http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, 2015; 20(5): 562–569 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.898657

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RESEARCH ARTICLE

Physicochemical stability of a new topical timolol 0.5% gel formulation for the treatment of infant hemangioma V. Merino-Boho´rquez1, M. Casas2, F. Caracuel1, M. Camea´n1, M. J. Ferna´ndez-Anguita3, G. Ramı´rez-Soto1, and M. J. Lucero2 1

Servicio de Farmacia, Hospital Universitario Virgen Macarena, Sevilla, Spain, 2Departamento de Farmacia y Tecnologı´a Farmace´utica, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain, and 3Servicio de Farmacia, Hospital Universitario Puerta del Mar, Ca´diz, Spain

Abstract

Keywords

Infant hemangioma (IH) is the most common tumor in infants, which affects 5–10% of white children. It is a tumor of vascular origin that appears in the first months of life. The indication for the treatment of the IH is not approved in the datasheet of the product, however it has been used in the infant hemangioma by topical administration as an alternative to oral propranolol, avoiding the main problems of the oral route (bradycardia and hypotension). The objective of this work is to study the physical and chemical (HPLC stability indicating method) stability of a 0.5% timolol gel for topical application during 60 days (considering the stability limit as 90% of initial concentration of timolol maleate). The gel was prepared with a polyacrylic acid derivative and the physical stability of the system was studied by visual control, rheological and mechanical characterization. The studied formulation guarantees the correct dose administering and stability after 60 days stored at 25 ± 2  C and light protected (tube of aluminum). We have developed an easy topical gel for the treatment of infant hemangioma with physical and chemical stability higher than those provided by the majority of hospitals.

Beta-blockers, compounding, HPLC, rheology

Introduction Infant hemangioma (IH) is a benign tumor which affects 5–10% of white children. It is a tumor of vascular origin which appears during the first months of life, involving two phases: a proliferative phase, with a rapid growth, followed by a slow involutional phase that can last for years. While most revert spontaneously, certain locations require early treatment to prevent the onset of sequelae1–3. This is the case of the periocular localization, since it has been associated to complications as the development of amblyopia, asymmetric astigmatism, proptosis, strabismus, or exposure keratitis4,5. Traditionally, the therapy has been oral and systemic corticosteroids6. Topical and intralesional corticoids, laser therapy, surgery and interferon a are corticosteroids alternatives7,8. Beta-blockers are also used for this disease because non cardioselective beta-blockers (propranolol, timolol, etc.) are able to inhibit the Vascular Endothelial Growth Factor, VEGF, involved in angiogenesis. Timolol is a non cardioselective beta-blocker used topically for various eye conditions9,10. Betablockers that have a relatively short duration of its effects will be given 2 or 3 times a day. Timolol is absorbed almost completely in the digestive tract, but has a moderate first-pass metabolism. Maximum plasma concentrations can be obtained around 1–2 h after a dose. Timolol is little or moderately fat-soluble. The plasma protein binding is low. A 4-h half-life of elimination

Address for correspondence: V. Merino-Boho´rquez, Servicio de Farmacia, Hospital Universitario Virgen Macarena, Avda. Dr. Fedriani S/N, 41009 Sevilla, Spain. Tel: +0034-9545008583/8586. Fax: +0034955008585. E-mail: [email protected]

History Received 17 January 2014 Revised 3 February 2014 Accepted 6 February 2014 Published online 22 July 2014

has been described. It is highly metabolized in the liver and the metabolites are excreted in the urine with part of timolol unchanged. Timolol is not dialyzable11. Timolol maleate (CAS 26921-17-5), whose chemical structure is described in Figure 1, is a white to almost white powder, odorless or almost odorless. It is soluble in water, alcohol or methanol, moderately soluble in chloroform and propylene glycol, insoluble in ether and cyclohexane12. Its main indications are hypertension, angina pectoris, acute myocardial infarction and the treatment of glaucoma. It is also used for prophylaxis of migraine11. The indication for the treatment of the IH is not approved in the datasheet of the product, however it has been used in the infant hemangioma by topical administration as an alternative to oral propranolol13–15. This was demonstrated by Le´aute´-Labre`ze et al. in 2008, becoming one of the most effective alternatives nowadays16. Some authors have used the ophthalmic formulation in gel form getting similar results to those obtained with oral propranolol and avoiding the main problems of the oral route (bradycardia and hypotension)17–19. In contrast to emulsions, gels generally do not comprise two immiscible phases of opposite lipophilicity. The consistency of gels is caused by gel former agents, which belong mainly to polymers. These polymers build up a three-dimensional network. Intermolecular forces bind the solvent molecules to this polymeric network and thus, due to the reduced mobility of these molecules in structured systems with increased viscosity, exhibit viscoelastic properties20. In the current literature there is no stability data of formulations of timolol maleate gel for topical application. For the above reasons, the objective of this work is to study the physical and chemical stability of a 0.5% timolol gel for topical application during 60 days. The gel was prepared with a polyacrylic acid derivative and the physical stability of the

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level concentration) precisely prepared and by determination of the relative standard deviation (RSD). Precision was assessed by testing the repeatability of three different standard solutions 15 determinations in the same day (intra-day) and by intermediate precision analyzing the same three standard solutions on different days (n ¼ 15) (inter-day). The detection and quantitation limits were based on the standard deviation of the response and slope. Sample preparation

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Figure 1. Timolol maleate.

system was studied by visual control, rheological and mechanical characterization.

Material and methods Reagents

One gram of timolol maleate powder was weighed and dissolved in 100 ml of mobile phase consisting of 65/35 v/v buffer phosphate (pH ¼ 2.8) and methanol. Subsequently an aliquot was taken, filtered (0.45 mm) and injected for HPLC analysis. At least, three samples were analyzed. Data analysis

Timolol maleate and excipients were pharmacopoeia grade and were provided by Acofarma (Barcelona, Spain). All other reagents were those of analytical grade and were purchased to Merck (Darmstadt, Germany) and JT Baker (Deventer, Nederland).

Data were analyzed to assess the percentage of recovery of the initial concentration of timolol maleate, considering the stability limit as 90% of recovery. The ChemStationÕ software (Agilent TechnologiesÕ , Waldbronn, Germany) enabled HPLC system control, acquisition and processing of data.

Gel production

Study of accelerated degradation

About 0.34 g of timolol maleate (content 0.25 g timolol) was weighed and dissolved in 33.41 g of water conservans (at room temperature). Then, 15 g of propylene glycol was added and mixed in a beaker. Subsequently, 1.25 g of CarbopolÕ 940 (Acofarma, Barcelona, Spain) was weighed and dispersed in the previous solution, leaving it to stand for 24 hours (pH ¼ 3.0). The following day it was neutralized to pH ¼ 7.0 with triethanolamine, getting the gel consistency. It was packed in a tube of aluminum (50 g) at ambient temperature (25  C). The final concentration of timolol maleate in the formulation was 0.5% (5 mg/g).

To verify the selectivity of the method, an accelerated degradation trial was carried out. We used an aqueous solution of timolol maleate 1 mg/ml, and besides, the finished product, i.e. 1 g of the gel. Both were exposed to the following degrading conditions for a total of 12 hours: acid hydrolysis (1 M HCl), basic hydrolysis (1 M NaOH), oxidizing (3% H2O2) and thermal hydrolysis (80 ± 2  C). Physical stability Visual control

Chemical stability HPLC stability indicating method validation The timolol concentrations were assayed using a highpressure liquid chromatography (HPLC-UV, Agilent InfinityÕ 1260,Waldbronn, Germany) equipped with a quaternary pump coupled to the degasifier, an automatic injector, a thermostatized column and an UV Diode Array Detector (UV-DAD). HPLC conditions were established according to the USP21 thus, flow was 1.5 mL/min, the mobile phase was a mixture of potassium dihydrogen phosphate buffer solution 50 mM and methanol grade HPLC (65/35 v/v), adjusted to pH 2.8 with ortho-phosphoric acid 85%, the injection volume was 15 mL and the temperature of the system was controlled at 40  C. According to the UV spectra obtained in the range 200–400 nm (Figure 2), the detector wavelength was adjusted to 295 nm. The RP-column was used AgilentÕ Zorbax Eclipse Plus C18 (4.6  100 mm, 3.5 mm). A method to quantify a timolol maleate gel by HPLC was validated22. It was carried out with a stock solution containing 10 mg/ml of timolol maleate (PhEur) and dissolved in ultrapure water (milli-Q Direct purification system). Stock solutions were prepared from it and diluted with the mobile phase. Linearity was tested within an interval of standard solutions (0.02–0.07 mg/mL of timolol maleate) in order to calculate the calibration curve. Each level of concentration was prepared in triplicate. The experimental results were graphically plotted, obtaining a calibration curve and carrying out the corresponding statistical study (ANOVA). Accuracy was tested by mean percentage recoveries of five samples of timolol maleate at three different concentrations (n ¼ 15, five replicates for each

In order to detect any changes in gel appearance (color, odor, consistency, etc), a visual control was made on each day of analysis. Rheological properties The gel was allowed to rest at room temperature for 24 h after its preparation before conducting any rheological test in order to avoid mechanical memory effects. Multi-step flow curve measurements were run using a controlled-stress rheometer, (AR-2000, TA Instruments, New Castle, DE), using a geometry of 60 mm diameter with flat surface. The experimental protocol consisted of applying every shear stress either until an approximation to the steady-state of 0.001 was reached or until a maximum time of 300 s per point. Small amplitude oscillatory shear experiments were carried out in the AR-2000 rheometer (TA Instruments, New Castle, DE) to determine the linear dynamic viscoelastic properties of samples. First of all, the linear viscoelastic region for the different systems was determined by stress sweeps at 1 Hz. With these results it is selected the stress amplitude to determine the mechanical spectra in a frequency range from 0.01 rad/s to 100 rad/s. The analysis of results is based on the storage (G0 ) and loss 00 (G ) moduli, which are related to the elastic and viscous component, respectively. Also, the complex dynamic viscosity (Z*) are used as viscoelastic function:  ¼ G =!

G ¼ G0 þ IG00 23

where ! is angular rate (rad/s) .

ð1Þ

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40 30 20 10 0 220

240

260

280

300

320

340

360

380

nm

Figure 2. 2D and 3D UV spectra of timolol maleate obtained in the range 200–400 nm.

On the other hand, flow curves and viscosity with rotational shear assay have also been determined. All measurements were made at 25 ± 1  C. Mechanical characterization Mechanical properties of the studied gel were determined by uniaxial compression with a load cell of 5 kg force. Texture analyzer TA.XT2i (Stable Micro Systems Ltd., Surrey, UK) was used and controlled by software. A probe (HDP/SR Stable Micro Systems, Surrey, UK) penetrated in complementary conical sample holder containing 7.5 ± 0.1 g of gel. The probe was put to a distance of 17 mm and move at 2 mm/s compression rate. Hardness, compressibility and adhesiveness were measured.

The force versus time data was converted to a true stress (s) and Hencky’s strain ("H), relationship using the following substitutions24,25. ¼

FðtÞHðtÞ Ao Ho

  HðtÞ " ¼ ln Ho

ð2Þ

ð3Þ

where F is the momentary force at time t, H is the height at time t, A0 is the cross-sectional area of the original gel (1521.504 mm2) and H0 is the initial length when the compression starts (17 mm). Stress (sf) and strain ("f) fractures were determined as the maximum point of the true stress–Hencky strain curve and the

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fracture work (Wf) was the area under curve of the fracture sone26.

Results Chemical stability

565

two peaks appeared in the gel (0.63 min and 0.88 min). Recovery of timolol was approximately 45% in both cases. Thermal hydrolysis (80 ± 2  C): Any degradation peaks were detected in solution or gel samples, and the recovery of timolol in both cases was around 80%.

The variance analysis (ANOVA) of the linear regression confirmed the linearity of the method through rejection of the null hypothesis of linearity deviation for a significance level of 0.05 ( ¼ 0.05); the coefficient of variation of the method was 0.82%. The equation of the regression lineal obtained corresponds to the following expression: y ¼ 6875.80 x  2.167 (n ¼ 18; R2 ¼ 0.9998) where y is the peak area and x is the timolol maleate concentration (mg/mL), with a residual standard error of 49.572. The validated method was accurate and precise in the whole range of linearity (Table 1). The detection and quantification limits, based on the standard deviation of the response and slope, were 0.56 mcg/mL and 1.70 mcg/mL, respectively. The results obtained show that the gel is stable (over 90% of initial concentration of the drug) within 60 days of the study (Figure 3). The time of retention (Tr) of timolol maleate was 2.08 min under the studied conditions. Study of accelerated degradation The study of accelerated degradation in different conditions showed the following results (Figure 4): 1 M NaOH: A single unidentified peak of degradation appeared at 1.28 min both in the solution of timolol maleate and the gel. The percentage of timolol recovery was 54.61% and 77.10%, respectively. 1 M HCL: Both the solution and the gel there were no detectable degradation peaks. The recovery in both systems was approximately 82%. 3% H2O2: The solution had several unidentified peaks of degradation at 0.63, 0.74 and 0.89 min. However, only Table 1. Accuracy and precision for low, medium and high concentration measured within-day (n ¼ 15) and between-day (n ¼ 15). Within-day mean (mg/ml) RSD (%) Level concentration

Low

Medium

0.019 (0.06)

0.050 (0.28)

Between-day mean (mg/ml) RSD (%)

High

Low

Medium

High

0.049 (0.85)

0.070 (0.58)

Physical stability Visual Control This is a transparent gel, easily extensible and with an excellent cosmetic appearance. There were no changes in color, odor or physical characteristics of the gel during the 60 days of study. Rheological properties Stress sweep of timolol gel can be seen in Figure 5. From these results we can determine the linear viscoelastic region of the gel. Elastic (G0 ) and viscous (G00 ) moduli are constant and independent of stress applied27. Thus, this independent behavior is observed up to 10 Pa, and from this point the sample begins to flow, critical stress. Figure 6 displays G0 and G00 versus frequency sweep. It can be appreciated the predominance of storage modulus (G0 ) relative to the loss one (G00 ) in the frequency range investigated. At the same time, the 0.1 value of tan  (tan  ¼ G00 /G0 ) indicates that the product is predominantly elastic. Therefore, the system is more elastic than viscous28 showing no dependence on the applied frequency, as expected, indicating that it is a three-dimensional and very structured gel29. Viscosity curves of timolol gel at 25  C are shown in Figure 7. The gel shows a characteristic behavior of pseudoplastic fluids in which the apparent viscosity decreases when shear rate increases. Finally, the data were fitted to mathematical rheological models, corroborating what was observed in the graphs. The best fit to the gel corresponded to the Cross model. Cross flow model30 describes the shear-thinning or pseudoplastic behavior by: _ ¼ ZðÞ

Z0 _ m 1 þ ðÞ

ð4Þ

90

where Z0 is the zero-shear rate viscosity (Pa s),  is a structural relaxation time or time constant (s) and m is a shear index dimensionless which indicated the degree of shear thinning (m ¼ 1  n). When m approaches zero the liquid is Newtonian, while the most shear thinning liquids have a value of m approaching unity31,32. These parameters are given in Table 2. Our system is a shear-thinning fluid because displays m values near 1. And, taking into account the higher value of zero-shear rate viscosity, our results agree with the very structured gel mentioned before. Finally, comparing the different curves carried out over time, it is observed that all they are superimposable and this indicates the maintenance of the structure over time, and therefore its physical stability.

85

Mechanical characterization

Timolol maleate

0.069 0.019 (0.29) (1.17)

110 % Remaining Timolol maleate

105

% Remaining

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HPLC analysis

100 95 Stability threshold

80 0

10

20

30

40

50

60

Time (days)

Figure 3. Evolution of remaining % of timolol.

70

Texture analysis studies or mechanical behavior evaluate the response of the system when subjected to a deformation caused by a normal force to the sample, in contrast to the rotational study, which is done by shearing or by a tangential force to the sample.

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2.083

566 mAU 70 60 50

Timolol gel at me 0 day

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1 M NaOH 30 1.282

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1 mAU

mAU 60

80ºC

50 40 30 20 10 0 1

2

3

Figure 4. Peaks degradation of timolol gel in accelerated conditions for 12 hours.

The profiles of texture analysis (force versus time and force versus distance) and the mechanical parameters are shown in Figure 8 and Table 2. High hardness values are in agreement with the structure of the gel and the zero-rate viscosity. On the other hand, it has higher compressibility than adhesiveness, which is interesting because the compressibility is a measure of the work required to break the physical connections within it, while the

adhesiveness is the capability to remove the probe from the holder. According to fracture ("f) and Hencky’s ("H) values, they indicate its solid structure, with higher values than other hydrogels33. Finally, variations observed in mechanical parameters with time are probably due to the relaxation of the physical bonds, but that do not constitute a commitment to the physical stability of the

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Figure 5. Stress sweep. Storage (G0 ) and loss (G00 ) moduli values of timolol gel at time 0, 30 and 60 days at 25  C.

Figure 6. Storage (G0 ) and loss (G00 ) moduli versus frequency sweep of timolol gel at time 0, 30 and 60 days at 25  C.

gel, since any significant change had resulted in rheological analysis during the days of study (Figure 6).

Discussion Community and Hospital Pharmacists are often challenged to provide a topical formulation for the treatment of infant hemangioma. In our work we have developed a new easy gel for

this disease, using commercial available materials. According to the current bibliography, we have chosen a gel because this is the only topical system, with good results, tested in children with infant hemangioma17–19. Probably, more clinical studies demonstrating the effectiveness of other beta-blockers and other topical forms are needed. Regarding the HPLC results, we can appreciate the high chemical stability of timolol gel, which remains stable within 60 days of the study. The slight increase

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Figure 7. Viscosity curves of timolol gel at time 0, 30 and 60 days at 25  C.

Table 2. Parameters obtained in mechanical characterization of timolol gel at time 0, 30 and 60 days. Parameters Hardness (N) Compressibility (N mm) Adhesiveness (N mm) Fracture strain (Pa) Hencky’s strain k1 (s) k2 (s)

Gel time 0

Gel time 30

Gel time 60

57.364 32.173 18.761 49 996 7.43 0.0006 1.0018

60.358 32.477 17.003 48 077 6.91 0.0004 1.0011

40.363 23.685 15.975 41 265 7.03 0.0005 1.0012

Figure 8. Typical mechanical curves obtained with the texture analyzer (force versus time and force versus displacement) to timolol gel at time 0, 30 and 60 days.

in percent recovery on 40 day may be due to a small deviation of the precision of the analytical method, rather than a real increase of timolol. Accelerated degradation tests show that oxidizing condition is the factor that most destabilizes the gel, followed in

order by alkaline, thermal and acid condition. With respect to the physical stability, rheological studies ensure the maintenance of the physical characteristics, since all profiles were superimposable with the time.

DOI: 10.3109/10837450.2014.898657

Conclusions We have developed a new timolol-based formulation for the treatment of infant hemangioma. This timolol maleate 0.5% gel remains stable both from physical and chemical point of view, during all the study. Therefore, we propose an expiration date of 60 days keeping at 25  C for the studied formulation.

Declaration of interest

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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Physicochemical stability of a new topical timolol 0.5% gel formulation for the treatment of infant hemangioma.

Infant hemangioma (IH) is the most common tumor in infants, which affects 5-10% of white children. It is a tumor of vascular origin that appears in th...
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