Solid dispersions of artemether and polyethylene glycol 6000 (PEG6000) were prepared in ratio 12 : 88 (group-1). Self-emulsified solid dispersions of artemether were prepared by using polyethylene glycol 6000, Cremophor-A25, olive oil, Transcutol, and hydroxypropyl methylcellulose (HPMC) in ratio 12 : 75 : 5 : 4 : 2 : 2, respectively (group-2). In third group, only Cremophor-A25 was replaced with Poloxamer 188 compared to group-2. The solid dispersions and self-emulsified solid dispersions were prepared by physical and freeze dried methods, respectively. All samples were characterized by X-ray diffraction, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimeter, scanning electron microscopy, and solubility, dissolution, and stability studies. X-ray diffraction pattern revealed artemether complete crystalline, whereas physical mixture and freeze-dried mixture of all three groups showed reduced peak intensities. In attenuated total reflectance Fourier transform infrared spectroscopy spectra, C–H stretching vibrations of artemether were masked in all prepared samples, while C–H stretching vibrations were representative of polyethylene glycol 6000, Cremophor-A25, and Poloxamer 188. Differential scanning calorimetry showed decreased melting endotherm and increased enthalpy change (
Malaria is the infection caused by protozoan parasites transmitted with female Anopheles mosquitoes belonging to the genus,
According to a wide survey of malaria, ninety-nine countries out of 106 malaria endemic countries had ongoing malaria transmission. About 3.3 billion people in the world were endangered of malaria according to estimation. Malaria is the infectious disease as well as most prevalent disease in the world which in each year affects 515–600 million humans. About 40% of world population was vulnerable to malarial infection. In 2010, an estimated 655,000 persons died because of malaria; out of them, 86% were only the children with age of less than five years [
In order to advance the cure rates and clinical responses as well as to slow the development of resistance of malaria parasite, WHO has suggested that artemisinin derivatives should be present in antimalarial regimens. Artemether (ARTM) which is artemisinin derivative reduces malaria transmission and may also reduce the gametocyte carriage [
In the literature, various technological strategies are reported such as solid dispersions, self-emulsifying drug delivery systems (SEDDSs), micronizations, and complex formation with cyclodextrins [
Structure of the ARTM [
The aim of this study was to prepare self-emulsified solid dispersions (SESDs) of ARTM by using PEG6000, Poloxamer 188, Cremophor-A25, Transcutol, olive oil, and HPMC in order to improve solubility and dissolution behavior of ARTM.
Artemether (ARTM) (Alchem, China), acetonitrile HPLC grade (Merck, Germany), analytical grade methanol (Merck, Germany), Cremophor-A25 (chemically known as polyethylene glycol 1100 mono(hexadecyl/octadecyl) ether, YunGou Chemicals, China), polyethylene glycol 6000 (PEG 6000, Fluka, USA), Poloxamer 188 (chemically known as poly(ethylene glycol)-
Physical mixtures (PMs) were prepared using weighed amount of ARTM and PEG6000 in ratio 12 : 88 named group-1 and ARTM, PEG6000, Cremophor-A25, olive oil, Transcutol, and HPMC with ratio 12 : 75 : 5 : 4 : 2 : 2, respectively, named group-2. Similarly, ARTM, PEG6000, Poloxamer 188, olive oil, Transcutol, and HPMC with ratio 12 : 75 : 5 : 4 : 2 : 2, respectively, was named group-3. These physical mixtures were dried in oven at 37°C and then, after complete drying, homogenous mixture was made by using pestle and mortar with soft grinding. These mixtures were passed through a sieve of 180
Via freeze drying method, soluble mixtures of weighed amount of ARTM and PEG6000 in ratio 12 : 88 (Group-1) and ARTM, PEG6000, Cremophor-A25, olive oil, Transcutol, and HPMC with ratio 12 : 75 : 5 : 4 : 2 : 2, respectively (group-2) were prepared. Similarly ARTM, PEG6000, Poloxamer 188, olive oil, Transcutol and HPMC with ratio 12 : 75 : 5 : 4 : 2 : 2, respectively (group-3) were mixed to prepare soluble mixture. According to these corresponding groups, solutions were transferred to round bottom flasks and shaked on orbit shaker (BioTechnics, India) for mixing. After proper mixing, solvents were evaporated by using rotary evaporator (Prolific Instruments, India). Then, small amount of deionize water was added, shaken well, and frozen at temperature of −70°C to −80°C in the electronic deep-freezer (Dawlance, Pakistan). The frozen form is then freeze-dried using lyophilizer (Labconco, England) at temperature of −42°C using vacuum of 0.100 mBar for complete removal of solvents. After complete drying, the freeze-dried (FD) mixtures were transferred to pestle and mortar, softly grinded, and passed through a sieve (180
The X-ray powder diffraction (XRD) study of all samples was done by using apparatus named Siemens D-500. The measurements and conditions of XRD consisted of the targeting of CuK
By using potassium bromide (KBr) disc method (i.e., 0.5–1% of the sample in 200 mg KBr disc), ATR-FTIR spectra of SESDs of ARTM were obtained through Perkin Elmer spectrum 1. The scanning was at 400–4000 cm−1 and a resolution was then 1 cm−1. Instrument calibration was occasionally repeated during these operations.
Differential scanning calorimetric (DSC) analysis of physical and freeze-dried mixtures of ARTM and excipients was performed by using Q2000 DSC (TA instrument, USA). The samples were heated at a rate of 5°C/min from 25 to 250°C under a dry nitrogen gas purge. Tzero aluminum was used to calibrate the cell constant. All measurements were conducted in sealed nonhermetic aluminum pans. The typical sample weight was 5–10 mg.
In order to identify and confirm the nature as well as surface topography of all formulated samples of ARTM, scanning electron microscopy (SEM, Perkin Elmer, USA) was used. SEM analysis was also performed to study the morphologies of pure drug as well as different self-emulsifying agents. For scanning electron photographs, an accelerating voltage of 5 kV was utilized and the resultant micrographs were then examined at magnifications of ×1000, ×1500, and ×2500.
For solubility in equilibrium studies, 0.4 g of each group was weighed properly and then transferred into test tubes containing 10 mL of the deionized water and mixed by using vortex mixture for period of about 1 to 2 min at 1400 revolutions per minutes (RPM). The prepared samples were fixed on orbit shaker for mixing and shaken for a period of 7 days at about 150 RPM at a temperature of 37°C. After a period of 7 days, each sample was then centrifuged at about 6000 RPM for 20 min. Then, upper layer of about 5 mL was decanted carefully by using micropipette and was then further diluted with 20 mL of deionized water. They were then analyzed on HPLC at 215 nm ultraviolet (UV) wavelength.
Tablets were prepared employing direct compression method using single punch tablet machine. To make tablets, the homogenous mixture of preformulated grains and lactose (quantity sufficient for 500 mg tablet weight) was prepared followed by passing through a sieve of 180
The dissolution studies of all formulations of ARTM were done by utilizing USP dissolution apparatus II (Digitek, Lahore, Pakistan) with stirring speed of 100 RPM at 37°C. Fasted state simulated gastric fluid (FaSSGF) with pH of 1.6 with composition of sodium taurocholate 80
For pure ARTM, SDs, and SESDs of ARTM, the stability tests in Hank’s balanced salt solutions were carried out at 37°C that indicated the dissolution test temperature. The Hank’s balanced salt solution was formulated with 0.40 gL−1 KCl, 8.00 gL−1 NaCl, 0.06 gL−1 KH2PO4, 0.35 gL−1 NaHCO3, 0.19 gL−1 CaCl2·2H2O, 0.05 gL−1 Na2HPO4, 0.09 gL−1 MgSO4, and 1.00 gL−1 glucose and the pH was adjusted to 7.4 (with NaHCO3, 3.8 mM, pH 11.2, solution). For stability tests in Hank’s balanced salt solutions (pH 7.4), ARTM and its SESDs solution with concentration of 100
The supernatant solutions of each group of SESDs of ARTM were withdrawn and then filtered through the cellulose acetates filters of 0.22
The validation data shows that the used HPLC method follows linearity in the range of 0.078 to 2.5 mg, as evident from the value of
In all cases, analysis of the data was carried out by applying one-way ANOVA with a probability of
The XRD patterns of artemether (ARTM) showed very strong characteristic diffraction peaks at 2
X-ray diffraction patterns of ARTM (A), PEG6000 (B), physical mixture of group-1 (C), freeze-dried mixture of group-1 (D), physical mixture of group-2 (E), freeze-dried mixture of group-2 (F), physical mixture of group-3 (G), and freeze-dried mixture of group-3 (H).
Poloxamer 188 is crystalline in nature and gives three characteristic peaks, that is, at 19°, 22°, and 23° [
The X-ray diffraction analysis of physical mixture of group-2 showed characteristic diffraction peaks at 2
When Poloxamer 188 was incorporated in place of Cremophor-A25 in SESDs compared to XRD of physical mixture of group-3, SESDs showed diffraction peaks at 2
ATR-FTIR spectra of artemether (ARTM) indicated the presence of four characteristic peaks of C–H stretching vibrations at 2844.99 cm−1, 2873.61 cm−1, 2914.58 cm−1, and 2936.97 cm−1, C–O–O–C bending vibrations at 1121.62 cm−1, C–O–C stretching vibrations at 1023.89 cm−1 and 1277.83 cm−1, and C–H bending vibrations at 1451.05 cm−1 (Figure
ATR-FTIR spectra of pure ARTM (a), PEG6000 (b), physical mixture of group-1 (c), freeze-dried mixture of group-1 (d), Cremophor-A25 (e), physical mixture of group-2 (f), freeze-dried mixture of group-2 (g), Poloxamer 188 (h), physical mixture of group-3 (i), and freeze-dried mixture of group-3 (j).
The ATR-FTIR spectra of PEG6000 showed characteristic bands of C–H stretching vibrations at 2882 cm−1, O–H bending vibrations at 1341.02 cm−1 and 359.52 cm−1, C–O stretching vibrations at 1059.97 cm−1 and 1278.91 cm−1, and C–H bending vibrations at 1466.38 cm−1 (Figure
Physical and freeze-dried mixtures of group-1 showed characteristic bands of C–H stretching vibrations in functional group region at 2882.76 cm−1 and 2883.43 cm−1 which was single broader peak instead of four peaks of ARTM alone; in the fingerprint region, C–O–O–C bending vibrations of both physical and freeze-dried mixtures of group-1 were unaltered. C–O–C stretching vibrations of physical mixtures of group-1 were red shifted at 1033.31 cm−1 and 1278.83 cm−1, while its freeze-dried mixtures were also red shifted at 1060.12 cm−1 and 1279.05 cm−1, respectively. C–H bending vibrations of physical mixtures of group-1 were red shifted at 1456.79 cm−1 and 1465.91 cm−1, while its freeze-dried mixtures were red shifted at 1456.91 cm−1 and 1465.74 cm−1, respectively. ATR-FTIR spectra tell about presence and absence of bonding interaction among ARTM and excipients due to mixing, grinding, and freeze drying (Figures
ATR-FTIR spectra of Cremophor-A25 showed characteristic bands of C–H stretching vibrations at 2885.69 cm−1 and 2915.24 cm−1, O–H bending vibrations at 1341.58 cm−1 and 1359.54 cm−1, and C–O–C stretching vibrations at 1060.60 cm−1 and 1279.38 cm−1. In group-2 of SESDs of ARTM, physical and freeze-dried mixture showed characteristic bands of C–H stretching vibrations in the functional group region at 2883.29 cm−1 and 2884.06 cm−1, respectively, which indicated that C–H stretching vibrations of ARTM were masked as compared to pure ARTM and the C–H stretching vibrations of both physical and freeze-dried mixtures showed characteristics bands of Cremophor-A25. Similarly, in the fingerprint region, the C–O–O–C bending vibrations were unaltered which showed that there was no change in trioxane ring that indicated that our SESDs retained their antimalarial activity. The C–O–C stretching vibrations of physical mixtures of group-2 were red shifted at 1030.21 cm−1 and 1278.76 cm−1, while its freeze-dried mixtures were red shifted at 1060.25 cm−1 and 1279.06 cm−1; C–H bending vibrations of physical mixture of group-2 were red shifted at 1465.92 cm−1 and its freeze-dried mixture was red shifted at 1466.15 cm−1 (Figures
ATR-FTIR spectra of Poloxamer 188 showed characteristic bands of C–H stretching vibrations at 2882.68 cm−1, O–H bending vibrations at 1341.58 cm−1 and 1359.38 cm−1, and C–O–C stretching vibrations at 1060.33 cm−1 and 1279.21 cm−1. When Poloxamer 188 was substituted with Cremophor-A25 in group-3 of SESDs of ARTM, the peak intensities and frequency of transmittance were not changed significantly. Physical and freeze-dried mixtures of group-3 showed characteristic bands of C–H stretching vibrations at 2883.06 cm−1 and 2884.08 cm−1 which indicated that C–H stretching vibrations of ARTM were masked and the C–H stretching vibrations of both physical and freeze-dried mixtures showed characteristics of Poloxamer 188. There was no change in C–O–O–C bending vibrations in physical and freeze-dried mixtures of group-3. C–O–C stretching vibrations of physical mixtures of group-3 were also red shifted at 1060 cm−1 and 1278.90 cm−1, whereas its freeze-dried mixture was red shifted at 1060.25 cm−1 and 1279.04 cm−1, respectively. C–H bending vibrations of physical and freeze-dried mixtures of group-3 were red shifted at 1466.13 cm−1 and 1466.11 cm−1, respectively (Figures
The disruption in crystalline structure was similar to that of DHA [
The DSC thermogram of ARTM showed typical characteristics of a crystalline substance having one endothermic peak at 86.64°C while melting onset temperature at 84.86°C. An enthalpy change (
DSC thermogram of ARTM (A), physical mixture of group-1 (B), freeze-dried mixture of group-1 (C), physical mixture of group-2 (D), freeze-dried mixture of group-2 (E), physical mixture of group-3 (F), and freeze-dried mixture of group-3 (G).
Physical mixture of group-1 showed melting onset at 65.90°C, peak temperature at 66.55°C, and enthalpy change of 186.5 J/g, while its freeze-dried mixture showed decreased melting onset at 56.84°C, peak temperature at 61.90°C, and
Physical mixture of group-2 showed melting onset temperature at 61.84°C, peak temperature at 66.31°C, and
Scanning electron microscopic photographs of artemether (ARTM) alone showed typical crystalline blocks of ARTM, while in group-1 SDs of ARTM, SEM showed that these crystalline structures of ARTM were decreased in size enormously having no sharp edges in both physical and freeze-dried mixtures of group-1. Scanning electron micrographs of physical mixture of group-2 showed formation of flakes representing amorphous agglomerates with smooth surfaces, whereas its freeze-dried mixture showed glassy appearance in addition to size reduction and embedment. SEM of physical mixture of group-3 in which Cremophor-A25 was substituted with Poloxamer 188, showed flakes having no smooth surface, while its freeze-dried mixture showed modified irregular shaped glassy appearance (Figure
SEM of ARTM (a), physical mixture of group-1 (b), freeze-dried mixture of group-1 (c), physical mixture of group-2 (d), freeze-dried mixture of group-2 (e), physical mixture of group-3 (f), and freeze-dried mixture of group-3 (g).
In group-1 of the prepared samples of artemether (ARTM), the solubility of physical mixture (PM) was increased up to 9 times (2.74 mg/mL) and solubility of its freeze-dried (FD) mixture was improved up to 15 times (4.74 mg/mL) as compared to ARTM alone (0.30 mg/mL). While in group-2 of SESDs, the solubility of physical mixture was increased up to 94 times (28.38 mg/mL) and its freeze-dried mixture was improved up to 121 times (36.33 mg/mL). In group-3 of SESDs when Cremophor-A25 was replaced with Poloxamer 188, the solubility of physical mixture was up to 65 times (19.49 mg/mL), while solubility of its freeze-dried mixture was increased further up to 135 times (40.56 mg/mL). In all cases, the solubility was in the decreasing order of FD > PM > ARTM (Figure
Dissolution profiles of ARTM alone and its formulations.
All the physical and freeze-dried mixtures of all samples showed a substantial increase in equilibrium solubility. The increase in solubility was due to amorphous nature of prepared samples or inhibition of crystallization by polymers as obtained earlier [
The quality control parameters of all prepared tablets were in accordance with official requirements [
From dissolution data (Table
Kinetic analysis of dissolution data.
Formulations | Zero order model | First order model | Higuchi model | Korsmeyer-Peppas model | |||
---|---|---|---|---|---|---|---|
|
|
|
|
|
|
| |
Pure ARTM | 0.013 | 0.7857 | 0.000 | 0.7913 | 0.171 | 0.9569 | 0.549 |
Physical mixture of group-1 | 0.036 | 0.5417 | 0.000 | 0.5622 | 0.478 | 0.8383 | 0.459 |
Freeze-dried mixture of group-1 | 0.044 | 0.5961 | 0.000 | 0.6199 | 0.589 | 0.8535 | 0.483 |
Physical mixture of group-2 | 0.181 | 0.5622 | 0.002 | 0.6719 | 2.403 | 0.8322 | 0.473 |
Freeze-dried mixture of group-2 | 0.194 | 0.5651 | 0.003 | 0.6839 | 2.573 | 0.8336 | 0.475 |
Physical mixture of group-3 | 0.115 | 0.5002 | 0.001 | 0.5739 | 1.546 | 0.8346 | 0.437 |
Freeze-dried mixture of group-3 | 0.227 | 0.5121 | 0.003 | 0.6677 | 3.035 | 0.8215 | 0.450 |
The rate of dissolution was increased by using Cremophor-A25 as well as Poloxamer 188 in addition to PEG6000, olive oil, Transcutol, and HPMC. By comparing physical and freeze-dried mixtures of SESDs of ARTM, the freeze-dried mixtures showed enhanced dissolution as compared to physical mixtures and ARTM alone. This enhanced dissolution of SESDs of ARTM was due to less crystalline structure and their conversion into amorphous form [
The stability of artemether (ARTM) in Hank’s balanced salt solution of pH 7.4 was very poor and only 8% of ARTM was left at the end of 6 hours. Therefore, Hank’s balanced salt solution pH 7.4 was chosen as medium for the stability analysis of ARTM in the SDs and SESDs at 37°C, as used previously for dihydroartemisinin [
The changes of ARTM concentration percentage as a function of time in Hank’s balanced salt solution (pH 7.4) at 37°C for ARTM alone (A), physical mixture of group-1 (B), and freeze-dried mixture of group-1 (C).
The changes of ARTM concentration percentage as a function of time in Hank’s balanced salt solution (pH 7.4) at 37°C for ARTM alone (A), physical mixture of group-2 (B), and freeze-dried mixture of group-2 (C).
The changes of ARTM concentration percentage as a function of time in Hank’s balanced salt solution (pH 7.4) at 37°C for ARTM alone (A), physical mixture of group-3 (B), and freeze-dried mixture of group-3 (C).
The degradation rate constant (
The rank order of the
It can be concluded from our results that solubility and dissolution profile of artemether (ARTM) can be increased by preparing their self-emulsified solid dispersions (SESDs) with PEG6000, Poloxamer188, Cremophor-A25, olive oil, HPMC, and Transcutol by using freeze-dried method. The increase in solubility and dissolution profile of SESDs of ARTM agreed with data of XRD, FTIR, DSC, and SEM, which indicated that self-emulsified solid dispersions by freeze-dried method improved the physicochemical properties of ARTM.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are thankful to Higher Education Commission (HEC) of Pakistan for providing funding for research project due to which this work was possible and Hamaz Pharmaceutical Company, Multan, Pakistan, for providing facility of some instruments.