Safe use of Dexamethasone in pediatrics: design and evaluation of a novel stable oral suspension

Introduction

Dexamethasone is a glucocorticoid with high anti-inflammatory activity and a slight mineralocorticoid effect used in pediatrics in treatment of croup and bronchopulmonary dysplasia at dose of 0.6 mg/Kg and 0.5–0.6 mg/Kg/day [1]. In some countries there are commercial oral liquid formulations of Dexamethasone for pediatrics [2], but they include a combination of inadequate excipients for this population such as preservatives (benzyl alcohol, benzoic acid, benzoates), sweeteners (sucrose, fructose, sorbitol, xylitol, aspartame), solvents (lactose, ethanol, propylene glycol) or colouring agents (azo dyes, quinolone dyes, triphenylmethane dyes, xanthene dyes) [3]. Therefore, this therapeutic gap must be covered. Dexamethasone is classified as a Biopharmaceutics Classification System-Class I/III active substance [4], authorized by oral, ophthalmic, otic and parenteral routes. Dexamethasone sodium phosphate is authorized for parenteral, ophthalmic and otic routes [5]. Dexamethasone phosphate lacks sufficient data on oral bioavailability [6], so it is not marketed as an oral formulation [7, 8]. Recently Binson et al. published a study about the preparation of an oral stable suspension using the acetate salt formulated with complex excipients as Ora-Sweet^® and Ora-Plus^® at a dose of 5 mg/mL [9]. This salt is authorized in France for oral solid formulations [10], but it is not authorized for this route of administration in other European countries as Spain [11] or USA [12]. When Dexamethasone needs to be administered orally at pediatric doses, a compounding preparation should be elaborated from Dexamethasone as active pharmaceutical ingredient (API) authorized for this route, rather than from an authorized dosage form not indicated for this use [13, 14]. A Standard Operating Procedure (SOP) must also be followed to ensure the quality of the final compounding. Also, if this preparation needs to be administered to a vulnerable population as pediatrics, the lowest possible number of excipients must be used and in the lowest proportions [15, 16]. When lower doses of Dexamethasone than the marketed form for adults are needed in clinical practice, what actually happens is that oral formulations are prepared by manipulation or compounding [17, 18] the authorized Dexamethasone tablets or intravenous formulations. The main disadvantage of using tablets is that excipients from this authorized dosage form are introduced into the new formulation. These excipients could be not suitable for pediatrics (sucrose, lactose, coloring, etc) [2, 5], difficult to disperse (magnesium stearate, starch, lactose, etc) [2, 5] and/or interfere with the distribution of the API, especially when it is formulated in low proportions [19]. In the case of using injectable forms, the Dexamethasone phosphate is administered orally as suspension using complex excipients (benzyl alcohol, sodium sulfite and citrate) [2, 5]. This is not necessary due to this salt being easily soluble in water [20, 21]. Toledo et al. conducted a pharmacokinetic comparison between Dexamethasone from a commercially available oral formulation and Dexamethasone phosphate from intravenous formulation administered orally to healthy volunteers. They concluded that these two formulations are not bioequivalent [22].

To avoid this non-suitable and non-authorized use of Dexamethasone, in this study we proposed the formulation of two oral 1 mg/ml suspensions. Both contain the API and the least number of excipients in the lowest possible proportions avoiding the use of preservatives. Strict monitoring of the SOP should ensure the quality of the final formulation. To do this, a validated chromatographic method was used to determine and quantify the Dexamethasone. It was subjected to the European Pharmacopoeia test for multi-dose containers [23], adapted for determining the declared API content in each dose. Since the stability of pediatric oral formulations of Dexamethasone is only tested on those compounded from an authorized dosage form [20, 21, 24], physical, chemical and microbiological stability was also tested following pharmacopoeia criteria.

Materials and Methods

Dexamethasone, sucrose, citric acid and sodium citrate were pharmacopoeia grade and were provided by Acofarma (Barcelona, Spain). Water was purified in our laboratory by a Milli-Q system. Acetonitrile and methanol were analytical grade (Sigma-Aldrich, Madrid, Spain).

Quality Control

pH

The pH was determined every 7 days for 60 days (7, 14, 21, 35, 42 and 60 days) for both formulations stored at 5, 25 and 40 °C. The samples were the same used for the chemical stability studies (see below). The pH was tested in duplicate using a Crison GLP 21 pHMeter (Barcelona, Spain).

Viscosimetry

The viscosity was measured in a programmable viscometer Brookfield^® LVDV-II (Middleboro, MA, USA) at 25 °C. A spindle SC4-18 was used so as to determine viscosities between 1.5 and 30,000 mPa.s with 8 ml as sample volume. Data were processed with the Wingather^® 32 program (Middleboro, MA, USA). All the measurements were taken with a torque between 10–90 %. Every characterization was in triplicate.

Dose content uniformity

The European Pharmacopoeia (EP) states that for multi-dose containers, 20 doses are individually weighed at random and the individual and average masses determined; no more than 2 of the individual masses may deviate from the average by more than 10 % and none may deviate 20 % [23]. This mass uniformity criterion only indicates the uniformity of mass extracted with respect to the average mass, and assumes a uniform distribution of the active ingredient in the formulation. To test this latter aspect, a modification of this test was additionally applied, the percentage of declared Dexamethasone content was determined instead of the average content in each preweighed dose, applying the same test criteria.

20 doses (5 ml) taken randomly from the elaborated formulations were weighed prior to determining each Dexamethasone content at day 0 and after stored 30 days at 5±2 °C. This dose volume was taken out with an 5 ml syringe (BD Discardit^TM II) for oral use after the formulations were manually shaken [30]. This device allows accurate dose measurement and controlled administration to the buccal cavity for all ages [3]. Afterwards, the samples were diluted adding 5 ml of methanol to promote the solubilization of Dexamethasone, and after purified water was added until 50 ml. Dilution to concentration 5 µg/ml were made with mobile phase. A Dexamethasone mass balance was determined from the volume remaining in the containers after removal of 20 doses.

Chemical stability studies

An accelerated study of Dexamethasone’s stability at a temperature of 60±0.1 °C (Heraeus UT 6060, Spain) and a concentration of 50 µg/ml at different pH conditions (citric/citrate buffer, pH 2–8) was performed. At time 0, 1, 18, 24, 42 and 48 h, the samples were diluted with mobile phase to a concentration of 5 µg/ml and were analysed immediately by UPLC.

F1 and F2 were checked for stability at different temperatures and storage conditions following the International Conference Harmonization (ICH) guidelines [31, 32]. At 5±0.1 °C (Fridge-stove P-selecta Welidow type, Spain), at 25.0±1.3 °C (dark chamber, own manufacture), and at 40±0.1 °C (Heraeus UT 6060, Spain). Samples (5 ml) from each Dexamethasone formulation batch were taken every 7 days in triplicate until 42 days (7, 14, 21, 35 and 42 days).

Physical stability studies

An additional test was performed to determine the physical stability of the dosage form and to indicate the importance of shake before removal of the dose [33]. A possible sedimentation of Dexamethasone could influence the uniformity of the dose extracted from different heights of the formulations elaborated as suspensions (F1 and F2). Each formulation in duplicate was poured into a 100 ml graduated cylinder to study the degree of flocculation and sedimentation of the suspensions and after manual shaking (10 times inverted 180°), doses were taken at heights ZI (100–80 ml) and ZIII (20–0 ml), see Figure 1.

Figure 1:

Heights (Z1 and Z3) at which the doses were taken from F1 and F2 suspensions at time 0 (a), after 30 days storage at 5 °C (b), and after shaking 180° 10 times (c).

Doses (5 ml) were taken, weighed and their Dexamethasone contents were determined. This process was done after 30 days stored at 5±0.1 °C (Fridge-stove P-selecta Welidow type, Spain).

From this test two parameters were calculated: Dmax, the maximum difference between doses taken at different heights, and Dmax/t, Dmax corrected for the standing time after shaking. Both parameters were expressed as a percentage of declared dose (DV %).

Microbiological stability studies

Microbiological tests of formulations were done at 0, 15 and 30 days of storage at room temperature (25 °C), simulating the conditions under which daily doses are removed. Both formulations were prepared at the compounding laboratories at two pharmacies following the SOPs proposed for F1 and F2. Tests were performed according to the EP and The United States Pharmacopoeia (USP) monograph of non-sterile products [34, 35] at a laboratory accredited as quality manager ISO 9001. The microbial count was considered to be the average number of colony forming units (cfu) found in the appropriate medium by plate-count method. Liquid oral formulations meet microbial requirements if the total aerobic microbial count was less than 102 cfu/ml, the total combined yeast/mold count less than 101 cfu/ml, and the absence of Escherichia coli was confirmed.

Results

Figure 2 shows the chromatogram obtained by the UPLC method for Dexamethasone as pure pattern, after its extraction from suspension F2 and after 2 days stored at 60 °C at pH 6.14, for testing the ability of the method to separate degradation products in the elaborated formulation. A peak with an elution time of approximately 1.4 minutes was detected. The chromatographic area of this peak decreased with time at 60 °C and a new peak appeared, identified as a degradation product at approximately 1.2 min [36, 37]. As can be seen, no interference with the excipients of the suspension was detected. The average extraction yield of Dexamethasone by the UPLC method was 102.5±4.3 % and 100.2±3.4 %, from F1 and F2 respectively.

Figure 2:

Dexamethasone pure pattem chromatographic peak as standard of 5 µg/ml (continuous bold line), Dexamethasone extracted from F2 suspension (discontinuous bold line) and Dexamethasone in pH 6.14 solution, after 48 h stored at 60 °C (discontinuous thin line).

The results of the accelerated stability test at pH 7.85 (Figure 3) reveal how rapidly Dexamethasone degraded, after 48 h leaving a remainder of less than 10 %. However, at pH 7.09 the molecule degraded more slowly, showing a greater decrease in the percentage remaining between 18 and 24 h. The pH with maximum stability for Dexamethasone was the most acidic (2.13) [26], with 100 % remaining at 42 h.

Figure 3:

Remaining percentage of Dexamethasone in different pH solutions stored at 60 °C.

Table 1 shows the pH and viscosity values obtained for both suspensions. As can be seen F2 has a more acid pH and lower viscosity as expected, since F2 is F1 diluted with citric/citrate buffer.

Figure 4 shows the pH variation during the 60 days of storage for both formulations at different temperatures. F2 remained with a more constant pH value than F1 for 60 days. The greatest variability was observed at 25 °C after the storage of the suspensions for 14 and 21 days, for F1 and F2 respectively.

Figure 4:

pH evolution at F1 (symbols with filler) and at F2 (symbols without filler) during the 60 days storage at 5, 25 and 40 °C.

Both suspensions showed Newtonian behaviour, see Figure 5, wherein the viscosity of each system remained constant with shear rate (Figure 5a), and the shear stress increased linearly with shear rate (Figure 5b).

Figure 5:

Variation in viscosity with shear rate (a), and in shear stress with shear rate (b) of F1 and F2.

Table 2 shows the mass uniformity tests performed for F1 and F2. Both formulations met the EP test for mass uniformity of multi-dose containers. If the same EP criteria are applied for the declared Dexamethasone content determined in each preweighed dose, see Table 3, both formulations also passed the adapted EP test. The Pharmacopoeia tests were also complied with after 30 days storage at 5±0.1 °C. In order to determine the physical stability of the dosage form and highlight the importance of shake before removal of the dose, we measured the variation in uniformity of doses taken from different heights of the same formulation. As seen in Table 4, the Dmax at 30 days gives the maximum difference between quantities of Dexamethasone in the sample dose taken from the two zones after storage of the formulations in the indicated conditions. This parameter gives us an idea of ​​the rate of sedimentation of the solid. In our case, the highest difference between areas was clearly F1 after 30 days of storage at 5±0.1 °C, taking the dose without previous shaking. F2 presented a sediment after 30 days, which was readily redispersible after shaking (Figure 1). Dmax 70 min and Dmax 90 min indicate the maximum difference between areas after 70 and 90 minutes of rest following 180° inversion 10 times. Dmax/t value was slightly smaller for F1 than for F2. A two-factor ANOVA with two samples per group was used to analyse these data. There were no statistically significant differences (p>0.05) between zones before and after shaking each formulation (Table 4).

After storage of the two formulations under different temperature conditions, Dexamethasone in F2 (Figure 6) was observed to maintain its average remaining percentage above 90 %, regardless of temperature. In addition, at 25 °C the average remaining percentage detected was higher than 95 % during 21 days, see Figure 6b.

Figure 6:

Percentage of Dexamethasone remaining in F1 a) and F2 b) after 60 days storage at 5, 25 and 40 °C. Dashed lines are the upper and lower limits established at 100±5 % and 100±10 %.

For both formulations prepared at two pharmaceutical compounding pharmacies which followed strictly the SOPs proposed, the total aerobic microorganism count was less than 10² cfu/ml and total combined yeast/molds was also less than 10¹ cfu/ml at 15 days of storage. During this time, the daily dose sampling was simulated and the storage temperature was the most favorable for microbial growth (25 °C). No E. coli contamination was observed during this time. Although these types of formulations usually have a 30 days period of validity, in this case is less due to mold growth.

Discussion

The UPLC method allowed us to detect and quantify Dexamethasone accurately and precisely, both as a pure pattern and after extraction from the suspensions studied (F1 and F2). In addition, this chromatographic method can be used as a stability-indicating method since it detects the degradation products of Dexamethasone.

Two 1 mg/ml suspensions of Dexamethasone as API, no using its salts, were easily prepared without preservatives as parabens, and with the least number of excipients and at their lowest possible proportions, suitable for pediatrics. Suspension F1 shows a higher viscosity and a neutral pH, in opposition to F2 that incorporates a citric/citrate buffer in order to acidify the pH to a more stability area of the API. Both formulations have a Newtonian rheological behavior, ensuring constant viscosity. Citric/citrate as buffer at formulation F2 decreased viscosity but did not change rheological behaviour. Both suspensions met the EP test for uniformity of mass, but this is insufficient information to know if drug content in each dose agrees with the declared one. If we apply the same criteria for dose uniformity with respect to the declared Dexamethasone content (1 mg/ml), F1 and F2 also met the criteria, recently prepared and after 30 days of storage at 5 °C. It must be noted that the variability of the doses measured as RSD decrease with storage time for both formulations in all uniformity tests. But it was lower in F2 which leads us to conclude that in this formulation the API is more uniformly distributed after shaking.

Then, the UPLC method is valid to study the uniformity content and its evolution with storage time.

The use of certain parameters (Dmax and Dmax/t) in extracted dose determination enabled us to analyze the sedimentation of the two suspensions. High values ​​of Dmax point to insufficient resuspension of the API after the inversion of the container. The uniformity of doses, taken from different heights of each formulation stored under the same conditions, reveals how F2 improved after shaking of the container. Analysis of the variance in dose content sampled at different heights confirms this conclusion. Considering all the above, although F2 presents a sediment in the storage conditions, it is easily redispersible after shaking and it is the formulation which Dexamethasone is more uniformly distributed.

With respect to chemical stability study, Dexamethasone average remaining percentage extracted from F2 at 21 days after storage at 25 °C was above 95 % of the declared content in each dose. At F2 Dexamethasone is more stable when stored at different temperatures, due to more acid environment. This was checked in the accelerated studies reported here, and in the literature [38, 39].

Microbiological stability reveals presence of mold at 30 days. F1 and F2 complies with EP and USP specifications for microbial examination of non-sterile products during 15 days period. These results indicate the validity of use of F2 during 15 days stored at 5 and 25 °C although the physical and chemical stability studies indicate more time. This period could be increased if F2 is prepared with simple syrup that contains parabens dissolved in the water in which it is prepared. The addition of parabens can be done avoiding the usual use of propylene glycol as co-solvent due to its toxic effect in pediatrics [40].

Conclusion

F2 is a novel and suitable Dexamethasone suspension for oral pediatric administration that, if it is strictly prepared following the proposed SOP, ensures mass and content uniformity in each dose, as well as physical, chemical and microbiological stability for 15 days stored at 5 and 25 °C. It is possible the safe oral administration of Dexamethasone as API in pediatrics using the minimum number and minimum proportion of excipients suitable for this population, without the use of preservatives. Avoiding the manipulation or compounding the intravenous formulations, with the sodium form not authorized for oral route, or the commercial tablets which introduces excipients not appropriated for oral liquid formulations.