Formulation, characterization and anti-malarial activity of homolipid-based artemether microparticles.

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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

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Formulation, characterization and anti-malarial activity of homolipid-based artemether microparticles

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Chukwuma O. Agubata a, * , Ifeanyi T. Nzekwe b , Anthony A. Attama c , Christel C. Mueller-Goymann d, Godswill C. Onunkwo a a

Department of Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria, Nsukka, Enugu State, Nigeria Department of Pharmaceutics and Pharmaceutical Technology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria Department of Pharmaceutics, University of Nigeria, Nsukka, Enugu State, Nigeria d Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu Braunschweig, Braunschweig, Germany b c

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A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 September 2014 Received in revised form 14 November 2014 Accepted 17 November 2014 Available online xxx

The anti-malarial activity of artemether is dependent on its bioavailability. The purpose of the research is to improve the solubility, bioavailability and therapeutic efficacy of lipophilic artemether using homolipid-based microparticles. Irvingia fat was extracted from Irvingia gabonensis var. excelsa (Irvingia wombolu), and its lipid matrices (LM) with Phospholipon1 90G (P90G) were characterized by differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). Solid lipid microparticles were formulated, characterized, filled and compressed into capsules and tablets, respectively, and drug release studied. In vivo anti-plasmodial activity of artemether SLMs was evaluated in mice. The crystallinity of the phyto-lipid reduced in the presence of P90G, which was integrated into the irvingia fat crystal lattice. SLM dispersions with 3:1 irvingia fat/P90G composition showed higher diffusion and permeability through dialysis membrane while lower proportion of P90G (9:1 LM) favored increased dissolution rate of artemether from capsules (p < 0.05). Significant increase (p < 0.05) in % plasmodial growth inhibition and reduced parasitemia were observed in mice administered with the SLM dispersions compared with the controls. Therefore, SLMs prepared with composite mixtures of a homolipid and P90G could be used to improve the solubility, dissolution, permeability, bioavailability and anti-malarial efficacy of artemether. ã 2014 Published by Elsevier B.V.

Chemical compounds studied in this article: Artemether (PubChem CID: 456408) Microcrystalline cellulose (PubChem CID: 14055602) Croscarmellose sodium (PubChem CID: 6328154) Starch from maize (PubChem CID: 439341) Keywords: Homolipid Microparticles Anti-malarial activity

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1. Introduction

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The oral route has been a major route of drug delivery. However, the oral delivery of lipophilic drugs presents a major challenge because of their low aqueous solubility (Mandawgade et al., 2008). The limited dissolution rate arising from low solubility frequently results in the low bioavailability of orally administered drugs, and compounds with aqueous solubility lower than 100 mg/ml usually present dissolution limited absorption (Horter and Dressman, 2001). In such a case, higher doses would be required until the blood drug concentration reaches the therapeutic drug concentration range. This dose escalation may cause local toxicity in the gastro-intestinal tract upon oral administration, and such toxicity could lead to a reduction in patient compliance. The poor aqueous solubility of these drug moieties is associated with low bioavailability, high inter- and

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* Corresponding author. Tel.: +234 8062404493. E-mail address: [email protected] (C.O. Agubata).

intra-subject variability and a lack of dose proportionality (Komuru et al., 2001). Solid lipid microparticles (SLM) are micro-scale drug carriers possessing matrix made from fatty acids, glyceride, fatty alcohol and solid wax with high melting points (Long et al., 2006). The solid lipid matrix protects loaded labile substances against degradation and can possibly offer controlled drug release and drug targeting. It can also protect the GIT mucosa from the harsh irritations of some drugs. The suitability of lipid particles as prolonged release formulation for lipophilic drugs has been demonstrated (Eradel et al., 2009). Moreover, lipid matrices (LM) can be structured with phospholipids for improved functionality (Bekerman et al., 2004; Elgart et al., 2012). Artemether is a methyl ether derivative of artemisinin, which is a sesquiterpene endoperoxide lactone isolated from the Chinese anti-malarial plant, Artemisia annua. Artemether is anti-malarial. The mode of anti-malarial action of artemether involves iron catalyzed generation of a carbon-centered free radical, followed by the alkylation of malaria specific proteins (Kamchonwongpaisan and Meshnick, 1996). Its mechanism of action involves the hememediated decomposition of the endoperoxide bridge to produce

http://dx.doi.org/10.1016/j.ijpharm.2014.11.044 0378-5173/ ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Agubata, C.O., et al., Formulation, characterization and anti-malarial activity of homolipid-based artemether microparticles. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.044

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carbon-centered free radicals, which generates alkylated heme and proteins (Meshnick, 2002). In the presence of intra-parasitic iron, these drugs are converted into free radicals and other electrophilic intermediates which then alkylate specific malaria target proteins (Meshnick et al., 1996). It has been shown that both heme and also free intracellular reduced iron species can lead to the bioactivation of artemisinin, a prerequisite for the drug to become covalently bound to macromolecules throughout the whole parasite. Artemether, therefore covalently modifies multiple targets (Muller and Hyde, 2010). The peroxide structure of artemether is therefore necessary for its activity. Irvingia gabonensis var. excelsa or Irvingia wombolu is a tropical tree, now generally recognized to belong to Irvingiaceae family. Irvingia species are commonly known as African mango, bush mango or wild mango and the nut commonly called dika nut. The seed or nut contains fat which can be used for food, pharmaceutical and cosmetic applications (Ejiofor et al., 1987). Fat derived from the nut is generally regarded as safe.

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2. Materials and methods

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2.1. Materials

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Artemether (Fig. 1) was obtained from Hangzhou Dayang Chemical (China). Phospholipon1 90G (P90G) was obtained from Phospholipid GmbH (Cologne, Germany). Labrasol1 (caprylocaproyl macrogol-8-glyceride) was a gift from Gattefosse (St. Priest, France). Other materials used were Avicel1 – microcrystalline cellulose, Ac-Di-Sol1 – croscarmellose sodium (FMC biopolymer, USA) and maize starch (BDH, England). Simulated gastric fluid (SGF), without pepsin, was prepared and titrated to pH 1.2. Irvingia fat was prepared in Department of Pharmaceutical Technology and Industrial Pharmacy Laboratory, University of Nigeria, Nsukka.

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2.2. Extraction of fat from I. gabonensis var. excelsa (I. wombolu)

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Irvingia fat was extracted from nuts of I. gabonensis var. excelsa with petroleum ether (40–60 C) as solvent, using column extraction and concentrated with a rotary evaporator. The nuts were dried and milled to coarse form before extraction. Further, purification was performed by heating a 2%w/w suspension of activated charcoal and bentonite (1:9 ratio) in the lipid at 50 C for 1 h. Thereafter, the slurry was vacuum-filtered using Buchner funnel. The yield of extracted irvingia fat (dika fat) was calculated using Eq. (1);

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Yield% ¼

Weightof extracted Irvingia fat 100 Weight of dried Irvingia nut

(1)

2.3. Characterization of extracted irvingia fat

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2.3.1. Differential scanning calorimetry (DSC) of irvingia fat Differential scanning calorimetry (DSC) was performed on irvingia fat sample using a DSC instrument (NETZSCH DSC 204 F1, Germany) at a temperature range of 30–300 C and heating rate of 10 K/min on an aluminum pan with a pierced lid. The DSC of the sample was used to assess its thermal property and crystallinity. A thermogram was obtained, while the peak/melting point and enthalpy were recorded.

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2.3.2. Wide angle X-ray diffraction (WAXD) of irvingia fat Wide angle X-ray diffraction was performed on the irvingia fat sample using the X-ray generator (PW3040/60 X’Pert PRO, Fabr. DY2171, PANalytical, Netherlands) connected to an X-ray tube (copper anode, PW3373/00 DK147726Cu LFF) that delivered X-ray of wavelength l = 0.1542 nm at a high voltage of 40 kV and anode current of 25 mA. The WAXD measurements were taken with a goniometer (PW3050/60 MPD-system, PANalytical Netherlands). The interlayer spacing, d, was determined from the diffraction peaks according to Bragg’s equation (Eq. (2)).

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nl ¼ 2dsinu

86 87 88 89 90 91 92

94 95 96 97 98 99 100 101 102

(2)

2.4. Thermal properties, crystallinity, stability and diffraction of lipid matrices

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2.4.1. Differential scanning calorimetry of artemether-loaded lipid matrices The thermal, crystallinity and stability of artemether lipid matrices were evaluated by DSC. The DSC was performed on the drug-loaded lipid matrices (LM) using a differential scanning calorimeter (DSC Star Excellence System Autosampler MettlerToledo1, Switzerland). The thermal properties were determined between 5 and 150 C at a scan rate of 5 K/min, while the DSC was controlled by the software, Stare SW 10.000. The crystallinity index (C.I) was used to evaluate the degree of crystallinity of the lipid matrices. This was determined from the enthalpy of the transition according to Eq. (3):

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C :ILM % ¼

EnthalpyLM ðJ=gÞ 100 Enthalpyirvingia fatðJ=gÞ

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106 107 108 109 110 111 112 113 114 115 116

(3)

CH3 H O H3C O

EnthalpyLM is the enthalpy of the lipid matrices in J/g while Enthalpyirvingiafat is the enthalpy of irvingia fat in J/g. In this study, enthalpies of the artemether-loaded lipid matrices and irvingia fat were used as EnthalpyLM and Enthalpyirvingiafat, respectively.

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2.4.2. WAXD of lipid matrices (LM) WAXD was performed on samples of drug-free and artemether lipid matrices using the X-ray generator (PW3040/60 X’Pert PRO, Fabr. DY2171, PANalytical, Netherlands) connected to an X-ray tube (copper anode, PW3373/00 DK147726Cu LFF) that delivered X-ray of wavelength l = 0.1542 nm at a high voltage of 40 kV and anode current of 25 mA. The WAXD measurements were taken with a goniometer (PW3050/60 MPD-system, PANalytical Netherlands). Interlayer spacing of the crystal lattice of the matrices were determined from Eq. (2).

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O H

H O

CH3 O H3C Fig. 1. Chemical structure of Artemether.


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2.5. Formulation of artemether solid lipid microparticles (SLM)

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Solid lipid microparticles containing artemether, were prepared by hot homogenization. Artemether-loaded SLMs were prepared by dissolving appropriate amount of artemether (2%w/w) in molten irvingia fat (5%w/w) at 60 C in a bottle while aqueous Labrasol 1 surfactant solution (1.5%w/w) was maintained at the same temperature in a bottle on an electronic thermo-regulated water tank. The surfactant solution was then added to the molten irvingia fat with gentle stirring. Thereafter, the mixture was homogenized at 20,000 rpm for 5 min with a homogenizer (Ultra-Turrax1 T25 basic digital, Ika Staufen, Germany) in the water bath. The resulting SLM dispersion (AD) was allowed to cool and subsequently stored in a refrigerator (4 C). The procedure was repeated using irvingia fat/P90G lipid matrices at 9:1, 4:1 and 3:1 mass ratios (the P90G was pre-heated at 80 C) to form ADP9, ADP4 and ADP3, respectively.

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2.6.1. Particle size and pH The particle sizes of the SLMs were studied by viewing thin films of the dispersions with a light microscope and images captured with a Motic1 camera and analyzed using Motic1 images software (Motic, Xiamen, China). The polydispersity index was calculated as ratio of standard deviation to the mean particle size of the SLM. The pH of the dispersions was evaluated using a validated pH meter (HANNA Instruments, Padova, Italy). The electrode component was immersed into 50 ml quantities of the dispersions and the reading recorded. Each measurement was performed in triplicate and the average calculated. 2.6.2. Drug encapsulation efficiency, loading capacity and yield The content of artemether in the microparticles was determined using spectrophotometric method. The dispersions were centrifuged at 3000 rpm for 20 min and the supernatant assayed with a UV–vis spectrophotometer (Spectrumlab, UK) at 254 nm. A laboratory desktop centrifuge (model SM 800B, Uniscope Surgifriend Medicals, England) was used for centrifugation. In order to facilitate phase separation, 2 ml aliquots of each SLM dispersion were mixed with 2 ml of distilled water and the mixtures refrigerated at 4 C for 5 min. Each test sample was then centrifuged and supernatant was assayed accordingly. Artemether lack strongly absorbing chromophores, therefore, absorbs weakly in the low wavelength region (ultraviolet). This UV spectrophotometric method assayed the HCl decomposition product of artemether, which absorbs in the UV region with lmax at 254 nm. Thereafter, 1 ml quantity of supernatant of the dispersions was treated with 25 ml volume of 1 N HCl for derivatization and heated at 80 C for 30 min in a water bath. The treated samples were then diluted to 100 ml with distilled water prior to absorbance reading. The drug encapsulation efficiency (EE %) of the loaded microparticles was calculated using Eqs. (4) and (5): Real drug loading 100 Theoretical drug loading

W W free 100 EE% ¼ total W total 183 182 184

The drug loading capacity (DLC %) was calculated using Eq. (6):

2.6. Characterization of SLMs

EE% ¼

3

(4)

(5)

where Wtotal = weight of drug added to the system. Wfree = weight of free drug dissolved in medium/supernatant.

DCL% ¼

W total W free 100 W total W free þ W lipid

(6)

where Wlipid is the weight of lipid added to the system. The percentage yield of the solid lipid microparticles after the hot homogenization process was calculated using Eq. (7): Yield% ¼

actual weight of SLM 100 theoretical weight of SLM

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(7)

2.7. Drug release and diffusion studies of solid lipid microparticle dispersions

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A 1 ml volume of each solid lipid microparticle dispersion equivalent to 20 mg of artemether was enclosed in dialysis membrane tubing (MWCO 5000 – 8000) with hermetically sealed ends. The diffusion surface area was maintained by using membranes with same length (3 cm) and width (2.5 cm) for all the tests. The enclosed dispersions were suspended in 900 ml of simulated gastric fluid (SGF) in a beaker mounted on a magnetic stirrer assembly and the medium was maintained at 37 1 C and stirred at 50 rpm. Series of 5 ml volumes of the test solutions were withdrawn at 30 min interval for 6 h, derivatized with 5 ml of 1 N HCl at 80 1 C for 30 min, diluted to 20 ml with distilled water and assayed at 254 nm using the UV–vis spectrophotometer. The kinetics and mechanism of drug release and diffusion of the SLMs from the membrane were determined using different models. The cumulative amount of drug released from the formulated dispersions at different time intervals were fitted to the following plots: zero order kinetic model using cumulative % drug release versus time or ‘Q vs t’ (Narashimhan et al., 1999); first order kinetic model using log cumulative of % drug remaining versus time or ‘log (100 Q) vs t’ (Dash et al., 2010); Higuchi model using cumulative % drug release versus square root of time or ‘Q vs t1/20 (Higuchi, 1963; Bravo et al., 2002). The linearity of these plots was determined by their R2 values and the plot (model) with the highest linearity was taken as that which described the kinetics and mechanism of drug release.

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2.8. Lyophilization (freeze-drying) of SLM dispersions

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The dispersions were lyophilized using a lyophilizer/freezedrier (York Scientific Industries Pvt., Ltd., model YSI 280, India). The dispersions were converted to powder at a temperature of 40 C. The lyophilized powders were stored in a cool, dry place.

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2.9. Formulation of tablets and capsules

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2.9.1. Powder blends for tablets and capsules The SLM powder samples were converted to tablets using direct compression technique. Appropriate amount of each freeze dried SLM powder batch (corresponding to 20 mg artemether per tablet) was separately mixed with 2%w/w croscarmellose sodium (Ac-DiSol1) and enough microcrystalline cellulose (Avicel1) for 5 min in a planetary mixer to produce 300 mg tablets after compression of the powder blends. Batches containing Phospholipon1 90G were difficult to compress to tablets due to extrusion from the die cavity. Therefore, batches ADP9, ADP4, ADP3 (containing P90G) and PD (without P90G) were individually mixed with appropriate diluents for capsule filling. A flow optimized blend of starch and

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microcrystalline cellulose at a weight ratio of 2:3 (Starch-MCC 2/3) was used as diluent. The required quantity of artemether-loaded SLM (equivalent to 20 mg artemether per capsule) from each batch and enough diluents were mixed for 5 min in a planetary mixer to fill hard gelatin capsules (200 mg powder content per capsule).

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2.9.2. Flow properties of powder mixtures The bulk density (rb) of the powder blends was determined as the quotient of the weight to the bulk volume of each batch. The tapped density (rt) was determined as the quotient of weights to the volumes of the SLM powders mixtures after tapping the

Fig. 2. DSC thermograms of irvingia fat (a) and Phospholipon1 90G (b).


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measuring cylinder containing the samples 500 times from a height of 1 cm from the bottom of the cylinder and recording the tapped volume. Hausner’s quotient was derived as the ratio of the tapped density to the bulk density (Hausner, 1967), while % compressibility was calculated as 100 times the ratio of the difference between the tapped density and bulk density to the tapped density (Carr, 1965). The flow rate of the SLM samples was determined simultaneously with the angle of repose using the fixed height funnel method (Makenna and Macafferty, 1982). A plastic funnel with an orifice diameter of 1 cm fitted firmly by means of a support (clamp and retort stand) with its tip 7.5 cm above a clean flat horizontal surface was used. The flow rate was determined by recording the time taken for 20 g of each powder sample to completely flow out through a plastic funnel orifice and calculated as weight of powder per unit time (in g/s). Angle of repose was obtained from the mean height of the powder heap and diameter of the base of the powder heap. The tangent of the angle of repose was then calculated from the geometry of the powder heap as the ratio of the height of powder heap to the radius of its base. The flow data were then compared with standards to assess the quality of flow obtained. 2.9.3. Compression and capsule filling The tablet powder blends were compressed to 300 mg tablets using a tablet press (Manesty, UK). The tablets were produced at 50 kgf compression force using upper and lower punches of 10 mm diameter. Tablets were stored in clean, dry, hermetically sealed plastic containers. Powder mixtures, for capsules, were filled into transparent hard gelatin capsules (no. 1 capsules with fill volume of 0.48 ml) to produce capsules containing 200 mg powder content

each. The capsules were stored in clean, dry, hermetically sealed plastic containers.

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2.10. Evaluation of tablets and capsules

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2.10.1. Physical and mechanical properties The thickness and diameter of the tablets produced from formulations AD (artemether-loaded irvingia fat microparticles) were determined using vernier calipers. For weight uniformity evaluation, twenty units were randomly selected from each batch of tablets and capsules. The tablets and capsules were weighed individually using an analytical weighing balance (OHAUS1 Adventurer, China). The mean weight and deviations were calculated for each of the batches. The weight uniformity of capsules was evaluated using powder content only. Monsanto Hardness tester (Monsanto, USA) was used to determine the force (crushing strength) required to diametrically break each of 10 randomly selected tablets from AD formulation containing artemether and irvingia fat matrix. Mean crushing strength values (in kgf) were obtained. For friability studies, 10 tablets selected randomly were dedusted and weighed using the analytical weighing balance. The tablets were introduced into a friabilator (Roche, Switzerland) and rotated for 4 min at 25 rpm, after which the tablets were dedusted and re-weighed. The percentage friability was calculated using Eq. (8):

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Percentage friability ¼

Weight loss 100 Initial weight

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7000

6000

5000

4000

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1000

0 5

10

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279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297

298

9000

0

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(8)

10000

Impulse (Cts)

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20

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30

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40

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° 2-Theta Fig. 3. Wide angle X-ray diffractogram of irvingia fat.


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The tensile strength Ts of the tablets were calculated using Eq. (9): Ts ¼

2P pdt

(9)

where P is applied force or pressure (crushing strength), d is diameter and t is thickness of the tablets. The equation defined tensile strength as being proportional to the diameter and thickness of the tablets.

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Fig. 4. DSC thermograms of pure artemether (a) and artemether-loaded irvingia fat (b).


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Fig. 5. DSC thermograms of artemether-loaded 3:1 (a) and 4:1 (b) lipid matrices.


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extrapolation from standard calibration curve (Beer’s plot). The experiment was repeated using artemether capsules (powder content only).

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2.10.2. Absolute drug content (content of active ingredient) of tablets and capsules The content of active ingredient for the artemether SLM tablets and capsules was determined using a modification of a validated UV-spectrophotometric method (Shrivastava et al., 2008). Twenty tablets selected at random were accurately weighed together and the average calculated. The tablets were crushed to powder in a mortar, mixed and an amount equivalent to 20 mg artemether (1 tablet) was weighed out into a volumetric flask. The test powder was mixed with 50 ml of methanol and the solution was sonicated for 10 min in a sonicator (Fisher Ultrasonics, USA), after which the test solution was diluted to 100 ml mark with methanol. The solution was filtered through Whatmann filter paper no. 42. Thereafter, 1 ml volume of the filtrate was then mixed with 5 ml of 1 M methanolic HCl in a closed flask shaken for few seconds and the solution was heated on the water bath for 3 h at 60 2 C. The solution was allowed to cool at room temperature after heating and then diluted to 10 ml mark with methanol. The absorbance of the test solution was measured at 254 nm against blank using the UV–vis spectrophotometer (Spectrumlab 752s, UK) and the average drug content of the artemether tablets was obtained by

2.10.3. Disintegration times of tablets and capsules The disintegration times of 6 randomly selected tablets and capsules from each batch were evaluated in 500 ml simulated gastric fluid (SGF – without pepsin) at 37 1 C using disintegration apparatus (Erweka, Germany). The B.P method was followed. The time for each tablet and capsule to completely break down was recorded. The mean value and standard deviation for the batches were calculated.

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2.10.4. Dissolution rates of tablets and capsules The test for dissolution rates of the tablets and capsules were performed in a dissolution apparatus (Veego, Mumbai, India) using the USP paddle method. For each batch, 900 ml of SGF was transferred into the jar, maintained at 37 1 C and stirred at 50 rpm. Each tablet and capsule was separately submerged inside the medium and timer started. Series of 5 ml volumes of test solutions were withdrawn at 5 min time interval for 1 h, filtered and assayed using the UV–vis spectrophotometer (Spectrumlab 752 s, UK). Each withdrawn sample was replaced with an equal volume of the SGF maintained at 37 1 C. Prior to assay, to each 5 ml of artemether test solution was added 5 ml of 1 N HCl and the

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a

Phospholipon 90 G

4000 3000

Impulse (Cts)

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The crushing strength–friability ratio (CSFR) was calculated by dividing the crushing strength by the friability of each tablet formulation.

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b Irvingia fat + Phospholipon (4:1)

12000 10000

Impulse (Cts)

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8000 6000 4000 2000 0 0

5

10

15

20 25 ° 2-Theta

30

35

40

45

c Irvingia fat + Phospholipon (3:1)

10000 Impulse (Cts)

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8000 6000 4000 2000 0 0

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Fig. 6. Wide angle X-ray diffractograms of P90G (a), LM (4:1) (b) and LM (3:1) (c).


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2.10.5. Kinetics and mechanism of drug release from solid dosage forms The kinetics and mechanisms of drug release from the tablets and capsules were studied using the following models: zero order kinetic, first order kinetic, Higuchi, Korsmeyer and Hixson–Crowell cube root models. The initial three models have been described in Section 2.7 in drug released from solid lipid microparticles. Korsmeyer model was plotted using log cumulative % drug release versus log time or ‘log Q vs log t’ while Hixson–Crowell cube root model was plotted using cube root of % drug remaining in matrix

a

versus time or ‘(100 Q)1/3 vs t’. The Korsmeyer plot characterizes drug release from cylindrical shaped matrices (Korsmeyer et al., 1983). Hixson–Crowell cube root model describes the release from systems where there is a change in surface area and diameter of particles or tablets (Hixson and Crowell, 1931).

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2.11. Evaluation of in vivo anti-plasmodial activity of artemether SLM

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The animals used in this study were cared for and all treatment protocols were performed in accordance with guidelines on animal ethics in Nigeria and University of Nigeria, which complied with EU directive for animal experiment. The in vivo anti-plasmodial activity of the artemether SLM dispersions was studied using 4 day suppressive test protocol

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Artemether

1500000 Impulse (Cts)

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1000000 500000 0 0

5

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° 2-Theta

b Impulse (Cts)

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mixture heated in a water bath for 30 min at a temperature of 80 2 C and subsequently cooled to room temperature and diluted with distilled water to 20 ml.

Artemether + Irvingia fat

15000 10000 5000 0 0

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20

25

° 2-Theta

c Artemether + Irvingia fat + Phospholipon (4:1)

15000 Impulse (Cts)

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° 2-Theta

d Impulse (Cts)

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Artemether + Irvingia fat + Phospholipon (3:1)

8000 6000 4000 2000 0 0

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25

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° 2-Theta Fig. 7. Wide angle X-ray diffractograms of artemether (a), artemether + irvingia fat (b), artemether + LM (4:1) (c) and artemether + LM (3:1) (d).


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Fig. 8. Photomicrographs of artemether SLMs prepared with irvingia fat (AD), lipid matrices 9:1 (ADP9), 4:1 (ADP4) and 3:1 (ADP3).

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(Peters et al., 1993). Plasmodium berghei (ANKA strain) malaria parasite employed for the study was obtained from the National Institute of Medical Research, Yaba, Lagos, Nigeria. The parasite was hosted by donor mice. Thirty five Wistar albino mice obtained from the Faculty of Veterinary Medicine, University of Nigeria, Nsukka, were used for the study and were maintained on food and water ad libitum. The mice were infected by intraperitoneal inoculation with 0.2 ml of donor mouse blood suspension on the first day. The animals were weighed and divided into 7 groups of 5 mice per group. Group 1 was treated with 4 mg/kg of artemether, Groups 2–5 were treated with drug-equivalent doses of SLM dispersions AD, ADP9, ADP4 and ADP3 respectively. Group 6 received the unloaded irvingia fat SLM formulation containing no drug (D, placebo) while Group 7 was infected but not treated.

Treatment commenced 24 h post-infection and the formulations were administered daily (24 hourly) for the next 3 days. The animals were subsequently evaluated for red blood cell (RBC) count and parasitemia. The RBC count was performed using hemocytometer method by placing blood solution (prepared by mixing 0.1 ml of animals blood with 0.9 ml of Hayem’s solution) in a charged counting chamber where the cells in 5 counting grids of the central counting area were counted as n and a multiplication of n by 10,000 was expressed in cells/mm3. The level of parasitemia was determined from thin blood smears of the tail blood of mice, fixed with methanol and stained with Giemsa. The number of parasitized erythrocytes was counted thrice and average calculated. The anti-malarial activity was expressed as percentage growth inhibition and determined using Eq. (10) as follows

Table 1 Physicochemical and drug loading properties of SLMs. Formulation

Particle size (mm) S.D

Polydispersity

pH

Drug encapsulation efficiency (%) S.D

Loading capacity S.D

Yield (%)

AD ADP9 ADP4 ADP3

11.0 2.0 11.0 2.1 9.1 2.0 8.5 1.8

0.18 0.19 0.22 0.21

5.6 5.0 5.5 5.7

95.6 2.5 96.9 2.5 80.0 1.8 80.3 2.0

27.7 0.7 27.9 0.7 24.2 0.5 24.3 0.6

92 90 92 88


392 393 394 395 396 397 398 399 400 401 402 403 404 405

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11

60

50

% drug released

40

30

20

10

0 0

50

100

150

200

250

300

350

400

Time (min) AD

ADP9

ADP4

ADP3

ARM

Fig. 9. Percent drug release profile of membrane-enclosed artemether solid lipid nanoparticles.

Table 2 Flow properties of powder mixtures for compression and capsule filling. Formulation

rb

rt

Hausner’s quotient

Carr’s compressibility index (%)

Flow rate (g/s)

Angle of repose ( )

0.36 0.38 0.44 0.35 0.37

0.41 0.50 0.50 0.46 0.45

1.14 1.31 1.13 1.32 1.22

12.50 23.80 11.20 24.35 17.98

8.00 7.60 7.50 7.80 7.80

32.00 25.30 25.20 25.35 25.30

(g/cm3)

AD1a ADP9 ADP4 ADP3 AD2a a

(g/cm3)

Codes with superscript 1 and 2 represent same lipid matrices for tablets and capsules respectively.


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(Tona et al., 2004);

endothermic peak signified thermally induced phase transition of the fat and heat capacity was at its maximum at this point. The sharpness and symmetry of the peak showed a relatively high level of purity of the extracted irvingia fat. The thermograms of artemether-loaded irvingia fat and lipid matrices (Figs. 4 and 5) showed the melting peak of the carrier matrix but without the drug peak, indicating that the drug may have changed from crystalline to its amorphous structure. The melting point of artemether was 86.91 C. The loading of artemether into irvingia fat and structured lipid matrices also resulted in a reduction of enthalpy and crystallinity. The thermal behavior of a lipid matrix changes in the presence of other substances such that the melting temperature and changes in enthalpy depend on the nature of interaction between the constituents. The presence of P90G reduced the crystallinity of the irvingia fat and this was observed as lower enthalpy and crystallinity index of the lipid matrices. Lower enthalpy and crystallinity could possibly cause a retention of an entrapped drug over time (Attama and Muller-Goymann, 2006). This lower crystallinity may imply imperfect lattices with pockets of spaces that can accommodate drugs. The crystallinity indices of artemether-loaded irvingia fat/P90G lipid matrices 4:1 (ADP4) and

%growth inhibition 408 407

parasitemia of negative control parasitemia of test sample ¼ parasitemia of negative control 100 (10) 409 410

Data obtained were expressed as mean standard deviation.

411

3. Results and discussion

412

3.1. Differential scanning calorimetry (DSC) and wide angle X-ray diffraction of irvingia fat and lipid matrices

414 415 416 417

The thermogram of irvingia fat (Fig. 2) showed a peak at 43.4 C (melting point), while the wide angle X-ray diffraction of irvingia fat (Fig. 3) showed high intensity reflections at 2u = 20.9 and 23.2 with intensities of 8603 and 7446 Cts respectively. The DSC

100

90

80

70

60

% drug released

413

50

40

30

20

10

0 0

10

20

30

40

50

60

70

Time (min) AD tab

AD cap

ADP9 cap

ADP4 cap

ADP3 cap

ARM tab

Fig. 10. Drug release profile of artemether-loaded SLM-based tablets and capsules.


418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438

G Model


440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

3:1 (ADP3) were 87.7% and 90.3% respectively. This showed a reduction in crystallinity of 12.3% and 9.7% respectively from a hypothetical 100% (artemether-loaded irvingia fat). The X-ray diffractogram of irvingia fat is a physical/chemical fingerprint that can be used for its identification. It also showed the crystal structure of the fat. The interlayer spacing, d, of irvingia fat (from Bragg’s equation) was 3.38 nm. The X-ray diffraction patterns (Figs. 3, 6 and 7 ) and calculations from Bragg’s equation, showed that the interlayer spacing (d) increased from 3.38 nm (irvingia fat) to 4.22 nm (DP3) and 4.25 nm (DP4) in lipid matrices 3:1 and 4:1, respectively. Artemether-loaded lipid matrices also had an increased interlayer spacing of 4.39 nm (ADP3), 4.18 nm (ADP4) and 3.41 nm (AD) in artemether-loaded lipid matrices 3:1, 4:1 and 1:0, respectively. This implied an intercalation of the phospholipid within the lattice of irvingia fat, causing a widening of the crystal lattice. From the wide angle X-ray diffractograms, the disappearance of the numerous, insignificant impulses of P90G (Fig. 6) in lipid matrices DP3 (3:1) and DP4 (4:1) indicated an integration of

P90G into the crystal lattice of irvingia fat. The diffractogram of P90G expressed its very low crystallinity, with the presence of ‘almost’ amorphous halo which is characteristic of P90G. The lipid matrices had similar diffractograms with irvingia fat, however 3:1 LM showed increased impulse at 2u of 7.77 which was close to a peak observed in P90G. The WAXD also lacked the distinctive very high intensity/impulse peak of artemether (2u = 9.76 ) within irvingia fat and the lipid matrices (Fig. 7). This further demonstrated that the drug had changed from its crystalline form to the amorphous state and was now molecularly dispersed within the carrier matrix. Interestingly, the peak at 2u of 7.7 which was observed in irvingia fat, P90G and their LMs showed significant increase (p < 0.05) in impulse in the presence of artemether. However, these later peaks (e.g., 2u = 7.7 ) occur close to the small angle region and may indicate poorly layered structures. The WAXD results showed that the irvingia fat and Phospholipon1 90G remained chemically stable after mixture and the drug did not undergo degradation since no new peak nor pattern was observed.

120

100 y = 0.8549x + 1.8409 R² = 0.9501 (AD tab)

y = 0.3157x + 62.364 R² = 0.1441 (AD cap)

80

% drug released (Q)

439

13

y = 0.3923x + 71.25 R² = 0.2407 (ADP9 cap)

60

y = 0.7962x + 25.5 R² = 0.7966 ADP4 cap) 40

y = 0.8801x + 22.273 R² = 0.8822 (ADP3 cap)

y = 0.9808x + 44.75 R² = 0.5588 (ARM tab)

20

0 0

10

20

30

40

50

60

70

me (min) AD tab ADP4 cap Linear (AD tab) Linear (ADP4 cap)

AD cap ADP3 cap Linear (AD cap) Linear (ADP3 cap)

ADP9 cap ARM tab Linear (ADP9 cap) Linear (ARM tab)

Fig. 11. Zero order kinetic model for artemether SLM tablets and capsules.


457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474

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475

3.2. Characterization of SLM

476

The SLM sizes were in micro-scale (Fig. 8, Table 1). The particle sizes of artemether SLMs decreased as the P90G concentration increased. The P90G in these artemether formulations might have caused the reduction in particle size by delayed and decreased lipid particle precipitation. The solid lipid microparticle dispersions were slightly acidic (pH 5.0–5.7) as observed in Table 1. This may be the result of fatty acid components of the lipids. The SLMs had encapsulation efficiencies (EE) of 80–97% and loading capacities were in the range of 24–28%, while yields of 88–92% were achieved. The drug encapsulation and loading were relatively high. These are presented in Table 1. The high encapsulation of artemether may be due to relatively high solubility of the drug in the lipid matrix (solubility of artemether in irvingia fat is 470 mg/g). The physical and chemical properties

478 479 480 481 482 483 484 485 486 487 488 489 490

2.5

y = -0.0053x + 2.0085 R² = 0.9613 (AD tab) 2

y = -0.0028x + 1.4833 R² = 0.0654 (AD cap) y = -0.006x + 1.3186 R² = 0.2192 (ADP9 cap)

1.5

y = -0.0069x + 1.8908 R² = 0.8305 (ADP4 cap)

Log (100-Q)

477

of artemether, lipid(s), surfactant and water may have affected the encapsulation or loading of the drugs. Also, the method and conditions of preparation could determine the extent of encapsulation. The higher temperatures used in hot homogenization, results in lower particle sizes due to the decreased viscosity of the inner lipid phase (Lander et al., 2000). Furthermore, irvingia fat and the structured lipid matrices contained fatty acids with different chain lengths which may have formed less perfect crystals that offered more spaces to ‘accommodate’ the drugs. The higher presence of P90G in the matrices of ADP4 (20%w/w of lipid) and ADP3 (25%w/w of lipid) may have reduced the saturation solubility of artemether in the lipid, thereby causing partial expulsion of the drug during the cooling and solidification phase of SLM production. This could have caused the reduction in drug encapsulation efficiency of these SLMs. The high SLM yield showed that the production process was cost-effective.

y = -0.0076x + 1.9149 R² = 0.9049 (ADP3 cap) y = -0.0151x + 1.7341 R² = 0.7887 (ARM tab)

1

0.5

0

0

10

20

30

40

50

60

70

AD tab

me (min) AD cap

ADP9 cap

ADP4 cap

ADP3 cap

ARM tab

Linear (AD tab)

Linear (AD cap)

Linear (ADP9 cap)

Linear (ADP4 cap)

Linear (ADP3 cap)

Linear (ARM tab)

Fig. 12. First order kinetic model for artemether SLM tablets and capsules.


491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507

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3.3. Drug release and diffusion of artemether solid lipid microparticles

509

The % release of artemether from the SLM dispersions is shown in Fig. 9. The initial release of artemether observed in all the formulations may be caused by un-encapsulated drugs and a little burst release of artemether at the periphery of the SLM. Artemether-loaded SLM prepared with 3:1 irvingia fat/P90G (ADP3) had the highest % drug release. This could be attributed to particle size, since ADP3 had significantly lowest particle size than other formulations (p < 0.05). Earlier works had highlighted that lower particle sizes increased drug release via increased surface area (Akiyama et al., 1993; Savolainen et al., 2002). Also, higher concentration of P90G may have facilitated increased adhesion of the SLMs to the wall of the dialysis membrane tubing,

511 512 513 514 515 516 517 518 519 520

causing improved drug permeation. Extrapolation from the graph showed that the T30 of ADP3, ARM (pure artemether), ADP4, ADP9 and AD were 68.57 min, 71.25 min, 105 min, 120 min and 210 min respectively. Therefore, the extent and rate of drug release from the microparticles could be ranked as follows: ADP3 > ARM > ADP4 > ADP9 > AD. Moreover, the low melting point of the SLMs (39–42 C) which was close to body temperature (37 C) might have facilitated release of embedded drug. The release of artemether from the dispersions did not fit into any of the kinetic models. The plots had low R2 values. This behavior of the artemether SLM dispersions could be attributed to the complexity of the release, diffusion and permeation process, whereas the theory of these kinetic models was based on simple defined release mechanisms and sometimes changing geometrics

120

y = 8.9271x - 19.03 R² = 0.9719 (AD tab)

100

y = 4.3676x + 48.821 R² = 0.2588 (AD cap)

80 y = 4.8598x + 57.513 R² = 0.3465 (ADP9 cap) % drug released (Q)

510

15

y = 8.4845x + 5.1324 R² = 0.8488 (ADP4 cap)

60

y = 9.3553x - 0.1134 R² = 0.9353 (ADP3 cap) 40

y = 11.31x + 14.983 R² = 0.6972 (ARM tab)

20

0 0

2

4

6

8

10

t1/2 (min1/2 ) AD tab ADP4 cap Linear (AD tab) Linear (ADP4 cap)



Fig. 13. Higuchi model for artemether SLM tablets and capsules.


521 522 523 524 525 526 527 528 529 530 531 532 533 534

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IJP 14483 1–21 16 535


538

and structure of solid matrices. Consequently, the release of artemether from the microparticles and through the membrane could not be satisfactorily explained using the three kinetic models.

539

3.4. Characterization of tablets and capsules

540

The flow properties of powder mix for capsules and tablets are presented in Table 2. The flow properties of the powder blends were generally good since their Hausner’s quotient values were around 1.2, angles of repose values were close to 25 , flow rate showed less resistance to flow and the Carr’s compressibility indices were mostly below 18%. The diameter, thickness, crushing strength, friability, tensile strength (Ts) and crushing strength–friability ratio of the tablets are 10.50 mm, 4.00 mm, 4.00 kgf, 0.68%, 0.06 kgf/mm2, 5.88 respectively.

537

541 542 543 544 545 546 547 548 549

3.5. Disintegration times of tablets and capsules

550

Capsules with irvingia fat and lipid matrices at 1:0, 9:1, 4:1 and 3:1 ratios disintegrated in 9.2 0.3, 8.0 0.3, 5.1 0.2 and 4.0 0.2 min, respectively. Capsules with lipid matrices containing no or less P90G had higher disintegration time. This occurred because P90G is an amphiphilic substance and would therefore allow more passage of water (causing softening) through powder mixtures where it is present. In fact, it can be concluded that the P90G created a valuable water ‘softened’ channel within the lipid matrix. P90G is a phospholipid and contains a phosphorylated diglyceride (hydrophobic tail) and a choline head group (hydrophilic). The hydrophilic component must have accepted enough water to form a soft channel within the fatty powder mixture of the capsules. This hydro-channel created by P90G might have reduced the disintegration time of the corresponding capsules in a concentration related pattern, facilitating the

551

2.5

y = 1.1397x - 0.2553 R² = 0.9602 (AD tab) 2 y = 0.2551x + 1.4855 R² = 0.4646 (AD cap)

y = 0.22x + 1.6023 R² = 0.4924 (ADP9 cap) 1.5 y = 0.488x + 0.99 R² = 0.8525 (ADP4 cap)

Log Q

536

y = 0.516x + 0.9446 R² = 0.9572 (ADP3 cap) 1

y = 0.6771x + 0.8748 R² = 0.6951 (ARM tab)

0.5

0 0

0.5

1

1.5

2

Log t AD tab ADP4 cap Linear (AD tab) Linear (ADP4 cap)



Fig. 14. Korsmeyer model for artemether SLM tablets and capsules.


552 553 554 555 556 557 558 559 560 561 562 563 564 565

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disentanglement of the contents of the capsules. The average disintegration time of AD tablets was 40 0.5 min.

568

3.6. Drug release profile of artemether SLM tablets and capsules

569

Capsules containing solid lipid microparticles (SLMs) formulated with 9:1 irvingia fat/P90G lipid matrix (ADP9) showed the highest drug release (Fig. 10). The initial high release could be attributed to its capsule form rather than tablet form. AD tablets containing irvingia fat as the only lipid component had the lowest artemether release. The fluctuations in drug release profile observed in some formulations were absent in ADP3 because of its high P90G content which seemed to control the release. Also, only ADP9 capsules showed high drug release than artemether tablets (ARM tab). SLMs containing 9:1 lipid matrix (ADP9) relatively showed burst release. Studies have shown that some model drugs formulated as solid lipid nanoparticles by hot homogenization method exhibited significant burst release (Zur Muhlen and Mehnert, 1998).

571 572 573 574 575 576 577 578 579 580 581 582 583

The profile obtained could be explained as partitioning effects of the drug between melted lipid phase and the aqueous surfactant phase during microparticle production. During solid lipid microparticle production by hot homogenization technique, the drug might have partitioned from the liquid oil phase to the aqueous water phase. The amount of drug partitioning to the water phase increased with the solubility of the drug in water at high production temperature. During the cooling of the produced O/W microemulsion, the solubility of the drug in the water phase decreased continuously with decreasing temperature of the water phase. This implies a re-partitioning of the drug into the lipid phase. However as the temperature continued to drop, recrystallization of the lipid started from the core with the formation of solid lipid core. Consequently the drug became ‘trapped’ and concentrated at the still liquid outer shell of the SLM and/or on the surface of the particles. The drug in the outer shell and on the particle surface was released in the form of a burst while the drug incorporated into the particle core was released in a prolonged pattern. This is the core–shell model, with drug enriched shell. The low melting point of irvingia fat could have facilitated the release process.

5

4.5

y = -0.0166x + 4.6485 R² = 0.9594 (AD tab)

4

y = -0.0079x + 3.1984 R² = 0.0911 (AD cap)

3.5

y = -0.0136x + 2.8521 R² = 0.2319 (ADP9 cap)

3 (100-Q)1/3

570

17

y = -0.0194x + 4.2427 R² = 0.8226 (ADP4 cap)

2.5

2

y = -0.0214x + 4.3144 R² = 0.8991 (ADP3 cap)

1.5 y = -0.0338x + 3.7693 R² = 0.7124 (ARM tab) 1

0.5

0 0

10

20

30

40

50

60

70

me (min) AD tab ADP4 cap Linear (AD tab) Linear (ADP4 cap)



Fig. 15. Hixson–Crowell cube root model for artemether SLM tablets and capsules.


584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603

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IJP 14483 1–21 18


100

90

80

% growth inhibition

70

60

50

40

30

20

10

0 AD

ADP9

ADP4

ADP3

D(placebo)

ARM +ve

untreated

Fig. 16. Anti-malarial activity of artemether-loaded solid lipid microparticles.

604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626

Artemether SLM capsules containing 9:1 LM (ADP9) had T75 and T50 of 8.75 min and 6.15 min respectively. Whereas, artemether tablets prepared without any fat (ARM) released 75% and 50% of its artemether content in 16.43 min and 9.88 min respectively. Furthermore, T75 and T50 of AD SLM capsules without P90G were 16.25 min and 7.78 min respectively. However, ADP4 (4:1 LM) and ADP3 (3:1 LM) capsules could not release 75% of the drug but had T50 of 22.96 min and 24.17 min respectively. Artemether SLM tablets containing only irvingia fat as its fat matrix (AD tab) could not release 75% and 50% of its artemether content within the 1 h study period. Drug encapsulation efficiencies of ADP9 (96.9%) and AD (95.6%) SLM dispersions were significantly higher (p < 0.05) than those of ADP4 (80.0%) and ADP3 (80.3%). This implied that more of the lipophilic artemether was in amorphous state, dissolved in the lipid matrices of the former dispersions and this may have improved the dissolution of artemether from the corresponding capsules. ADP4 and ADP3 may have contained more free but crystalline poorly aqueous soluble drug. In summary, the drug release of the solid dosage forms could be ranked thus: ADP9 cap > ARM tab > AD cap > ADP4 cap > ADP3 cap > AD tab. The generally fast disintegration of the capsules implied that differences in disintegration times of the batches did not significantly affect the relativity in the dissolution rates.

3.7. Kinetics and mechanism of artemether release from lipid based solid dosage forms

627

Only AD tablets (with LM 1:0) showed R2 values > 0.9 for all the 5 models studied while ADP3 capsules (with LM 3:1) only had R2 values > 0.9 for 3 models (Figs. 11–15). Therefore only the drug released from these two batches could be described by the kinetic models. The kinetics of release of artemether from AD tablets fitted most with Higuchi model (R2 = 0.9719) while ADP3 cap was best described by Korsmeyer plot (R2 = 0.9572). The result showed that

629

Table 3 The % parasitemia, RBC count and % plasmodial growth inhibition after administration of SLM dispersions. Formulations

Parasitemia (%)

RBC count (106)

% growth inhibition

AD ADP9 ADP4 ADP3 D (placebo) Artemether (+ve) Untreated (ve)

2.5 3.1 3.3 2.4 14 8.3 19

5.72 5.37 5.33 5.41 3.12 5.6 2.9

86.84 1.6 83.68 1.5 82.63 1.4 87.37 1.5 26.32 2.0 56.32 1.8


628

630 631 632 633 634 635

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AD tablets released artemether from their matrices through a predominantly Higuchi kinetic model and fickian diffusion mechanism. The release of the artemether might have depended strongly on the porosity of the tablets. Higuchi model have been used to explain drug release from different geometrics and porous systems (Grassi and Grassi, 2005). Furthermore, the relatively low melting points of irvingia fat and AD SLM (42.06 C and 39.5 C respectively) could have aided the development of pores within the tablet matrices. In order to determine the mechanism of drug release from ADP3 cap, the first 60% of drug release was fitted into Korsmeyer–Peppas model. The value of n characterized the mechanism of release. The n values of 0.5407 (for first 60% release) and 0.5160 (for 100% release) are greater than 0.45 but less than 0.89, which signified that the mechanism of release from ADP3 cap might be non-fickian transport. 3.8. In vivo anti-plasmodial activity of artemether SLM All the artemether-loaded SLMs showed significantly (p < 0.05) greater inhibition of plasmodial growth than artemether alone and unloaded SLM (placebo). The anti-plasmodial activity of the formulations is presented as % growth inhibition in Table 3 and Fig. 16. Karbwang et al. (1997) have shown that the therapeutic potential of artemether is considerably hampered due to its low bioavailability (40%). This low bioavailability of artemether is because of its poor aqueous solubility. Literature indicates that bioavailability of artemether increases with the administration of fatty meals (Lefèvre and Thomsen, 1999). Therefore the

19

formulation of artemether in fatty microparticles could have improved its bioavailability. Also, the solid lipid microparticles could have protected encapsulated artemether against degradation. Moreover, the SLM might have enhanced the trans-mucosal permeation of artemether or lymphatic transport of the drug. The fatty acid contents of irvingia fat have been described as absorption enhancers. Oleic acid has been shown to decrease the phase transition temperatures of membrane lipids with resultant increase in rotational freedom or fluidity of these lipids (Golden et al., 1987). Lauric, palmitic and myristic acids have also been described to have penetration enhancing effects. Lauric acid, as its salt sodium laurate, has been shown to improve the oral absorption of insulin in rats (Aungst and Rogers, 1989). Since myristic acid is a component of membrane phospholipids, a more effective interaction might have occurred between irvingia fat (containing myristic acid) and the mice membrane. Furthermore, phospholipids including P90G, have been shown to disrupt membranes (Kakemi et al., 1970). In addition, Labrasol1, which is a non-ionic surfactant, might have improved the permeability of artemether. It has been observed that some tensioactive agents might influence tight junction permeability (Anderberg and Artursson, 1993). Reports have shown that Labrasol1 can cause fluidization of intestinal cell membranes (Rama Prasad et al., 2003; Koga et al., 2006). Futhermore, studies have also revealed opening of tight junctions by Labrasol1 (Chang and Shojaei, 2004; Sha et al., 2005). The irvingia fat based SLM also has the advantage of its melting point (39–41 C) being close to body temperature, thereby promoting SLM flexibility and fluidity in vivo.

Fig. 17. Red blood cells of infected mice: untreated (a) and treated with AD (b), ADP9 (c) and ADP4 (d).


663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691

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The high solubility of artemether in irvingia fat (470 mg/g) and entrapment within lipid matrices (irvingia fat and P90G) might have improved drug absorption via multiple pathways like trans-cellular, paracellular, partitioning, lipid digestive channels (lipolysis) and lymphatic transport. Furthermore, artemether SLM formulations might have provided a more prolonged release of the artemether which was effective, considering the short half life of artemether (1 h in humans). Interestingly, small % growth inhibition was observed in mice group receiving unloaded SLM (placebo) relative to the untreated group. This could be because of the nutritive value of the irvingia fat. Also, the membrane activity of the fatty acids in irvingia fat might have inhibited the parasite’s infestation of erythrocytes. Fatty acids have been reported to cause degeneration in intraerythrocytic stages of P. falciparum in vitro (Kumaratilake et al., 1992). It was reported that fatty acids and their methyl esters can kill P. falciparum by interfering with the fatty acid biosynthetic pathway of the parasite. The fatty acids could also activate the neutrophils and their effector cells thereby enhancing their antimalarial properties. In fact another study reported anti-plasmodial effect of a series of C18 fatty acids against the FCR3 strain of P. falciparum (Krugliak et al., 1995). Images of the red blood cells (erythrocytes) of the mice are presented in Figs. 17 and 18. Small granules observed in the plasma of infected blood could be neutrophiles and white blood cells

responding to infection. Small stacks of red blood cells were also observed in parasitized blood, indicating onset of infection. The artemether-loaded SLM dispersions (AD, ADP9, ADP4 and ADP3) showed higher percentage reduction in parasitemia than the artemether suspension (+ve control) and placebo. The SLM dispersions improved the solubility of artemether and might have provided a prolonged and controlled delivery of the drug. Moreover the onset of action is not really delayed since gradual melting of the solid lipid serves as a trigger. Chemical protection of artemether within the GIT of the animals was also achieved. These advantages might have reduced parasitemia, increased % growth inhibition and consequently improved the anti-malarial efficacy of the artemether-loaded solid lipid microparticle dispersions. Although the artemether suspension (+ve control) produced lower plasmodial suppression, the red blood cell counts of the treated mice were similar to that of those treated with the test formulations. This showed that artemether suspension (standard) prevented the rupture of the red blood cells even though more erythrocytes were parasitized, therefore, these intact but parasitized cells could have been counted in the RBC count. Formulations prepared with 3:1 lipid matrix (ADP3) and irvingia fat (AD, 1:0 lipid ratio) showed the highest anti-malarial activity with % growth inhibition of 87.37% and 86.84% respectively. ADP9 and ADP4 formulations also caused high plasmodial inhibition (83.68 and 82.63% respectively). All the artemether-loaded SLMs showed significantly greater plasmodial growth inhibition (p < 0.05) than artemether alone and placebo.

717

4. Conclusion

744

Solid lipid microparticles (SLMs) formulated with irvingia fat/Phospholipon1 90G at 3:1 ratio, improved the diffusion and permeability of artemether across dialysis membrane while, 9:1 lipid matrix showed increased dissolution rate of drug from capsules (p < 0.05). Artemether-loaded SLMs significantly increased (p < 0.05) plasmodial growth inhibition and reduced parasitemia in test mice population. Malaria is a common disease in the tropical region of Africa and the abundance of the plant, I. gabonensis var. excelsa (I. wombolu), would facilitate the commercial production of these lipid matrices. Therefore, SLMs formulated with fat derived from I. gabonensis var. excelsa (I. wombolu) and Phospholipon1 90G could be used to improve the solubility, dissolution, permeability, bioavailability and antimalarial efficacy of artemether.

745

Uncited reference Yoshikawa et al. (2003).

Fig. 18. Red blood cells of infected mice treated with ADP3 (e), ARM (f) and unloaded irvingia SLM (placebo) (g).

718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743

746 747 748 749 750 751 752 753 754 755 756 757 758

Q7 759 760

Acknowledgements

761

We appreciate the assistance of Manuela Handt and Ursula Jahn for the DSC and X-ray diffraction measurements.

762

References

764

Akiyama, Y., Yoshioka, M., Horibe, H., Hirai, S., Kitamori, N., Toguchi, H., 1993. Novel oral controlled-release microspheres using polyglycerol esters of fatty acids. J. Control. Release 26, 1–10. Anderberg, E.K., Artursson, P., 1993. Epithelial transport of drugs in cell culture 8. Effects of sodium dodecylsulfate on cell membrane and tight junction permeability in human intestinal epithelial (Caco-2) cells. J. Pharm. Sci. 82, 392–398. Attama, A.A., Muller-Goymann, C.C., 2006. A critical study of novel physically structured lipid matrices composed of a homolipid from Capra hircus and theobroma oil. Int. J. Pharm. 322, 67–78. Aungst, B.J., Rogers, N.J., 1989. Comparison of the effect of various transmucosal absorption promoters on buccal insulin delivery. Int. J. Pharm. 53, 227–235.

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G Model

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Pharmacokinetics of a novel sublingual spray formulation of the antimalarial drug artemether in healthy adults.

Enhanced antimalarial activity by a novel artemether-lumefantrine lipid emulsion for parenteral administration.

Formulation and characterization of hydrochlorothiazide solid lipid microparticles based on lipid matrices of Irvingia fat.

Impact of surfactant selection on the formulation and characterization of microparticles for pulmonary drug delivery.

Discs Containing Metformin Hydrochloride.

Formulation Development, Process Optimization, and In Vitro Characterization of Spray-Dried Lansoprazole Enteric Microparticles.

Formulation and characterization of atropine sulfate in albumin-chitosan microparticles for in vivo ocular drug delivery.

lumefantrine in a mouse model of Plasmodium berghei.

Formulation of nanotized curcumin and demonstration of its antimalarial efficacy.

Development of a specific monoclonal antibody-based ELISA to measure the artemether content of antimalarial drugs.

Reply to van der Pluijm et al., "Antimalarial Resistance Unlikely To Explain U.K. Artemether-Lumefantrine Failures".

Thermoporometry characterization of silica microparticles and nanowires.

Antimalarial Drug Artemether Inhibits Neuroinflammation in BV2 Microglia Through Nrf2-Dependent Mechanisms.

Characterization of Novel Antimalarial Compound ACT-451840: Preclinical Assessment of Activity and Dose-Efficacy Modeling.

Novel Pullulan-Eudragit® S100 blend microparticles for oral delivery of risedronate: formulation, in vitro evaluation and tableting of blend microparticles.

Efficacy of a novel sublingual spray formulation of artemether in African children with Plasmodium falciparum malaria.

Development of artemether-loaded nanostructured lipid carrier (NLC) formulation for topical application.

Tailoring a Pediatric Formulation of Artemether-Lumefantrine for Treatment of Plasmodium falciparum Malaria.

Mechanical characterization of microparticles by scattered ultrasound.

Transdermal microemulsions of Boswellia carterii Bird: formulation, characterization and in vivo evaluation of anti-inflammatory activity.

Antimalarial Activity of Small-Molecule Benzothiazole Hydrazones.

Antimalarial activity of Tanzanian medicinal plants.

Antimalarial activity of some natural peroxides.

Antimalarial activity of diethyldithiocarbamate. Potentiation by copper.