Physicochemical Characterization and In-Vitro Dissolution Behavior of Artemether and Lumefantrine: Hydroxypropyl-Β-Cyclodextrin Inclusion Complex

Sanjesh Rathi*, Dhaval Patel, Shrenik Shah

Department of Pharmaceutics, Saraswati Institute of Pharmaceutical Sciences, Gujarat, India-382355

*Corresponding Author E-mail: rathi.sanjesh@gmail.com

ABSTRACT:

The objectives of this research were to prepare and characterize inclusion complexes of Artemether and Lumefantrine with hydroxypropyl-β-cyclodextrin (HP-b-CD) and to study the effect of complexation on the dissolution rate of Artemether and Lumefantrine, water-insoluble drugs. The stoichiometric ratio determined by phase solubility analysis for inclusion complexes of Artemether and Lumefantrine with HP-b-CD was 1:1.5. Binary complexes were prepared by different methods such as kneading and physical mixing method and were further characterized using DSC and FT-IR. These studies indicated that a complex prepared by kneading method had successful inclusion of the Artemether and Lumefantrine molecule into the cyclodextrin cavity. The mean dissolution time for Artemether and Lumefantrine decreased significantly after preparing complexes using kneading method compare to its physical mixture and plain form. The similarity factor indicated a significant difference between the release profiles of Artemether and Lumefantrine from complexes and to its physical mixture and plain form. Hard gelatin capsules containing single dose of Artemether and Lumefantrine (20-120mg) with cyclodextrins had significant improvement in the release profile of both drugs as compared to market formulation containing multiple dose (80-480 mg tablet) of Artemether and Lumefantrine without cyclodextrin.

KEYWORDS: Artemether, Lumefantrine, hydroxypropyl-β-cyclodextrin, inclusion complexation, DSC and FT-IR.

1. INTRODUCTION:

Malaria is caused by protozoan parasite plasmodium and transmitted by mosquitoes. There were nearly millions of malarial deaths in every year, of which 85% were children under age of five. In the face of growing drug resistance, the WHO has issued recommendations strongly encouraging the use of combination therapies of combat uncomplicated malaria. Amongst the most effective treatments are those which combine artemisinin derivatives with a longer acting component (Chadha, R et al., 2010, Natarajan, S et al., 2012, Ali S, et al, 2010).

Artemether (ART), a rapidly acting antimalarial drug is potent, efficient against acute and severe P. falciparum malaria. WHO has listed it as an essential drug for the treatment of severe multiple resistant malaria. Both artemether and lumefantrine act as blood schizontocides. Artemether is a sesquiterpene lactone derived from artemisinin to treat fever from ancient times. Artemether is absorbed rapidly with peak plasma concentrations reached about 2 h after dosing. Lumefantrine (LUM) is slowly absorbed with an estimated half life of 5 h. The maximum concentration is reached in approximately 10 h. ART and LUM are poorly water soluble and crystalline drugs so exhibit highly erratic and very low dissolution profile. Also, both the drugs are lipophilic in nature therefore not suitable for intravenous use. The therapeutic efficacy of ART and LUM are greatly hampered due to its high crystallinity and low aqueous solubility (Afosah, D, 2010, Patel R, 2010).

One of the approaches to overcome these problems is to use cyclodextrins (CDs) as drug carriers. They are known for their ability to molecularly encapsulate a wide variety of drugs into their hydrophobic cavity without the formation of any covalent bonds. The binding forces within these inclusion complexes may involve hydrophobic, van der Waals, hydrogen bonding, or dipole interactions (Martin Del, 2003, Rasheed, A, 2008). Hydroxypropyl-β-cyclodextrin (HP-β-CD), a chemical derivative of β-CD, is the most accepted representative of hydroxyalkylated derivatives as a hydrophilic drug carrier because of its amorphousness, high water solubility and solubilizing power, and low cost and toxicity (Raymond, R, 2006, Patel R, et al, 2008)

2. MATERIAL AND METHODS:

Artemether and Lumefantrine were generous gift from West-Coast Pharmaceutical work limited, Ahmedabad. The complexing agent HP-b-CD was purchased from Cadila Pharmaceuticals limited, Ahmedabad. Talc and Magnesium stearate were procured from Purvi enterprise Ltd., Ahmedabad. Methanol, Acetonitrile, Water were procured as HPLC grade from SD fine chemicals, Mumbai. All other chemicals and solvents used were of pharmaceutical and analytical grade. Double distilled water was used throughout the study for all the experimental procedures.

PHASE SOLUBILITY STUDY:

Phase solubility study of LUM and ART:

The stoichiometric ratio and quantitative expression of stability constant were determined by using phase solubility method (Higuchi.T, 1965). Excess quantities of the LUM and ART (10mg) were transferred to 10 ml of aqueous solution HP-b-CD in various molar concentrations (1.0-14.0mM/L for LUM and (2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0 and 16.0mM/L for ART) contained in 50ml glass beaker. These solutions were stirred on electromagnetic stirrer at constant temperature 37°C±0.1°C for 24 h and 400rpm. After 24 h, samples were filtered through a 0.22µm membrane filter. The filtrate was suitably diluted and analyzed for LUM and ART at 335 nm using spectrophotometer (U.V. visible spectrophotometer, Shimazdu-1601) and using HPLC method at 210nm using PDA detector respectively.

Preparation of Inclusion complexes:

Complexes of LUM and ART with HP-β-CD were prepared in the molar ratio of 1:0.5, 1:1, 1:1.25, 1:1.5 and 1:1, 1:1.25, 1:1.5 by different methods such as kneading and physical mixture. Abbreviations given in Table I and Table II are used to designate samples of LUM and ART prepared with HP-b-CD by different methods throughout this work (Yang B et al, 2009).

Table 1: Abbreviations used to designate different samples of LUM

Type of CDs

Molar Ratio

Drug: HP-b-CD

Method of preparation

Name of sample

HPb-CD

1:0.5

1:1

1.25

1:1.5

Physical mixture

PMH-0.5

PMH-1

PMH-1.25

PMH-1.5

HPb-CD

1:0.5

1:1

1.25

1:1.5

Kneading Method

KNH-0.5

KNH-1

KNH-1.25

KNH-1.5

Table 2: Abbreviations used to designate different samples of art

Type of CDs

Molar Ratio

Drug: HPb-CD

Method of preparation

Name of sample

HPb-CD

1:1

1.25

1:1.5

Physical mixture

PMH-1

PMH-1.25

PMH-1.5

HPb-CD

1:1

1.25

1:1.5

Kneading Method

KNH-1

KNH-1.25

KNH-1.5

3. RESULTS AND DISCUSSIONS:

Phase solubility analysis of LUM and ART:

The phase solubility curve of LUM and ART in the presence of HP-β-CD is shown in Figure 1 and 2. From this curve, it can be seen that the solubility of LUM and ART increases due to the formation of an inclusion complex between LUM/ART and HP-β-CD. An improving the aqueous solubility was observed into water when 8mM/L to 14 mM/L of HP-b-CD was used for LUM and 2mM/L to 16 mM/L of HP-b-CD for ART. Solubility of LUM and ART was increased by 5 fold at 14mM/L and 9 fold at 16 mM/L at 37°C concentration of HP-b-CD. Increased solubility may be due to improved dissolution of LUM and ART particles in water by HP-b-CD.

The stoichiometric ratio at which optimum complexation occurs was confirmed by phase solubility analysis. The phase solubility plots revealed an A_L type for HP-β-CD (Figure 4 & 5), which indicated that a 1:1.5 (LUM-HP-β-CD, ART- HP-β-CD) inclusion complex was formed in solution. The apparent stability constant (K_1:1.5) 115.4M^-1for the LUM: HP-β-CD complex and 1560.7 M^-1 for the ART: HP-β-CD complex was found from the solubility data. An indication of the process of transfer of LUM and ART from pure water to aqueous solution of HP-β-CD was obtained from the values of Gibbs free energy change. The values of Gibbs free energy obtained are shown in Table IV. ΔG_tr° values were all negative for HP-β-CD at various concentrations, indicating the spontaneous nature of LUM and ART solubilization, and decreased with an increase in its concentration, demonstrating that the reaction became more favorable as the concentration of HP-β-CD increased (Fule, R, 2012). The endothermic heats of solution further explain the increase in solubility with temperature.

Evaluation complexes:

Drug content in drug: HP-β-CD (1:1.5) complex:

The drug content of the LUM and ART were found out to be 95.69% and 96.79% respectively, which approximately corresponds to stoichiometric ratio of the complex KNH-1.5 and indicate chemical stability and content uniformity of LUM and ART in its complex form (Jagdale, S, et al 2011).

Table 3: Percentage drug content and saturation solubility of drug: HP-β-CD (1:1.5) complex.

Complexes	% Drug content		Solubility (µg/ml)		Fold increase in solubility
Complexes	ART	LUM	ART	LUM	ART	LUM
Artemether	-	-	101.12	-	-	-
Lumefantrine	-	-	-	24.10	-	-
Kneading Method	96. 79%	95. 69%	261. 66	179. 12	258. 76%	746. 33%

Saturation solubility study:

The saturation solubility data for drug and complexes are given in Table V. The kneading complex shows maximum saturation solubility of Artemether (261.66 μg/mL) and for Lumefantrine (179.12μg/mL).

Characterization of inclusion complexes using FTIR

The FT-IR spectra of HP-β-CD, ART, LUM, LUM+ART, and inclusion complex are shown in Figure 3. FTIR spectra of pure ART indicated the presence of characteristic peaks of O-H stretching [3393.6cm^-1], C-H stretching [2934.0cm^-1], and C-O-O-C bending vibrations [1157.3cm^-1]. C=O stretching at [1655cm^-1] and C-H bending at [1373.8 cm^-1]. FTIR spectra of pure LUM indicated the presence of characteristic peaks of O-H stretching [3393.8cm^-1], C-H stretching [2951.0-2854.9cm^-1], and C-O-O-C bending vibrations [1152.5 cm^-1]. C=O stretching at [1652.8cm^-1] and C-H bending at [1402.5cm^-1], C-Cl stretching [834.9-894.4cm^-1]. The FT-IR spectrum of the HP-b-CD is characterized by intense bands at 3300–3500cm⁻¹due to O–H stretching vibrations. The vibration of the –CH and CH₂ groups appears in the 2800–3000cm⁻¹ region. The presence or absence of characteristic peaks associated with specific structural groups of the drug molecule was noted. Any sign of interaction would be reflected by changes in the characteristic peaks of LUM and ART, depending on the extent of interaction.

The FT-IR spectra of KNH showed more similarity to FT-IR spectra HP-b-CD. FT-IR spectra of KNH also showed the absence of most characteristic peak of LUM and ART at 834-894cm^-1(C-Cl stretching) and at 1655cm^-1(C=O stretching). The FT-IR spectras of LUM+ART and inclusion complex showed no peaks other than those of HP-b-CD. These results indicate entrapment of LUM and ART inside the HP-b-CD cavity.

DSC:

Differential scanning calorimetry enables the quantitative detection of all processes in which energy is required or produced (i.e., endothermic or exothermic phase transformations). The thermograms of HP-β-CD, ART, LUM, LUM+ART, and inclusion complex are presented in Figure 4. The ART showed a melting peak at 87.28°C.

Figure 3: FTIR curves of HP-β-CD (A), Artemether (B), Lumefantrine(C) Artemether and Lumefantrine mixture (D), Inclusion Complex (E).

In-vitro dissolution study of complexes:

The improvement in dissolution of LUM and ART by kneading with HP-β-CD is presented in Figure 5 and 6. From in-vitro release study, it was found that complex prepared as 1.5 using kneading method showed an improvement in dissolution behavior (43.69% for LUM and 92.15% for ART as compared to plain LUM (10.98%) and ART (67.9%) at 3 h in phosphate buffer pH 6.8. Even PMs of LUM and ART with HP-β-CD not significantly enhanced dissolution of LUM and ART in dissolution medium compare to its pure form.

Figure 5: Comparison of Dissolution behaviour of pure Lumefantrine, PMH and KNH complex in different ratio.

Figure 6: Comparison of Dissolution behavior of pure Artemether, PMH and KNH complex in different ratio.

4. CONCLUSION:

The inclusion complex of ART and LUM with HP-β-CD has been prepared in 1:1.5 molar ratio using methods such as physical mixing and kneading method. There was a significant, linear increase in the aqueous solubility of ART and LUM with increasing concentration of HP-β-CD. FTIR and DSC studies indicated that kneading was the best method to prepare inclusion complexes because the complexes prepared using this method showed trapping of the ART and LUM inside the cyclodextrins cavity. The in-vitro drug release of complexes prepared by kneading method was significantly improved the dissolution compared to the physical mixture and pure drugs. These findings suggested that the drawback of poor dissolution profile of ART and LUM could be overcome by preparing its inclusion complexes with HP-β-CD. From this it was concluded that only in single dose formulation treated with HP-β-CD by kneading method was shown better in-vitro dissolution behavior compare to its conventional multi-dose formulation. The above studies conclude that the complexation of ART and LUM with HP-β-CD lends an ample credence in enhancing their dissolution behavior, which in turn has the potential to produce rapid onset of action and will also be needful in dose reduction.

5. AKNOWLEDGEMENT:

It is immense pleasure to express my deepest sense of gratitude and sincere thanks to my highly respected and esteemed, Principal, Dr Shrenik Shah, Director and HOD of PG Department Dr Ankit B Chaudhary for his unflinching support, guidance and pearls of wisdom to enable me to complete this paper.

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Received on 06.07.2019 Modified on 04.09.2019

Research J. Pharm. and Tech 2020; 13(3): 1137-1141.

DOI: 10.5958/0974-360X.2020.00209.7