied and optimized extemporaneous suspension was xanthan gum-containing suspension (F2; 0.5%). The latter gave the best dissolution profile among all the extemporaneous formulations. It was an easily pourable viscous suspension with no sedimentation. Therefore, xanthan gum-containing extemporaneous suspension (F2) was used in the stability and bioavailability studies.
Accelerated stability studies
Forced degradation studies facilitate development, manufacturing, and packaging of pharmaceutical products [15]. A stress or accelerated stability study was done to study the impact of several environmental factors such as temperature, humidity, light, and oxidizing agents on the selected extemporaneous bisoprolol suspension (F2) as well as to predict its beyond-use date.
Chemical Stability
Changes in temperature, moisture, and light have sometimes drastic effect on the stability of drugs [68]. Chemical interactions between drug and excipients may decrease the drug stability [69-71]. Therefore, stress testing should include the impact of variety of environmental factors such as temperature, humidity, light, oxidizing agents as well as susceptibility across a wide range of pH values. The degradation behavior of bisoprolol under different stress conditions was studied by analyzing the tested samples at different time intervals.
For each stress condition, three chromatograms are shown in Fig. 2. Bisoprolol was quite stable in water for 6 months despite higher temperature, since UPLC chromatograms showed almost no change in peak area (Fig. 2a). Degradation of bisoprolol was more affected by 0.1 N HCl, 0.1 N NaOH, and 3% H2O2 (Fig. 2 b & c). After exposure to long period of time, more than 15% and 25% of the bisoprolol was degraded by keeping it in 0.1 N HCl, 0.1 N NaOH for 72 h. Bisoprolol showed high degree of instability towards oxidation at room temperature (Fig. 2d). A significant decrease in bisoprolol concentration was obtained in 3% H2O2 (v/v) all over the period of the experiment (72 h). Drug degradation was clearly observed where more than 60% of the drug was decomposed by 3% H2O2 after 72 h. For photostability study, bisoprolol was stable in in dark and light. The % degradation was 0.98% when suspension was subjected to light for 72 h. There were no changes in drug concentration nor retention time in different samples kept in the dark or subjected to light for the period of the experiment (one month). A similar results was reported and explained for the same drug [72, 73].
Physical Stability
The results of changes in pH and viscosity of F2 stored for 6 months at 25, 45 and 4 °C are shown in Fig. 3. No apparent change in mean pH nor viscosity occurred, regardless of whether they were stored at room temperature or in the refrigerator. The sedimentation, precipitation, and floating of suspensions was noted. No visible changes in color, odor, taste or smell were noticed during the study period. No sedimentation was noticed in F2 suspension throughout the study period, since sediment was prevented by the high viscosity value (217 mPa s) (Table 3).
Fourier transform infrared spectrophotometry (FT-IR)
An FT-IR spectra of pure bisoprolol fumarate, xanthan gum, glycerol, F2 suspension prepared from commercial tablets and pure bisoprolol are shown in the Fig. 4. The pure bisoprolol fumarate spectrum showed several absorption bands (Fig. 4a). The peaks at 3505 and 3418 cm-1 were assigned for aromatic C-H stretching while, the peak at 2973 cm-1 was attributed to aliphatic C-H stretching vibration. A biforked absorption band for C=O stretching vibration was shown at 1612 cm-1.The peak corresponding to the aromatic C=C stretching vibration was shown at 1550 cm-1. The symmetric CH3 and CH2 bending vibrations were clear at 1350 and 1400 cm-1. Peaks at 950-630 cm-1 revealed a bending vibration for skeletal vibration bands, out-of-plane aromatic C-H. A similar absorption bands for bisoprolol fumarate were reported previously [74].
FT-IR spectrum of xanthan gum revealed a vibration bands at 3413 cm-1 that was specified to OH group. Peaks at 1621 and 1407 cm-1 were specified for asymmetric– and symmetric–COO−stretching vibrations of pyruvate and glucuronate groups, respectively whereas, the peak at 1060 cm-1 was related to stretching vibration of C-O-C group (Fig. 4b). A similar absorption bands concurred with those published earlier for the same polymer [74,75] (Fig. 3b). In case of glycerol, the spectrum showed OH stretching frequency at 3391 cm-1, while C-H stretching was due to the peak at 2935 cm-1 (Fig. 3c). Bending of the C-O-H group was revealed by the peak at 1454 cm-1 of the C-O stretching of the primary alcohol, which was shown at 1115 cm-1 (Fig. 3c). The absorption bands were similar to those published by Danish et al, 2016 [77]. Similar absorption bands were shown in the spectra of xanthan gum-containing extemporaneous suspensions prepared from crushed tablets (F2) and pure bisoprolol powder (F2-unprocessed), which clearly indicated that no interaction coexisted between bisoprolol and additives for both extemporaneous suspensions prepared from crushed tablets (F2) or pure drug powder (F2-unprocessed).
Bioavailability study
The absorption and therapeutic efficacy of a bisoprolol in a suspension compounded from crushed tablets is unlikely to differ from that of the commercial tablets used in its compounding. To confirm this, the bioavailability of the selected bisoprolol extemporaneous suspension (F2) was evaluated. The pharmacokinetic parameters obtained with R and F2 are shown in Table 5. Fig. 4 shows the mean plasma concentration-time profiles of bisoprolol absorbed in humans. Two similar profiles were observed (Fig. 4). No significant difference in pharmacokinetic parameters between F2 and R (Table 5). The pharmacokinetic parameters were concurred with that published earlier [78]. The bioavailability of bisoprolol from F2 suspension was 107.7% compared to R tablets. The 90% CIs of the F2/R ratios for Cmax, AUC0-t, and AUC0-∞, of bisoprolol were within the acceptance range for bioequivalence.