Engineering of microcomplex of artemether and lumefantrine for effective drug treatment in malaria

Abstract

The objective of the present work was to engineer and characterize stable citric acid cross-linked microcomplex of the inclusion complexes of artemether with β-cyclodextrin and Kollidon VA 64^® with lumefantrine to release the drugs in controlled manner for effective combinational drug treatment in malaria. The microcomplex had a hydrodynamic diameter of 1047 ± 147 nm with surface charge of −19.7 ± 0.5 mV. The microcomplex showed high encapsulation efficiencies 85.6 ± 1.78% for artemether and 91.16 ± 2.21% for lumefantrine due to the lipophilic nature of drugs. In-vitro and in-vivo drug release studies showed the controlled release of artemether and lumefantrine for a period of 24 h.

Graphical Abstract

Keywords:

• Artemether
• lumefantrine
• microcomplex
• inclusion complex

Introduction

Malaria is an infectious disease caused by the parasitic protozoans belonging to genus Plasmodium. The symptoms of malaria are fever, fatigue, vomiting and headache. In serious cases, it causes seizures, coma and death. The drugs which have been used in the treatment of malaria are artemisinin, mefloquine, quinine, doxycycline, chloroquine, etc. (WHO 2010). Malarial infection in human beings is caused by five different species of Plasmodium: P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. The distribution of the pathogenic species of Plasmodium, show the majority of P. falciparum infection in Africa, while P. vivax triumphs in South America. Plasmodium falciparum and P. vivax are dominant in south-eastern Asia and western Pacific. The prevalence of P. malariae is very low but its co-infection with P. falciparum is sometimes faced in tropical African regions. Plasmodium ovale infection is chiefly observed in tropical Africa whereas P. knowlesi infection occurs in certain regions of South-East Asia (Autino et al. 2012). According to WHO list of essential medicines, the medicines used for the treatment of malaria are amodiaquine, artemether, lumefantrine, artesunate, mefloquine, chloroquine, doxycycline, primaquine, quinine, sulfadoxine, pyremethamine and proguanil (WHO list of essential medicines 2013). Resistance among the parasites has been developed to many antimalarial medicines like chloroquine resistant P. falciparum malaria has spread to many countries. This raises global concern for the long-term efficacy of artemisinin-based combination therapy (ACT) (Malaria Fact Sheet 2014). The artemisinin-based combination therapy includes treatment using artesunate-amodiaquine and artemether-lumefatrine. The problem regarding safety of ACT can be overcome by artemether-lumefantrine combination therapy. Artemether is chemically semisynthetic derivative of artemisinin. Mechanism of action shows artemether interferes with parasite transport proteins, produces disruption of mitochondrial function, inhibits and modulates host immune function. Lumefantrine interacts with the heme in the parasite’s food vacuole and results in the generation of toxic-free radicals causing death of the parasite.

The tablets for this combination are sold under the brand name Coartem^®, Riamet^® and Falcynate-LF^®. In January 2009, Novartis and Medicines for Malaria Venture (MMV) launched Coartem Dispersible^®, an artemisinin-based combination therapy developed specifically for children with malaria. The syrups available in the market are sold under the brand names Gnate-L, Lumether and Zemayl. In the area of novel drug delivery system (NDDS) the artemether-lumefantrine has been formulated as lipospheres, microparticles and nanostructured lipocarriers (Ali et al. 2016). Lumefantrine in the form of self-nanoemulsifying ionic complex, transferrin-modified-artemether lipid nanospheres are the drug delivery systems where the formulation has been developed for individual drugs. Artemether and lumefantrine both are practically insoluble in water and artemether acts by interacting with ferriprotoporphyrin IX (heme) and generates toxic radicals resulting in proteolysis of haemoglobin. Lumefantrine acts by inhibiting the formation of β-hematin thereby inhibiting nucleic acid and protein synthesis.

So the objective of the present work was to engineer and characterize stable citric acid cross-linked microcomplex of the inclusion complexes of artemether with β-cyclodextrin and Kollidon VA 64^® with lumefantrine to release the drugs in controlled manner for effective combinational drug treatment in malaria. Such microparticulate drug delivery system minimizes the side effects associated with the combination therapy along with reduced dose and dosing frequency for better patient compliance.

Materials and methods

Materials

Artemether and lumefantrine were purchased from S. Kant Healthcare Ltd., Vapi, India. β-Cyclodextrin was procured from SD Fine Chem Ltd., Mumbai, India. Kollidon VA 64^® was gifted from BASF, Germany. Citric acid was obtained from Fischer Corporation, USA. All other chemicals and reagents used were of analytical grade.

Methods

Preparation of inclusion complex of artemether and β-cyclodextrin

By kneading method, the mixture of artemether and β-cyclodextrin was taken in the mortar by molar ratio of 1:1 and triturated with small volume of water-methanol solution (1:2 v/v). The slurry so formed was kneaded for 10 min and then dried in an oven at 45°C. β-cyclodextrin acted as a complexing agent for artemether. The dried mass was then triturated in a mortar and passed through sieve number 120 (Shende et al. 2013). The formulation is abbreviated as F1.

Preparation of complex of lumefantrine

By hot melt technique, accurately weighed lumefantrine was complexed with Kollidon VA 64^® in the molar ratio of 1:3. Kollidon VA 64^® acted as a complex forming agent for lumefantrine (Fule et al. 2013). Lumefantrine and Kollidon VA 64^® were triturated in a mortar and then heated at 110 °C to obtain a homogeneous mass. The dried mass was passed through sieve number 120. The formulation is abbreviated as F2.

Preparation of microcomplex

The formulations F1 and F2 were mixed in a mortar by trituration using aqueous citric acid solution (0.1% w/v). This damp mass was then dried overnight at ambient temperature and then pulverized. Citric acid acted as a crosslinking agent for formulations F1 and F2. The formulation is abbreviated as F3.

Characterization

Particle size and zeta potential

The particle size and zeta potential of artemether, lumefantrine and formulations (F1, F2 and F3) were performed using Nano Zetasizer^® (Malvern, Worcestershire, UK) by preparing suitable dilutions with distilled water (Shende et al. 2013).

Scanning electron microscopy (SEM)

The shape and surface morphology of the F1, F2 and F3 were examined using SEM (Hitachi^®, Tokyo, Japan).

Solubility

Accurately weighed artemether, lumefantrine and formulations (F1, F2 and F3) were transferred to a conical flask containing 10 mL of distilled water and then placed on a rotary shaker (EXPO HI-TECH^®, Mumbai, India) for 24 h. The samples were filtered and analyzed using a UV-Vis spectrophotometer (Perkin Elmer^®, Waltham, MA) at 211 nm and 342 nm for artemether and lumefantrine, respectively.

In-vitro release study

In-vitro release studies of artemether and lumefantrine from formulations of F1, F2 and F3 were performed using a dialysis membrane (Himedia, Mumbai, India, Dialysis membrane 150^®) with 30 mL of phosphate buffer pH 6.8 (containing 1% Sodium Lauryl Sulphate, i.e., SLS) at a stirring rate 100 rpm and temperature 37 ± 0.5 °C. The samples were withdrawn at scheduled time points 1, 2, 4, 8 and 24 h. The aliquots withdrawn were replaced with fresh medium to maintain sink condition. The aliquots were then filtered and analyzed on UV-VIS spectrophotometer at 211 nm and 342 nm for artemether and lumefantrine, respectively.

Fourier transform infrared (FTIR) spectroscopy

FTIR analyses were performed on artemether, lumefantrine, and formulations (F1, F2 and F3) using a FTIR spectroscopy (Perkin Elmer^®, Waltham, MA) by KBr pellet method over a range of 4000–400 cm⁻¹.

Differential scanning calorimetry (DSC)

The drugs and formulations (F1, F2 and F3) were heated at a rate of 10 °C/min. Accurately weighed samples were crimped into aluminum pans and then used for the analyses of DSC (Mettler Toledo^®, Urdorf, Switzerland). An empty crimped aluminum pan was used as reference cell. The entire study was carried out in dry nitrogen atmosphere with a flow rate of 40 ml/min over a temperature range of 20 °C–340 °C.

Stability

The formulations (F1, F2 and F3) were subjected to accelerated stability conditions at 40 °C ± 2 °C and 75 ± 5% RH for a period of 1 month (Shende et al. 2012).

Based on the results of the solubility, entrapment efficiency, in-vitro drug release and stability studies of the formulations F1, F2 and F3, the formulation F3 was selected for the development of the dosage form.

Formulation of hard gelatin capsule for microcomplex

Accurately weighed amount of microcomplex of formulation F3 was transferred in hard gelatin capsule, sealed and used for evaluation. The formulation is abbreviated as F4.

Evaluation of hard gelatin capsule

Disintegration test

Disintegration time of the hard gelatin capsules was measured in phosphate buffer pH 6.8 at 37 °C using a disintegration tester (Campbell Electronics, Mumbai, India). This test was performed in triplicate.

Dissolution study

A dissolution system (Electrolab^®, Mumbai, India) with the USP apparatus II was used for the dissolution study of the formulation F4. The study was conducted in vessel at 50 rpm containing 900 ml phosphate buffer pH 6.8 (containing 1% SLS) at a temperature 37 °C as reported (Margret et al. 2014). The sinkers were used to prevent floating of the capsule. The samples were withdrawn at time intervals 1 h, 2 h, 4 h, 8 h and 24 h. The contents of drug were analyzed by UV-Vis spectrophotometer at 211 and 342 nm for artemether and lumefantrine, respectively.

High performance liquid chromatography (HPLC) method for estimation of artemether and lumefantrine

The estimation of artemether and lumefantrine was carried out using HPLC system (Perkin Elmer^®, EP 200 series, Waltham, MA) equipped with PDA detector (Perkin Elmer^®, Waltham, MA). The results were processed using Total Chrome Navigator software.

For the determination of artemether and lumefantrine in plasma, the plasma samples were collected from albino Wistar rats and the samples were prepared by extraction method. The separation was carried out on inertsil C18 column as stationary phase and acetonitrile phosphate buffer (0.1M) (70:30%v/v) containing trifluoroacetic acid (0.1%) as mobile phase. The drug peak was observed at 216 nm (Cesar and Pernetti 2009).

The stock solutions of artemether and lumefantrine were prepared by dissolving 10 mg of both the drugs in 100 ml methanol. The sample was prepared by dissolving the microcomplex in methanol. The samples were then injected into the HPLC system. The working standards were prepared by dilution in methanol. Twenty microliters of the working standard was added to 960 μl of plasma in order to get drug concentration of 96 μg/ml for lumefantrine and 16 μg/ml of artemether. The samples were stored in a freezer at −20 °C for further analysis (Patel et al. 2012).

Sample preparation

The plasma samples stored at subzero temperature were brought at room temperature before the analysis. The samples were then centrifuged after addition of acetonitrile for 5 min at 10,000 rpm × g. The supernatant was evaporated to dryness and reconstituted by mobile phase made of methanol and ammonium acetate. The sample was then injected into the column (Sandhya et al. 2015).

Experimental protocol

Albino Wistar rats (180–200 g) were housed under controlled environment (25 °C; relative humidity 60%) in a cage made of polypropylene. The rats were provided regular diet and purified water. The rats were kept in a fasting state during the study.

In- vivo drug release study

The rats were divided into two groups, three animals in each group. One group received formulation F4 at a dose of 1.14 mg/kg/d and 6.86 mg/kg/d for artemether and lumefantrine, respectively. The second group received plain artemether and lumefantrine at a dose of 1 mg/kg/day and 5 mg/kg/day respectively. The plasma samples were collected at time intervals of 1, 2, 4, 8 and 24 h after administration of formulation. The blood samples were centrifuged at 4000 rpm for 15 min. The plasma concentration of the artemether and lumefantrine was estimated by HPLC method at 335 nm (Olumide & Raji 2011).

Results and discussion

Particle size and zeta potential

The particle sizes of the drugs were 1143 ± 22.67 nm and 2551 ± 27.3 nm for artemether and lumefantrine, respectively. The particle size of the formulation (F1, F2 and F3) was decreased in comparison to the size of the individual drug molecule. Formation of the inclusion complexes by bonding of the drugs like artemether and lumefantrine with the carrier molecules β-cyclodextrin and Kollidon VA 64^® respectively resulted in decrease of the size of the microcomplex as shown in Table 1.

Table 1. Particle size and zeta potential.

Sample	Particle size (nm ± SD)	Zeta potential (mV ± SD)
Artemether	1143 ± 22.67	–21 ± 1.74
Lumefantrine	2551 ± 27.3	–12.7 ± 3.74
F1	694.4 ± 17.82	–10.4 ± 2.76
F2	1894 ± 6.4	–10.4 ± 1.22
F3	1047 ± 11.50	–19.7 ± 2.49

The size of the formulation F1 was found to be smaller because of the entrapment of artemether inside the β-cyclodextrin cavity. The reduction in the zeta potential is attributed to the cross-linking of the inclusion complexes of drugs with citric acid (Yang et al. 2009). The microcomplex showed negative zeta potential probably due to the presence of chloride (–Cl) and hydroxyl (–OH) groups.

SEM

Photomicrographs of SEM confirmed that the inclusion complexation of artemether and β-cyclodextrin led to the decrease in the particle size as shown in Figure 1. The hot-melt method of preparing inclusion complexes resulted in the decrease in the particle size of the complex. SEM micrograph of F2 formulation showed block like structures with rough edges. The SEM micrograph of F1 formulation demonstrated plate like structures due to interaction between polymer and drugs. The SEM micrograph of formulation F3 revealed almost circular particles. The cross-linking of the inclusion complexes of artemether and lumefantrine with citric acid showed surface charges on the microcomplex formulation.

Figure 1. SEM photographs of formulations (A) F1, (B) F2 and (C) F3.

Solubility

The solubility study of formulations F1, F2 and F3 is shown in Figure 2. The solubility of formulation (F1) was increased (1.25 folds) due to the complex formation with β-cyclodextrin whereas the solubility of formulation (F2) increased (6 folds) due to the hydrogen bonding between the drug and Kollidon VA 64^®. In case of formulation F3, the increase in solubility of artemether (1.4 folds) was due to the entrapment of artemether into the cavity of β-cyclodextrin. The drug undergoes a hydrogen bond formation with the methoxy (–OCH₃) groups of β-cyclodextrin and this results in a stable inclusion complex. Due to transformation of crystalline structure of lumefantrine particles to the amorphous form, it required lesser energy to solubilize and thus results in enhanced solubility of lumefantrine (6.4 folds) (Yang et al. 2009).

Figure 2. Solubility studies of drugs and formulations.

Encapsulation efficiency

The encapsulation efficiencies of artemether and lumefantrine were found to be 62.2 ± 1.78% and 58.48 ± 2.21% in the formulations F1 and F2, respectively. The encapsulation efficiencies of artemether and lumefantrine in formulation F3 were found to be 85.6 ± 1.78% and 91.16 ± 2.21%, respectively. The interaction between β-cyclodextrin and artemether occurs when the drug molecule approaches the hydrophobic cavity of β-cyclodextrin. This causes an upward chemical shift of the protons of artemether and downward chemical shift of carbons of β-cyclodextrin. Steric hindrance is also a deciding factor for the formation of the complex with cyclodextrin. The size of artemether is compatible with the size of the β-cyclodextrin cavity hence the formation of inclusion complex was possible. The complex of lumefantrine and Kollidon VA 64^® was prepared in a higher polymer concentration which balanced the energy released by H-bond formation due to their interactions. This resulted in a higher encapsulation for lumefantrine into the complex. It was also found that citric acid increased the solubility and the encapsulation of artemether and lumefantrine in formulation F3. This might be due to formation of salt and inclusion complex (Hanaor et al. 2012).

In-vitro drug release study

In-vitro drug release profile of formulation F1 showed a total release of 85.13 ± 1.45% for artemether and 94.64 ± 1.89% for lumefantrine over a period of 24 h. The release of artemether and lumefantrine from the microcomplex was found to be 87.24 ± 1.89% and 93.19 ± 1.45%, respectively, as shown in Figure 3. In the formulation F1, the drug was released slowly and in a controlled manner over a period of 24 h whereas in the formulation F2 only 2% of drug was released till 8 h and thereafter the drug was released at a controlled rate. In case of formulation F1, the controlled release of the drug was a result of the entrapment of artemether inside the cavity of β-cyclodextrin, forming a strong inclusion complex. In formulation F2, the hydrogen bond formed between Kollidon VA 64^® and lumefantrine provides sufficient strength to the complex and also facilitates the solubilisation and release of drug. The formulation F3 showed a controlled release for both the drugs as a result of the cross-linking of the inclusion complexes with citric acid. This result showed that citric acid is not involved in release of drugs.

Figure 3. In-vitro drug release profile of artemether and lumefantrine from formulations F1, F2 and F3.

Figure 4. FTIR Spectra of formulations (A) F1, (B) F2 and (C) F3.

Figure 5. DSC thermographs of formulations (A) F1, (B) F2 and (C) F3.

FTIR

The FTIR spectra of β-cyclodextrin (data not shown) and F1 formulation were found to be almost similar. This phenomenon occurred due to the coinciding absorption of the drug and the host carrier molecule. The groups that were masked by the cyclodextrin cavity are seen as peaks of β-cyclodextrin (1251.13 cm⁻¹, 1225.83 cm⁻¹, 1201.94 cm⁻¹, 1189.88 cm⁻¹ and 1177.30 cm⁻¹). The presence of a medium peak (H bonded –OH group) at 3371 cm⁻¹ showed that the bond formed was hydrogen bond. The FTIR spectrum of lumefantrine showed sharp peaks at 1673 cm⁻¹, 1736.85 cm⁻¹, 426 cm⁻¹, 1244.71 cm⁻¹ and 3471 cm⁻¹ as shown in Figure 4. The free –N and C = O groups forms hydrogen bonding with lumefantrine in the complex. Kollidon VA 64^® has two groups (=N and C = O) that form hydrogen bonds with lumefantrine in the complex. In the formulation F2, the intensity of the –OH bands decreased. This indicates interaction between the (–Cl and –OH) groups of lumefantrine and the (C = O) group of Kollidon VA 64^®. The peaks at 1085 cm⁻¹ was diminished in the F3 formulation which showed H-bond formation at the (C-O) bond of citric acid with the complexes.

DSC

The DSC thermograph of artemether showed a sharp endothermic peak at 86.77°C which relates to the melting point of the drug (Figure 5). No peak was observed in the DSC thermogram of F1 formulation which indicated the formation of the inclusion complex. Also the decomposition was shifted to a higher temperature which indicated the increased stability of the formed complex. The DSC thermograph of pure lumefantrine showed a sharp endothermic peak at 124.91°C which relates to the melting point of the drug. The peak was shortened in the DSC thermograph of the formulation F2 which indicates the formation of bond between lumefantrine and Kollidon VA 64^®. The DSC thermograph of formulation F3 showed no sharp peak which indicated the succesful formation of the microcomplex.

Stability study

The formulations F1, F2 and F3 showed no significant change in the particle size, % entrapment efficiency and in-vitro drug release studies for 1 month accelerated stability testing as shown in Table 2.

Table 2. Stability study of formulations F1, F2 and F3.

Time point	Formulation	Particle size (μm ± S.D.)	% Entrapment Efficiency	% In-vitro drug release
1 month	F1	692.4 ± 13.56	61.5 ± 1.68%	83.45 ± 1.32%
	F2	1888.1 ± 5.7	57.65 ± 2.17%	92.44 ± 1.75%
	F3	1039.2 ± 10.70	85.3 ± 1.88% for artemether and 90.87 ± 2.17% for lumefantrine	85.55 ± 1.32% for artemether and 90.26 ± 1.75% for lumefantrine

Evaluation of hard gelatin capsule

Disintegration test

The disintegration time of formulation F4 was found to be 11.8 ± 0.5 min.

Dissolution study

In-vitro drug release profile of the formulation F4 showed a total release of 85.44 ± 1.65% and 91.11 ± 2.31% for artemether and lumefantrine respectively for a period of 24 h as shown in Figure 6. The release profile for the drugs showed a controlled release pattern similar to the in-vitro drug release profile of the formulation F3. This might be due to the entrapment of artemether inside the cavity of β-cyclodextrin forming an inclusion complex and the H-bond formation between lumefantrine and Kollidon VA 64^®. The complex formation facilitates the enhanced drug release.

Figure 6. In-vitro drug release profile of artemether and lumefantrine from formulation F4.

In-vivo drug release study

In-vivo drug release study of formulation F4 showed a total release of 89.34 ± 1.67% and 94.17 ± 1.32% for artemether and lumefantrine, respectively, over a period of 24 h as shown in Figure 7. The release of artemether and lumefantrine for the pure drug was found to be 63.62% and 54.44%, respectively. The release profile of the formulation F4 was found to be better compared to the pure drug samples. The in-vitro drug release profile showed the microcomplex formulation to be a good candidate for effective treatment (Laxmi et al. 2014). The drugs artemether and lumefantrine are declared as class 4/3 according to the BCS system by WHO. (WHO Technical Report Series 937) The complexation of artemether with β-cyclodextrin and lumefantrine with Kollidon VA 64^® displayed the improvement of solubility of drugs in the in-vitro release study which supports the better in-vivo profile shown by the formulation F4.

Figure 7. In-vivo drug release profile of formulations.

Conclusions

The microcomplex formulation F3 was stable due to the adequate entrapment of drug in polymer structures. The increase in solubility of artemether in formulation F3 (1.4 folds) was due to the entrapment of artemether into the cavity of β-cyclodextrin. The drug undergoes a hydrogen bond formation with the methoxy (–OCH₃) groups of β-cyclodextrin and this results in a stable inclusion complex. Due to the transformation of crystalline structure of lumefantrine particles to the amorphous form, it required lesser energy to solubilize and thus results in enhanced solubility of lumefntrine (6.4 folds).The formulation F4 showed better in-vitro and in-vivo drug release profiles compared to pure drugs. Such stable microcomplex acts as an effective alternative treatment for drug resistant malaria by reducing the dosing frequency, side effects and for better patient compliance.

Disclosure statement

The authors have no conflict of interest.