Novel Glyceryl Monostearate- and Polyethylene Glycol 6000-Based Ibuprofen Pellets Prepared by Hot-Melt Extrusion: Evaluation and Stability Assessment

To prepare stable sustained-release (SR) pellets, containing high ibuprofen (IBU) loading, by hot-melt extrusion (HME) technique using polyethylene glycol 6000 (PEG 6000) and glyceryl monostearate (GMS). HME pellets (60% w/w IBU) were prepared using PEG 6000, GMS, and mixture of both polymers (1:1). Stability studies were performed under stress conditions (40 °C and relative humidity “RH” of 75%) for 6 months and at room temperature for 12 months. Fresh and stored IBU pellets were evaluated by drug content (HPLC), release rate study (USP apparatus IV), DSC, and XRD. HME succeeded to produce SR-IBU pellets with high drug loading. PEG 6000 gave higher IBU release rate and relatively unstable formula after storage. PEG 6000/GMS mixture gave prolonged IBU release up to 4 h with stable formula for 12 months at room temperature. While, IBU/GMS pellets gave SR profile up to 6 h and a stable formula under both testing conditions. These advantages of IBU/GMS pellets could be an excellent candidate for SR-IBU product. DSC and XRD analysis data (enthalpy and counts) for IBU and polymers gave a mirror image for IBU release profiles of the studied HME pellets, for both fresh and stored samples. Stable SR-IBU/GMS HME pellets with high IBU loading (60% w/w) were successfully produced, for the first time, without any other excipients.


Introduction
Many drug delivery systems are formulated employing the hot-melt extrusion (HME) technique.HME provides numerous advantages: fabrication is carried out without solvents; scaling-up and production are smooth; and the potential of enhancing drug bioavailability could be improved.Blending of a drug with other excipients (plasticizers and polymers) is carried out in HME, concurrently with fast melting resulting in a solid dispersion (SD) of the drug in the polymer.
The molten mass is extruded and cooled, providing a new physical solid form with different properties (drug SD) [1].
Ibuprofen (IBU) is a safe NSAID which is generally applied for the treatment of inflammation, pain, arthritis, and dysmenorrhea [2].It possesses a short half-life of elimination (t 1/2 = 2-3 h) [2]; therefore, high dosing frequency is required to get stable therapeutic blood levels.Accordingly, IBU sustained-release (SR) formulations will allow for decreasing dosing frequency, maintaining therapeutic blood levels, reducing GIT side effects, and hence increasing the patient's compliance [3].
Plasticizers are usually used in HME for their role in reducing the viscosity and glass transition temperature (Tg) of the employed polymers [4].Plasticizers act by increasing the free volume, decreasing the friction among polymer chains and consequently improving polymer chain mobility, which results in reduction in the degradation of the drug and carriers, hence, stability profile improvement [5].It was previously reported that IBU (M.P. 78 °C) had a known plasticizing effect which increased by increasing IBU loading with different polymers, like Kollidon® SR [6,7].Thus, IBU is supposed to be perfect for HME techniques.
Improving IBU dissolution employing a SD technique was previously reported.Various carriers were applied, in SDs prepared by fusion, to improve IBU dissolution and hence absorption such as PEG 6000 [8,9], mixture of Tween 80 and Span 80 [10], and PEG 8000 [11].
Previously, Emara et al. [12] studied, for the first time, the effect of different storage conditions on the physical stability and dissolution behavior of IBU/Sucroester®WE15 SD prepared by HME compared to physical-mixture and fusion techniques.HME was reported to give superior properties.The dissolution behavior was evaluated by the well-established in vitro release test employing the flow through cell (FTC) "dissolution apparatus USP IV" which was proven to be reproducible, sensitive, and valid for proper discrimination among formulations [12,14].Emara et al. [14] reported a drug release study employing the FTC method at the optimum conditions with achieving the highest reproducibility of the results through elimination of the errors resulted from the release data because of pellets spreading to undefined sites of the cell [14].So, validation of the selected in vitro dissolution method is important for accurate monitoring of changes that might affect product performance and hence its bioavailability.
Hydrophilic polymers such as polyethylene glycols (PEGs) of different molecular weights have been widely utilized in HME methods to enhance solubility of poorly soluble drugs and hence improve their bioavailability [1].
The present study aimed to develop SR IBU pellets employing the HME technique using PEG 6000, GMS, and "PEG 6000:GMS mixture" as extrudable carriers.The developed pellets were evaluated for IBU content, release rate, DSC, and XRD.Moreover, the stability of the HME pellets was carried out for 6 months under accelerated conditions (40 °C and relative humidity (RH) of 75%) and longterm bench study (12 months at room temperature).

Preparation of Solid Dispersions by Hot-Melt Extrusion
Processing of SDs of IBU/PEG 6000, IBU/ "PEG 6000:GMS, 1:1," and IBU/GMS was carried out employing HME with 60% IBU loading ratio for HME-1, HME-2 and HME-3, respectively.Each formulation contained 600 mg IBU/g (Table 1).Single-screw microtruder (¼ in.single screw extruder with a single rod die, Randcastle Microtruder RC-025, Randcastle Extrusion Systems, Inc., USA) was employed for HME.The conditions and steps performed for the preparation of HME pellets were reported before [14] with small changes.The rotation of the screw was adjusted at 25 rpm.The temperature range of the 4 zones was adjusted at 55-65 °C.The final HME pellets were made by manually cutting the produced extrudates to have 1.5 ± 0.1 mm length and 0.5 ± 0.1 mm width.

Determination of Percent Drug Content by HPLC
HME pellet amount (equivalent to IBU dose) was accurately weighed, dissolved (25 mL acetonitrile), vortexed, and filtered (0.45 µm Millex, Millipore, USA).Dilution of the filtrate was

In Vitro Drug Release Studies
IBU release from the prepared HME pellets was in vitro studied.In vitro release study was carried out in phosphate buffer (pH 7.2) [22] compared to IBU powder.The release studies, for both fresh and stored samples, were carried out employing FTC closed loop setup (dissolution apparatus USP IV, a Dissotest CE-6 equipped with a CY 7-50 piston pump, Sotax, Switzerland) as reported before [14].The efficiency to attain the optimal conditions for studying IBU release and discrimination ability between different HME pellet formulations was proven for the selected design of FTC [14].The experiment was carried out in sextuplicate.
Comparison of IBU release from stored against fresh samples was carried out by using the similarity factor (ƒ2) [23], following the equation: where n is the number of time points, and R t and T t are cumulative percentage release at the selected n time point of the reference and test, respectively.
The f 2 (a measure of the similarity between two release curves and its value ranges from 0 to 100) was suggested by FDA [24] to be in the value range of 50-100 for any compared two release patterns to be considered similar [23].

Differential Scanning Calorimetry (DSC)
Assessment of any possible incompatibility between the drug and polymers in the prepared HME pellets was carried out through investigation of the thermal behavior of drug and polymer in both prepared and pure forms.Samples of IBU powder, PEG 6000, "PEG 6000:GMS, 1:1", GMS, and the prepared HME pellets were powdered and evaluated employing DSC (differential scanning calorimeter DSC-50, Shimadzu, Japan).Each sample (5 mg) was put in an aluminum pan and the heating ramp was carried at 25 to 200 °C with scaling up rate of 10 °C/min and 20 mL/min nitrogen purge.Fresh and stored samples were investigated.

X-Ray Diffraction (XRD)
Fresh and stored samples were inspected for their patterns of X-ray diffraction and compared to pure forms of drug and polymers using an Empyrean diffractometer.Monochromatized Cu-Kα radiation was used for irradiation of samples.Analysis was carried out between 2θ of 3° and 80°, through step-size 0.026°.The voltages, current, and time per step were 45 kV, 30 mA, and 18.87 s, respectively.

Stability Studies
The prepared HME pellets were subjected to stability studies following ICH guidelines [25].The stability of the pellets was assessed in regard to drug release characteristics, chemical stability by HPLC, DSC, and XRD.Stability samples of the formulated HME pellets were stored in tightly closed amber glass containers.They were subjected to both accelerated (6 months) and long-term (12 months) conditions.Accelerated stability studies were performed under stress conditions (at temperature of 40 °C ± 0.5 °C and relative humidity (RH) of 75% maintained by the use of saturated sodium chloride solution in a thermostatically controlled oven [26]), while long-term stability studies were carried out by storage of the samples at room temperature (25 °C).

Drug Content
Drug content percentage of the prepared HME pellets were within the range of 96.87 ± 7.03% to 103.94 ± 6.23%.These results were in accordance with the accepted pharmacopoeial limits [27].

In Vitro Release Study for Fresh Samples
The in vitro release evaluation was performed using a previously described method [14], which proved to be sensitive in discrimination among various formulations and confirmation of high reproducibility of the in vitro release results and hence the ability for the detection of any difference that may appear after storage.Figure 1 shows release profiles of the three IBU HME pellets compared to IBU powder.It was found that the release rate was pronouncedly affected by both polymers.
While GMS sustained the IBU release up to 6 h, PEG 6000 was almost behaving as IBU powder.Meanwhile, mixing the two polymers (1:1) prolonged the IBU release up to 4 h.It was reported earlier that PEG 6000 was used as a dissolution enhancer when prepared as SDs using conventional methods (nifedipine [28] and IBU [8,9]), while GMS was used as a dissolution retardant for IBU in SDs prepared by microwave irradiation [19] and IBU beads prepared using the melt solidification technique [20].
In the current study, the hydrophilic polymer (PEG 6000) was used to investigate the effect on the release of IBU from HME pellets, while GMS (hydrophobic polymer) was used to control IBU release rate.Therefore, mixture of both polymers gave intermediate release rate as showed in case of HME-2.These results could be considered as the reason for the fast release rate of IBU from HME-1 and the delayed release of HME-3 pellets (Fig. 1).Accordingly, it could be possible to tailor the formula by changing the GMS:PEG 6000 ratios to design the required release profile.Also, it could be possible to formulate IR and SR preparations according to the therapeutic needs and patient compliance.Ding et al. [29] studied the pharmacokinetics of IR and SR of a single-dose (600 mg IBU) preparation.They reported that the values of AUC (area under IBU plasmaconcentration-time curve) for both formulations were almost the same, while C max (maximum IBU plasma concentration) value of IR preparation was almost double the C max value of SR preparation, while t 1/2 values (time required for half the quantity of a drug to be eliminated) of IR were almost half the t 1/2 values of SR preparation.

Stability Study
Stability of pharmaceutical dosage form is a pre-requisite to conclude its performance.Few stability studies of IBU HME preparations were reported using Sucroester [12], HPMC, and Eudragit [17].Emara et al. [12] studied stability of sustained-release HME SDs of IBU (30 and 60% w/w) with Sucroester® WE15 under accelerated and long-term stability studies.The study reported that only 30% w/w IBU HME preparation provided a stable formula under long-term storage conditions, where 60% w/w IBU HME preparation was not stable in both stability testing conditions [12].
Yang et al. [17] studied the stability of IBU-HME SDs prepared with Eudragit® E PO and hydroxypropylmethylcellulose E5 (HPMC E5).They demonstrated that the dissolution profiles and physical states of both IBU-HPMC (20% w/w) and IBU-EPO HME (10% w/w) SDs remained similar to the fresh samples, after 30-day storage at 60 °C/0% RH.It is worthy to mention, here, that Yang et al. prepared 10% and 20% IBU as maximum possible drug loading with accepted extrudate characteristics [17].
Therefore, the preparation and stability of 60% IBUloaded PEG 6000 and GMS preparations could be considered a great challenge to reach the ideal production of IBU-HME without a need for other excipients.
Figure 2 shows the physical appearance of extrudates of IBU-HME before and after storage.All fresh IBU-HME extrudates were in the form of white rods (Fig. 2a).These rods were cut into pellets of 1.5 ± 0.1 mm length and 0.5 ± 0.1 mm width (Fig. 2b).After 6 months at 40 °C/75% RH, HME-1 pellets were deformed (melted) and lost its "rod-like" shape (Fig. 2c).In such case, after the elimination of the stress conditions, the melt mass was solidified after an hour at room temperature, grinded, and analyzed by DSC, FTIR, and in vitro release test.On the other hand, after 12 months at bench, no change in the physical appearance was observed (Fig. 2d), while HME-2 and HME-3 pellets did not change their physical appearance either after 6 months (40 °C/75% RH) or 12 months of bench storage conditions (Fig. 2d).This means that the presence of GSM is a key factor for restoring the product physical stability.
The results of HPLC analysis of drug content of different IBU-HME pellets provided the percent of drug in all stored samples which was in the range of 98.23-102.95%.This indicated the chemical stability of IBU (relative standard deviation (RSD) [30] in the range 0.45-2.97%).
Table 2 and Fig. 3 summarize the similarity factor (f 2 ) and the release profiles of stored samples compared with initial data of fresh formulations.Figure 3a, b shows that presence of PEG 6000 (HME-1 and HME-2) pronouncedly and significantly decreased the percentage of IBU released from stored HME pellets under stress conditions for 6 months, as depicted by ƒ2 values (i.e.ƒ2 ˂ 50, Table 2).This decrease might be due to partial recrystallization of IBU during storage and/or change of polymer properties.Moreover, the percentage of IBU release was significantly increased from HME-1 pellets (containing PEG 6000) after storage for 12 months on bench, while HME-2 pellets (containing PEG 6000 and GMS) gave similar release profile, as depicted by similarity factor (ƒ2 values ˂ 50 and ƒ2 values ≥ 50, respectively; Table 2).
On the other hand, Fig. 3c shows that the presence of GMS gave significantly similar IBU release profiles from HME-3 stored pellets, as proved by similarity factor (ƒ2 values ≥ 50; Table 2).The most interesting result in this study is that GMS (HME-3 pellets) gave stable formula after storage in both accelerated and bench conditions.These results suggested that, addition of GMS is a powerful tool to produce a stable and prolonged release IBU preparation(s) (Fig. 3c).The current results clearly confirmed the superiority of GMS-based HME pellets for preparation of stable IBU/SDs over PEG 6000 with respect to release properties and the stability of the final product, with a high IBU loading (60%).
To understand the possible reasons for the changes observed before and after storage, DSC and XRD were carried out.

DSC
The main target for carrying DSC analysis was to investigate the stability of 60% loaded IBU-HME pellets for the first time.Figure 4 and Table 3 show IBU crystallinity and drug-carrier interaction, if any, with PEG 6000, GMS, PEG 6000:GMS (1:1) mixture, and IBU-HME pellets.The DSC thermograms showed the crystalline IBU with a single, sharp endothermic peak at 74.15 °C which represents the melting of the drug with an enthalpy (∆H) of −88.53 J/g (Fig. 4a and Table 3).The DSC thermogram of PEG 6000 and GMS shows endothermic peaks at 62.65 and 69.33 °C, respectively, which represents melting points of the polymers (Fig. 4a).It is worthy to mention that PEG 6000:GMS (1:1) melts at a lower temperature (60.11 °C) than both individual polymers (i.e., eutectic mixture).
DSC thermograms of fresh IBU-HME samples showed the disappearance of all characteristic melting endothermic peaks of IBU or polymers.Where, a depression of melting point(s) of IBU-HME pellets were recorded and new characteristic peak(s) appeared (Fig. 4 and Table 3), where these distinctive peaks were a single peak at 48.39 °C for IBU-HME-1 (Fig. 4b), three adjacent peaks at 44.44, 56.45, and 63.72 °C for IBU-HME-2 (Fig. 4c), and two adjacent peaks at 59.74 and 65.93 °C for IBU-HME-3 (Fig. 4d).This could be due to drug inclusion complexation between the components and/or incorporation of IBU between parts of the crystal lattice of the carrier, leading to certain physical changes, a probable drug/carrier interaction, and change of IBU to an amorphous form [12]. Hence, the new physical form of IBU-HME pellets gave different release profiles, greatly affected by the used polymer characteristics.
Under stress conditions, the characteristic endothermic peak of HME-1 pellets (Fig. 4b) was split into two peaks at 58.98 °C (endothermic) and 85.48 °C (exothermic).While, upon storage for 12 months at room temperature, HME-1 pellets maintained its single endothermic characteristic peak, but with relatively high shifting from 48.39 °C (∆H = −152.81J/g) to 55.01 °C (∆H = −117.6J/g) (Table 3 and Fig. 4b).These data agreed with release profile results, where HME-1 (PEG 6000) samples stored under stress condition gave a significant lower release rate than fresh samples, while 12-month stored samples gave higher release rate compared to the data of the fresh pellets with a significantly different release profile (Tables 2 and 3 and Fig. 3a).These results confirmed the instability of the preparation.
Under stress condition, the characteristic 3 adjacent peaks (2 endothermic and 1 exothermic) of HME-2 pellets (Fig. 4c) were substituted by a single endothermic peak with   3).This change was reflected by the significantly different release profiles (ƒ2 values ˂ 50) between fresh and stored HME-2 pellets (Table 2 and Fig. 3b), while upon storage for 12 months at room temperature, almost showed the same DSC thermogram as fresh pellets with only slight shifting of    the 3 adjacent peaks (retained 2 endothermic and 1 exothermic) (Fig. 4c).This was proved by the significantly similar release profiles (ƒ2 values ≥ 50) of fresh and stored samples (Tables 1 and 2 and Fig. 3b).Under stress conditions and 12 months of storage at room temperature, the characteristic 2 adjacent endothermic peaks of HME-3 pellets were only slightly changed (Table 3 and Fig. 4d).In the meantime, the IBU release profiles were significantly similar (ƒ2 values ≥ 50) for all stored HME-3 pellets compared to fresh ones (Table 2 and Fig. 3c).Therefore, only HME-3 preparation succeeded to produce a stable preparation under both storage conditions, with high IBU loading (60% w/w).Moreover, it was found that the DSC data gave a mirror image for IBU release profiles for fresh and stored samples.

XRD
Figure 5 shows the diffractograms of drug, carrier, and fresh and stored IBU-HME pellets.IBU showed several characteristic sharp and intense peaks at different angles of diffraction (2θ) which proved the crystalline nature of the drug (Fig. 5a).The major distinctive peaks of IBU, PEG 6000, GMS, and PEG 6000/GMS were at 16.5591, 19.0031, 19.4191, and 19.0551 (2θ), respectively (Table 4 and Fig. 5).The positions and counts of these major peaks of all samples and their intensities are listed in Table 4.The fresh HME-1, HME-2, and HME-3 samples showed that some IBU characteristic peaks were absent and others appeared with markedly reduced intensity (Fig. 5).
The positions of peaks of individual components (IBU, PEG 6000, and GMS) (Fig. 5a) compared to fresh and stored IBU-HME samples were not changed considerably (Fig. 5b-d).However, their impact on product performance might have its meaning on the solid-state changes (crystallineamorphous ratios and polymorphism) of all components proposed (Table 4).On the other hand, there were differences in peak counts (peak intensity) recorded after storage.
For HME-1 samples, the counts (peak intensity) of IBU major peak were highly decreased after storage (Table 4) from 1476.4832 (at 2θ of 16.6029) to 287.6423 (at 2θ of 16.6198) for 6-month stored samples under stress condition and 704.9457 (at 2θ of 16.6072) for 12-month bench stored samples (Fig. 5b).This could be the reason of the significantly different IBU release rates of stored samples compared to fresh samples (ƒ2 values ˂ 50) (Figs.3a and 5b and Tables 1 and 3).
For HME-2 samples, after 12-month bench-storage, a little change in IBU major peak counts (peak intensity) was recorded, from 1095.2613 (at 2θ of 16.5170) to 864.0314 (at 2θ of 16.6290) (Fig. 5c).This might be the reason of significantly similar release profiles (ƒ2 values ≥ 50) compared to fresh samples, while after 6-month storage under stress conditions, a pronounced decrease in IBU major peak counts was observed (716.8441 at 2θ of 16.6610) which could be the reason for the significantly different release profiles (ƒ2 values ˂ 50, Figs.3b and 5c and Tables 1 and 3).
It was found that the HME-3 pellets gave higher counts of IBU major peak (903.9400 and 1214.3541 after 6-month and 12-month storage, respectively), compared to all other IBU stored pellets (HME-1 and HME-2, Table 4).Moreover, HME-3 pellets gave similar release rates of IBU after storage at different conditions (ƒ2 values ≥ 50, Table 2).It was interesting to notice that as long as the major IBU peak (at ≈ 16.5591) gave counts that exceeded the value of 800 (Table 4), a stable formula after storage was produced, as shown by the similar release profiles of fresh and stored samples.
This current stability study might throw light on the different results obtained under stress conditions (40 °C and relative humidity of 75%) and long-term bench storage (e.g., 1 year at room temperature 25 °C).Also, differences reported between the two stability protocols pointed out an important question.A previous study [26] reported the instability of diltiazem hydrochloride SR poly(ethylene oxide) matrix tablets under stress conditions, while the same formula was stable for several years at room temperature [31].The program of shelf-life extension was raised up in 1986 by US-FDA [32] to re-evaluate the expiration dates of drug products.Therefore, acceptable long-term stability data must be documented [12,[33][34][35].

Conclusion
Stable formula containing 60% (w/w) IBU prepared by hotmelt extrusion with GMS was successfully produced, for the first time, without any other excipients.The stability study of IBU pellets based on PEG 6000, GMS, and their combination prepared via HME was not reported until now.This study highlights the use of PEG 6000, GMS, and their combination, as an extrudable carrier, in HME technique.Moreover, IBU was an excellent candidate for preparation of SD by HME technique due to its known plasticizing effect.GMS-based HME was found to be superior in sustaining the IBU release rate with excellent stability along with allowing incredibly high drug loading (60% w/w) in an acceptable physical characteristic.The recorded DSC and XRD data were valuable tools to understand the effect of different polymers on the system performance for both fresh and stored samples.HME-3 was a promising stable formula, which adds a value to this advanced technique.This formula deserves to be tested in vivo on healthy human volunteers.

Fig. 1 Fig. 2
Fig. 1 Release profiles of fresh IBU pellets prepared by HME

Table 2
Similarity factor (f 2 ) between the release rate data of IBU from different stored samples against fresh ones * 6 months (40 °C/75% RH) ** 12 months at room temperature

Table 4
XRD data of fresh and stored samples *IBU/PEG 6000