http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.949270
RESEARCH ARTICLE
Formulation, in vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Nyu Medical Center on 08/05/15 For personal use only.
John D. N. Ogbonna, Franklin C. Kenechukwu, Chinekwu S. Nwobi, Onochie S. Chibueze, and Anthony A. Attama Drug Delivery Research Unit, Department of Pharmaceutics, University of Nigeria, Nsukka, Nigeria
Abstract
Keywords
Context: Formulation, characterization, in vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles (SLMs). Objective: The objective of the study was to formulate and evaluate halofantrine-loaded SLMs. Materials and methods: Formulations of halofantrine-loaded SLMs were prepared by hot homogenization and thereafter lyophilized and characterized using particle size, pH stability, loading capacity (LC) and encapsulation efficiency (EE). In vitro release of halofantrine (Hf) from the optimized SLMs was performed in SIF and SGF. In vivo study using Peter’s Four day suppressive protocol in mice and the mice thereafter subjected to histological studies in kidney and liver. Results: Results obtained indicated that EE of 76.32% and 61.43% were obtained for the SLMs containing 7% and 3% of Hf respectively. The SLMs loaded with 3% of Hf had the highest yield of 73.33%. Time-dependent pH stability analysis showed little variations in pH ranging from 3.49 ± 0.04 to 4.03 ± 0.05. Discussion: The SLMs showed pH-dependent release profile; in SIF (43.5% of the drug for each of H2 and H3) compared with SGF (13 and 18% for H2 and H3 respectively) after 8 h. The optimized SLMs formulation and HalfanÕ produced a percentage reduction in parasitemia of 72.96% and 85.71% respectively. The histological studies revealed that the SLMs formulations have no harmful effects on the kidney and liver. Conclusion: SLMs formulations might be an alternative for patients with parasitemia as there were no harmful effects on vital organs of the mice.
Hematological parameters, halofantrine, histological studies, parasitemia, solid–lipid microparticles
Introduction 1
Malaria, a major public health problem in endemic regions affects about 500 million people annually and is a leading cause of mortality and morbidity in the developing World. The global impact of malaria parasite, the development of resistance especially to chloroquine by Plasmodium falciparum (P. falciparum) coupled with the emergence of new parasites all translate into incessant scientific challenge of drug discovery and formulation development. Over the past decades many researchers have focused on development of new antimalarials that are able to target important therapeutic processes. Treatment failures have been linked majorly to the development of resistance of the malaria parasite to standard antimalarial agents2,3. Effective treatment of malaria had been a great challenge to medicine and this has had a great impact on man’s health and economy (Sachs and Malaney, 2002). Halofantrine hydrochloride, a 9-phenanthrenemethanol is one of the most active antimalarial drugs against P. falciparum with a long half-life which lessens the need for repeated administration. The antimalarial activity is mediated partly by the parent drug and partly by its N-desbutyl metabolite4. It has a place in treatment of
Address for correspondence: John D. N. Ogbonna, Drug Delivery Research Unit, Department of Pharmaceutics, University of Nigeria, Nsukka 410001, Nigeria. Tel: +2348063674303. E-mail: johnixus@ yahoo.com, [email protected]
History Received 7 May 2014 Revised 7 July 2014 Accepted 22 July 2014 Published online 18 August 2014
multiresistant and chloroquine-resistant strains of P. falciparum when administered orally. However halofantrine has serious cardiotoxicity, prolonging the Q-T interval, which also causes the cardiac side effects of quinine and mefloquine. Owing to these adverse effects, including severe local irritation with strong erythema (probably related to the toxicity of the solvents used in preparation) as well as serious cardiac side effects (Q-T interval prolongation), which worsens in intravenous administration4 its oral use is a better formulation strategy, hence the need for novel formulation such as solid–lipid microparticles (SLMs). The oral route is generally the most widely and preferred route of drug administration because it improves patient compliance, avoids pain and discomfort as well as the possibility of infections associated with injections5. Most lipid drug delivery systems used as drug carriers have high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances and feasibility of variable routes of administration, including oral application and inhalation6. SLMs have the advantages of liposomes (possibility of controlled drug release and drug targeting), in addition to these advantages (use of non organic solvent, non-toxicity of the carrier system, chemical and physical storage stability for both the carrier and the drug, low cost of ingredients, not difficult in preparation and high scale-up potential)7–9. The most important limitation of SLMs is that drugs to be incorporated into SLMs must be lipophilic enough so as to ensure high-entrapment efficiency10 and to overcome this problem, solid reversed micellar solution
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(SRMS)-based carriers have been investigated, and successfully employed to achieve controlled release of a hydrophilic drug, zidovudine11. The aim of the present work was to formulate Hf-loaded SLMs and assess the activity of the formulations both in vitro and in vivo in Plasmodium berghei infected mice and compare their activity with a commercial sample, HalfanÕ tablets.
Materials and methods
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Materials The materials used were HalfanÕ tablets (GlaxoSmithKline, Lagos, Nigeria), PhospholiponÕ 90H (Phospholipid GmbH, Ko¨ln, Germany), sorbic acid, sorbitol, Polysorbate 80 (Merck, Darmstadt, Germany), distilled water (Lion Water, Nigeria), goat fat (obtained from a batch processed in our Laboratory). All other reagents and solvents were of analytical grade and were used without further purification. Parasites P. berghei NK-65, a strain free of contamination with Eperythrozoon coccoides and sensitive to chloroquine, was used for antimalarial evaluation in vivo. This strain is known to induce high mortality in mice, providing a good model to estimate antimalarial efficacy in reducing parasitemia, and is sensitive to all currently used antimalarial drugs. It was obtained from Nigerian Institute of Medical Research (NIMR), Yaba, Lagos. Animals Animal experiments were carried out according to the Principles of Laboratory Animal Care and legislation in force in Nigeria. Eperythrozoon-free Swiss albino mice (CD1) weighing 20 to 25 g were obtained from Department of Pharmacology and Toxicology, University of Nigeria. Extraction of goat fat The lipid (goat fat) used in the formulation was first extracted from goat fat (Capra hircus) using wet rendering method12 after which the lipid matrix was prepared using the fusion technique.
(to 100%w/w). The lipid matrix consisted of goat fat and PhospholiponÕ 90H. For each batch, the lipid matrix was placed in a stainless steel bowl and heated at 60 C until it had completely melted. The remaining excipients were weighed out appropriately and mixed with the corresponding quantity of water at 70 C. The excipients mixture with water at 70 C was poured into the lipid matrix-drug mixture and homogenized at 5000 rpm for 10 min with an Ultra-Turrax homogenizer (IKAÕ T25, Basic Digital, Werke Staufen, Germany). The hot emulsion was then poured into a bottle and allowed to recrystallize at room temperature for 24 h. The same procedure was adopted for the drug-loaded SLMs with varying quantities of the drug (halofantrine in concentrations of 3%, 5%, and 7%) as shown in Table 1 but the drug was poured into the melted matrix and mixed. The SLMs obtained after cooling at room temperature were lyophilized using a freeze-dryer (Amsco/Finn-Aqua Lyovac GTZ, Germany)8. Briefly, lyophilisates of the SLMs are obtained by freezing the formulations at a pressure of 2.7 Pa and temperature of 30 C; sublimation and drying at 15–25 C and all these operations took 6–12 h. SLMs containing no drug (unloaded SLMs) which served as negative control were also formulated. Characterization of the formulated SLMs Particle size analysis Particle size analysis was carried out on the SLMs after formulation. A 5 mg of the SLMs from each batch was dispersed in distilled water and smeared on a microscope slide using a glass rod. The mixture was covered with a cover slip and viewed using a polarized photomicroscope (HundÕ , Weltzlar, Germany), attached with a Motic image analyzer which is an Automated imaging system at a magnification of 100. Triplicate readings were taken. Determination of the loading capacity of the formulated SLMs Loading capacity (LC) expresses the ratio between the entrapped active pharmaceutical ingredient (API) and total weight of the lipids15. The loading capacity of the formulated SLMs was determined using the formula below:
Preparation of lipid matrix
Loading capacity ¼ 13
The lipid matrix was prepared according to Friedrich et al., ; Attama et al.,14. Briefly, a 70 g quantity of the prepared goat fat was weighed and melted in a beaker placed in a water bath at a temperature of 60 C, then 30 g of PhospholiponÕ 90H was added to the melted goat fat and stirred using a magnetic stirrer and hot plate (Jenway 400, EU), until an even mix was obtained. The molten lipid matrix was then placed in a cold water bath for 30 min at room temperature until solidification to obtain the solidified reverse micellar solution (SRMS).
Determination of the percentage yield of the SLMs After lyophilization, the water-free SLMs from all the batches were weighed. The yield of SLMs (% w/w) was calculated according to the following formula12.
Preparation of SLMs
W1 100 ðW2 þ W3Þ
H0 H1 H2 H3
ð2Þ
where, W1 ¼ weight of SLMs formulated (g), W2 ¼ weight of drug added (g), W3 ¼ weighed of the lipid matrix + polysorbate 80 + sorbitol + sorbic acid (g).
Table 1. Formula and composition of halofantrine SLMs. Batches
ð1Þ
where, W1 ¼ weight of lipid added in the formulation and W ¼ amount of API entrapped by the lipid.
% Recovery ¼ The solid lipid microparticles were prepared to contain: lipid matrix (17% w/w), halofantrine (0, 3, 5, 7%w/w), Polysorbate 80 (1.5%)), sorbic acid (0.05%), sorbitol (4%w/w) and water
W 100 W1
Halofantrine (%)
Lipid matrix (%)
Polysorbate 80 (%)
Sorbitol (%)
Sorbic acid (%)
Distilled water q.s (%)
0 3 5 7
17 17 17 17
1.5 1.5 1.5 1.5
4 4 4 4
0.05 0.05 0.05 0.05
100 100 100 100
In vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles
DOI: 10.3109/10837450.2014.949270
Determination of encapsulation efficiency of the SLMs
Evaluation of anti-malaria activity
A 50 mg of the SLMs from a batch was placed in a beaker containing 100 ml of distilled water. The dispersion was shaken properly and then filtered. The filtrate was then analyzed spectrophotometrically at a wavelength of 340 nm using a UV-VIS spectrophotometer (Unico 2102 PC UV/Vis Spectrophotometer, East Norwalk, CT). The encapsulation efficiency EE% was calculated using the formula16.
Preparation of the animals
Encapsulation efficiency ð%Þ ¼
actual drug content 100 theoritical drug content ð3Þ
3
The animal experimental protocols were in accordance with the guidelines for conducing animal experiments stipulated by our Institution’s Animal Ethics Committee and in compliance with the Federation of European Laboratory Animal Science Association and the European Community Council Directive of November 24, 1986 (86/609/EEC). Twelve healthy, non-pregnant adult Swiss albino mice were selected and divided into three groups of four mice per group. The mice were allowed water and food ad libitum and allowed to acclimatize for seven days.
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In vivo studies Time dependent pH stability studies of the formulations The formulations were subjected to pH analysis according to the method of Chime et al.,17 with slight modification; using a pH meter (Jenway 3510, EU) on days 0, 7, 60 and 90 to check the effect of storage with time on the pH stability of SLMs. The particles were stored as lyophilized powder and resuspended at the programme time point. Briefly, pH of 20 ml dispersion of SLMs from each batch was determined and triplicates readings taken. In vitro drug release studies The dissolution medium consisted of 200 ml of freshly prepared simulated gastric fluid (SGF, pH ¼ 1.2) 37 ± 1 C. The dialysis membrane (MWCO 6000–8000 Spectrum Labs, The Netherlands) was pre-treated by immersing it in the dissolution medium for 24 h, prior to the commencement of each release procedure. In each case about 500 mg of the formulation containing 5%, 7% of Hf and the commercial sample HalfanÕ was enclosed in a dialysis membrane containing 3 ml of the dissolution medium securely tied with a thermo-resistant thread and immersed in the dissolution medium containing 100 ml under agitation by a stirrer at 100 rpm. At predetermined time intervals, 5 ml aliquots of the dissolution medium (SGF, pH ¼ 1.2) was withdrawn and immediately replaced with 5 ml of fresh SGF and analyzed spectrophotometrically (Unico 2102 PC UV/Vis Spectrophotometer, East Norwalk, CT) at 340 nm. The amount of drug released at each time interval was determined using the standard Beer’s plot for halofantrine at 340 nm. The same procedure was repeated using formulations containing 5%, 7% of halofantrine and the commercial sample HalfanÕ with simulated intestinal fluid (SIF, pH ¼ 7.4)) as the dissolution medium.
The parasite, a chloroquine-sensitive strain of Plasmodium berghei NK 65 which was maintained in mice was obtained from the Nigerian Institute of Medical Research (NIMR), Yaba, Lagos. The 4-day test was performed as described by Peters et al.20. Each mouse was inoculated intraperitoneally (i.p) with 0.2 mL of infected blood containing about 10 000 000 Plasmodium berghei parasitized erythrocytes. The animals were left for four days; on day 4 after parasitic inoculation, parasitemia levels were measured and average parasitemia calculated for each group. Group E received SLMs containing Hf; group F received commercial sample (Halfan) and group G received no treatment at all and the treatment doses were based on body weight. The baseline packed cell volume (PCV), haemoglobin content (Hb), white blood cell content (WBC) and red blood cell content (RBC) were taken, these parameters were also determined before treatment after parasite inoculation and post treatment. The parasite count was done 4 days after infection and also post treatment from thin blood smears of the tail blood of mice, fixed with methanol and stained with Giemsa’s stain. The efficacy of the developed formulation was determined by monitoring the mean percentage parasitemia suppression activity against time as well as the animal survival period. Percentage parasitemia was calculated based on the parasite count pre-treatment and post-treatment using the formula; % parasitemia ðAv pretreatmentAv posttreatmentÞ parasitemia 100 ¼ Average pretreatment parasitemia
ð6Þ
Histological studies In vitro release kinetics The dissolution data for the SLMs were analyzed to determine the in vitro release kinetics using two kinetic models of zero order equation and Higuchi square root equation models. The zero order rate Equation describes the systems where the drug release rate is independent of its concentration18. C ¼ K0 t
ð4Þ
where, K0 is zero-order rate constant expressed in units of concentration/time and t is the time. According to Higuchi relationship, the amount of drug released per unit surface area is proportional to the square root of time. This equation explains diffusion release rate as indicated below: Qt ¼ KH t
1=2
ð5Þ
where, KH is Higuchi rate constant, Qt has same meaning as defined earlier19.
The mice were sacrificed seven days post treatment and the liver and kidney of a mouse from each group subjected to histological studies. Tissue sections of the liver and kidney of mouse from each group (E–G) were fixed in 10% normal saline and dehydrated in ascending grades of ethanol. Thereafter, the tissues were cleared in chloroform overnight, infiltrated and embedded in molten paraffin wax. The blocks were later trimmed and sectioned at 5–6 mm. The sections were deparaffinized in xylene, rinsed with water and subsequently stained with Haematoxylin and Eosin (H and E) and fixed for viewing which was done with a moticam (D-MOTICAM 580, U.S) fitted to the polarized photomicroscope. Statistical analysis Statistical analysis was done using SPSS version 14.0 (SPSS Inc., Chicago, IL). All values were expressed as mean ± SD. Data were analyzed by one-way ANOVA. Differences between means were assessed by a two-tailed Student’s t-test. p50.05 was considered statistically significant.
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Table 2. Particle size, yield, loading capacity, Encapsulation efficiency and pH stability studies.
Batches H0 H1 H2 H3
MPS*,y
% Yield*,y
12.41 ± 0.40 16.85 ± 0.10 22.75 ± 0.10 30.35 ± 0.33
87.74 ± 1.10 73.33 ± 0.70 60.48 ± 0.56 61.56 ± 0.51
EE (%)*,y
pH Day 0
stability Day 14
Study*,y Day 60
Day 90
61.43 ± 0.01 70.30 ± 0.30 76.32 ± 0.02
5.30 ± 0.02 3.60 ± 0.04 3.70 ± 0.01 3.70 ± 0.05
5.40 ± 0.02 3.49 ± 0.04 3.60 ± 0.01 3.65 ± 0.05
4.83 ± 0.05 3.77 ± 0.10 4.02 ± 0.07 4.03 ± 0.10
4.87 ± 0.03 3.74 ± 0.01 4.03 ± 0.05 3.70 ± 0.10
LC (%)*,y 10.84 ± 0.17 20.68 ± 0.41 31.43 ± 0.39
MPS: Mean particle size, LC: loading capacity, EE: encapsulation efficiency, *Mean_SD. yn ¼ 3.
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Results SLMs are a suitable carrier for Hf because the free base is very soluble in oil (4180 mg/ml) and has a favorable oil-water partition coefficient (log p ¼ 6.5)21. A high drug concentration was obtained (Table 2), and the drug was encapsulated with a yield of 61.56–73.33%; EE of 61.43–76.32%; mean diameter of the particle sizes were 16.85–30.35 mm. Percentage recovery of SLMs The results of percentage recovery of the SLMs presented in Table 2 showed that percentage recovery values of unloaded SLMs (H0) was 87.74%, while the recovery values for Hf-loaded SLMs ranged from 60.48 to 73.33%.
Figure 1. Release profile of the SLMs formulations and commercial sample in SIF.
Mean particle diameter of SLMs From the values of mean particle diameter presented in Table 2, it can be seen that halofantrine-loaded SLMs prepared with the lipid matrix, (H1–H3) exhibited the highest mean particle diameter and the SLMs were mostly irregular in shape. Encapsulation efficiency (EE%) and loading capacity (LC) Table 2 shows the EE% and the LC of various batches of formulated Hf-loaded SLMs. EE% ranged from 61.43% for batch H1 to 76.32% for batch H3 SLMs. Lipid matrices containing 7% of Hf exhibited the highest encapsulation efficiency of 76.32% for SLMs formulated with SRMS, which was followed by lipid matrix containing 5% drug loading. The lipid matrix with 3% drug loading had the least encapsulation efficiency, however LC increased with increase in drug loading as shown in Table 2. Batch H3, with 7% halofantrine, exhibited the highest loading capacity of 31.43 g of halofantrine/100 lipid. Time-dependent pH stability studies of the formulations In Table 2, the SLMs containing no drug (H0) had a relatively variable pH of 4.87–5.30. However, the pH of the SLMs formulated with 3% halofantrine (H1) exhibited a relatively stable pH; H2 exhibited a significant increase in pH from approximately 3.70 to 4.03 at 3 months (p50.05) while the pH of batch H3 exhibited variable values within the 3 months of the stability study. In vitro drug release studies The results of in vitro release presented in Figures 1 and 2 showed the release profile in SIF and SGF respectively of halofantrineloaded SLMs and Halfan. In vitro kinetics release The result of the in vitro release of the different batches was fitted into zero order, first order, Higuchi and Peppas models to determine the kinetics of release as shown in Table 3. These dependent methods are based on differential mathematical
Figure 2. Release profile of the SLMs formulations and commercial sample in SGF. Table 3. Kinetic models for the release studies. Zero Batch H2 in SIF H2 in SGF H3 in SIF H3 in SGF Halfan in SIF Halfan in SGF
2
–0.750 –0.760 –0.360 0.514 –1.270 –0.460
r
Order
Higuchi
K0
r
2
Kh
7.392 2.155 6.408 2.541 3.019 0.266
–4.580 –5.460 –1.230 0.874 –7.280 –1.400
18.850 5.481 16.610 6.245 7.822 0.674
Key: r2 ¼ regression coefficient, K0 ¼ Zero order Kh ¼ Higuchi rate constant.
rate constant,
functions which describe the dissolution profile. Determining the regression coefficients assessed the fitness of the data into various kinetic models of the batches. The rate constants for the respective models were calculated from their slopes22. The statistical analysis showed a high significance difference (p50.05) between the halofantrine-loaded SLMs and the Halfan tablet. In vivo studies The hematological parameters of the mice (packed cell volume, hemoglobin content, red blood cell count, white blood cell count)
In vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles
DOI: 10.3109/10837450.2014.949270
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Table 4. Heamatological parameters. Baseline
pretreatment
posttreatment
Group
PCV
Hb
RBC
WBC
PCV
Hb
RBC
WBC
PCV
Hb
RBC
WBC
E F G
38.0 34.0 36.0
10.8 14.2 14.5
9.9 9.3 8.7
10875 9150 8700
32.0 30.8 28.0
9.3 8.3 10.4
7.3 5.7 6.6
11075 9825 10725
37.5 31.5 21.33
13.7 14 11.5
8.6 8.8 5.5
7950 8650 7250
PCV (packed cell volume), Hb (heamoglobin content), RBC (red blood cell count), WBC (white blood cell count).
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Table 5. Percentage parasitemia reduction.
Groups E F G
AWA (g)
4 days post-infection treatment
Pre-TP
Post-TP
PP (%)
27.50 ± 0.06 30.70 ± 0.09 31.40 ± 0.07
3 doses of 0.4 ml of 0.5 mg/ml 3 doses of 0.1 ml of 2.5 mg/ml No treatment
27.00 ± 0.2 31.50 ± 0.5 54.40 ± 0.5
7.33 ± 0.14 4.50 ± 0.04 46.31 ± 0.62
72.96 ± 1.95 85.71 ± 2.11 14.89 ± 0.12
Group E received SLM containing halofantrine, group F received commercial sample of halofantrine and group G received no treatment; AWA is average animal weight (g), Pre-TP is Pre-treatment parasitemia, Post-TP is Posttreatment parasitemia, PP (%) is Percentage parasitemia (%).
and the effect of drug treatment (percentage parasitemia reduction) are presented in Tables 4 and 5, respectively. Peter’s Four day suppressive protocol studies were carried out in order to investigate the in vivo properties of the formulations and compare with the in vitro drug release and the reference drug. The result of anti-malaria properties of Hf-loaded SLMs presented in Tables 4 and 5 showed that halofantrine-loaded SLMs based on SRMS had a high parasitemia reduction. Histological studies The effect of the formulations in the liver and kidney of the mice are shown in Figures 3 and 4. The information from the study will give insight on the toxic effect of the halofantrine-loaded SLMs and the commercial tablet formulations (HalfanÕ ) on the vital organs of the body studied.
Discussion The percentage of SLMs recovered from the formulation showed that the unloaded SLMs had higher percentage recovery than the loaded SLMs while 5% Hf-loaded SLMs had the least percentage recovery of 60.48% (Table 2). This implies that the formulation technique adopted was reliable since more than 50% of the formulations were recovered. The SLMs containing 7% halofantrine (H3) had the highest particle size of 30.35 mm while the batch containing 3% (H1) had the least particle size of 16.85 mm which suggests that increased drug loading resulted in larger particle sizes of the SLMs Table 2. The SLMs showed stable, discrete, polydispersed, poorly flowable, mostly irregularly shaped Hf HCl-loaded SLMs with size range 16.85 ± 0.10 to 30.35 ± 0.33 mm were successfully prepared with the lipid matrices. The sizes of the SLMs were all within the micrometer range, indicating that the production was able to achieve the intended end point for oral administration as the size influences the rate of drug release and subsequent pharmacokinetics. This is because the rate of absorption, the speed of onset of effect and the duration of therapeutic response can all be determined by particle size for most route of administration; therefore the deliberate manipulation of particle size leads to a measure of control of activity and side effects of the formulations21. The time-dependent pH stability test was carried out to determine the pH stability of the SLMs formulations when stored
at room temperature at different time intervals. The H0 batch which contains drug unloaded SLMs formulation varied in pH from a value of 5.30 ± 0.02 on the day of formulation to 4.87 ± 0.03 after 90 days. The H1 batch which contains 3% of Hf varied from pH of 3.60 ± 0.04 on the day of formulation to 3.74 ± 0.01 after 90 days. The H2 batch which contains 5% of Hf varied from pH of 3.70 ± 0.01 on the day of formulation to 4.03 ± 0.05 after 90 days. The H3 batch which contains 7% of Hf varied from pH of 3.70 ± 0.05 on the day of formulation to 3.70 ± 0.10 after 90 days. The pH change upon storage for a period of 3 months as presented in Table 2, was insignificant hence there was little drug and excipient degradation during the storage period. Change in pH of a liquid drug formulation could be a function of degradation of the drug or the excipients. A prior stable drug may be affected by degradation of excipients with storage through generation of an unfavorable pH (increase or decrease) or reactive species for the drug14. However, the slight decline in the pH values in the formulations was not attributed to drug degradation since there was also a fall in the pH of the unloaded SLMs14. The pH decrease may be due to release of fatty acids from the lipid matrix. However, the little decrease in pH of the Hf-loaded SLMs indicated that the preparation would need a buffer to keep the pH more stable. The difference in mean particle size between the unloaded SLM (H0) and loaded SLMs (H1, H2 and H3) was quite significant; and the same applies for the percentage yield. The result of encapsulation efficiency and the loading capacity presented in Table 2 showed that the encapsulation efficiency and the loading capacity increased with increasing drug loading, SLMs containing 7% halofantrine gave the highest encapsulation efficiency and the loading capacity of 76.32% and 31.43% respectively, while the batch containing 3% gave the least encapsulation efficiency and the loading capacity of 61.43% and 10.84% respectively. The in vitro drug release study showed greater drug release from SLMs than from the commercial tablet formulation (HalfanÕ ) which could be attributed to the granular nature of the tablet as it has to dissolve before releasing the drug. There was also greater drug release in SIF than SGF (Figures 1 and 2). The zero order model is based on drug dissolution from dosage forms that do not disaggregate and release the drug slowly such as from matrix tablets with low soluble drugs in coated forms, osmotic systems etc23. None of the halofantrine loaded SLMs or
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Figure 3. Photomicrographs of liver sections of mice in groups E, F, G and H that is SLMs formulation, HalfanÕ , no treatment group and uninfected animals respectively. H and E 400.
Pharm Dev Technol, Early Online: 1–8
E
F
G
Figure 4. Photomicrographs of kidney sections of mice in groups E, F, G and H that is SLMs formulation, HalfanÕ , no treatment group and uninfected animals respectively. H and E 400.
H
E
F
G
H
the commercial tablet formulation (HalfanÕ ) showed zero order release kinetics. The first example of a mathematical model aimed to describe the drug release from a matrix system was proposed by Higuchi in 196114,24. It is based on a hypothesis that (i) initial drug concentration in the matrix is much higher than drug solubility; (ii) drug diffusion takes place only in one dimension; (iii) drug particles are much smaller than system thickness; (iv) matrix swelling and dissolution are negligible (v) drug diffusivity is constant; and (vi) perfect sink conditions are always attained in
the release environment23. The drug release kinetics showed that Higuchi plot of amount of drug release against square root of time for none of the batches of halofantrine-loaded SLMs were linear (r2 6¼ 0.9) except H3 which contains 7% of halofantrine-loaded SLMs in SGF as a medium. However, plot of log Q against log t according to Higuchi gave n value of 0.5 in most of the batches, indicating that diffusion-controlled process was predominant in these batches. All the mice infected with Plasmodium berghei exhibited the following signs: dullness, rough hair coat, paleness of the visible
DOI: 10.3109/10837450.2014.949270
In vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles
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Figure 5. Micrographs of the blood work of mice in group E (Bar represents 40 mm).
Figure 6. Micrographs of the blood work of mice in group F (Bar represents 40 mm).
Figure 7. Micrographs of the blood work of mice in group G (Bar represents 40 mm).
mucous membranes, weakness, diarrhea and thirst in accordance to the exhibited sign in the work of Saganuwan et al25. The in vivo studies showed that there were variations in the hematological parameters (PCV, Hb, RBC and WBC) due to the various effect of different treatments administered to the different groups as illustrated in Table 3. The data in Table 4 showed that group A which received SLMs containing halofantrine had 72.96% parasite clearance; group B which received commercial formulation of halofantrine had 85.71% parasite clearance while group C which received no treatment had 14.89% parasite clearance. The percentage parasitemia reduction properties of the SLMs formulation were comparable to that of the reference formulation. The in vivo pharmacodynamic properties of halofantrine-loaded SLMs were comparable to the in vitro release of halofantrine from SLMs. The formulated SLMs and commercial sample treatments yielded a more significant reduction in parasitemia compared to the negative control (group G). It has been reported that a change in relative or absolute weight of an organ after drug administration is an indication of the toxic effects of that drug26,27. The ability of
a drug to produce liver damage in vivo often results from the interaction of the uptake, biotransformation and elimination of potentially toxic compounds24,28. The observation from the histological studies conducted on the liver of the mice from various groups, showed varying degrees of hepatitis on the liver of the mice in groups E, F and G. Also, studies on the kidneys of various animals from various groups revealed different cases of mild tubular dilatation and very mild tubular degeneration for the mice in group G. Photomicrograph of liver section of mice from experimental groups E, F, G showing varying degrees of periportal mononuclear infiltration of cells-periportal hepatitis (arrow) while the group H shows normal portal area and hepatocytes (H and E 400). Photomicrograph of kidney section of mice from experimental groups E, F and H showing apparently normal glomerulus (GM) and renal tubules (white arrow) but mild tubular degeneration in G (black arrow) (H and E 400). The results of this study showed that short-term administration of halofantrine did not cause any significant changes in the body regarding the relative organs’ morphology of the treated mice.
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This implies that short-term oral administration of this drug had no negative effects on somatic growth. The micrographs of the fixed blood of mice are shown in Figures 5–7 where the parasitic infection are shown in black dots in the red blood cells in groups E–G.
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Conclusion It can be concluded that SLMs are good candidates for oral administration of halofantrine. The parasitemia reduction on the halofantrine-loaded SLMs was comparable with the reduction from the commercial sample, Halfan. The histological studies carried out also established the safety of the SLMs formulations as the results showed no evidence of disastrous side effect on the organs of mice used.
Declaration of interest This research work was funded by the International Foundation for Science (IFS), Sweden (IFS No. F/4467-1) and Organization for the Prohibition of Chemical Weapons (OPCW), The Hague. Dr. A. A. Attama is highly grateful to the Foundation and the Organization.
References 1. Breman JG, Alilio MS, Mills A. Conquering the in-tolerable burden of malaria: what’s new, what’s needed: a summary. Am J Trop Med Hyg 2004;71:1–15. 2. Djimde´ A, Doumbo OK, Cortese JF, et al. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 2001;344: 257–263. 3. Ogutu BR, Smoak BL, Nduati RW, et al. The efficacy of pyrimethamine-sulfadoxine (Fansidar) in the treatment of uncomplicated Plasmodium falciparum malaria in Kenyan children. Trans R Soc Trop Med Hyg 2000;94:83–84. 4. Vanessa CFM, Philippe ML, Christian B, et al. Efficacy and pharmacokinetics of intravenous nanocapsule formulations of halofantrine in Plasmodium berghei-infected mice. Antimicrob Agents Chemother 2004;48:1222–1228. 5. Etienne M, Josias H, Lissinda du P, et al. EudragitÕ L100/NTrimethylchitosan Chloride Microspheres for Oral Insulin Delivery. Molecules 2013;18:6734–6747. 6. Chime SA, Attama AA, Agubata CO, et al. Micromeritic and antinociceptive properties of lyophilized indomethacinloaded SLMs based on solidified reverse micellar solutions. J Pharma Res 2012;5: 3410–3416. 7. Umeyor CE, Kenechukwu FC, Ogbonna JDN, et al. Preparation of novel solid lipid microparticles loaded with gentamicin and its evaluation in vitro and in vivo. J Microencapsul 2012;29:296–307. 8. Jaspert S, Bertholet P, Piel G, et al. Solid lipid microparticles as sustained release system for pulmonary drug delivery. Eur J Pharm Biopharm 2007;65:47–56. 9. Milak S, Medicott N, Tucker IG. Solid lipid micro particles containing loratadine prepared using a micro mixer. J Microencapsul 2006;23:823–831. 10. Long C, Zhang L, Qian Y. Preparation and crystal modification of ibuprofen loaded solid lipid microparticles. Chin J Chem Eng 2006; 14:518–525. 11. Uronnachi EM, Ogbonna JDN, Kenechukwu FC, et al. Formulation and release characteristics of zidovudine-loaded solidified lipid microparticles. Tropical J Pharm Res 2014;13:199–204.
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12. Attama AA, Nkemnele MO. In vitro evaluation of drug release from selfemulsifying drug delivery systems using a biodegradable homolipid from Capra hircus. Int J Pharm 2005;304:4–10. 13. Friedrich I, Reichl S, Muller-Goymann CC. Drug release and permeation studies of nanosuspensions based on solidified reverse micellar solutions (SRMS). Int J Pharm 2005;305:167–175. 14. Attama AA, Okafor CE, Builders PF, Okorie O. Formulation and in vitro evaluation of a PEGylated microscopic lipospheres delivery system for ceftriaxone sodium. Drug Deliv 2009;16:448–616. 15. Doktorovova S, Souto EB. Nanostructured lipid carrier-based hydrogel formulations for drug delivery: a comprehensive review. Expert Opin Drug Deliv 2009;6:165–176. 16. Morkhade DM, Joshi SB. Evaluation of gum dammar as a novel microencapsulating material for ibuprofen and diltiazem hydrochloride. Indian J Pharm Sci 2007;67:263–268. 17. Chime SA, Attama AA, Onunkwo GC. Sustained release indomethacin-loaded solid lipid microparticles based on solidified reverse micellar solution (SRMS): in vitro and in vivo evaluation. J Drug Del Sci Tech 2012;22:485–492. 18. Kalam MA, Humayun M, Parvez N, et al. Release kinetics of modified pharmaceutical dosage forms: a review. Continental J Pharm Sci 2007;1:30–35. 19. Singh J, Gupta S, Kaur H. Prediction of in vitro drug release mechanisms from extended release matrix tablet using SSR/SR2 techniques. Trends App Sci Res 2011;6:400–409. 20. Peters W, Ze-Lin L, Robinson BL, Warhurst DC. The chemotherapy of rodent malaria, XL. Ann Trop Med Parasitol 1986;80: 483–489. 21. Florence AT, Attwood D. Physicochemical principles of pharmacy. 4th ed. UK: RPS Publishing. Pharmaceutical Press; 2009. 22. Pachuau L, Sarkar S, Mazumdar B. Formulation and evaluation of matrix microspheres for simultaneous delivery of salbutamol sulphate and theophylline. Tropical J. Pharm Res 2008;7:995–1002. 23. Suvakanta D, Padala NM, Lilakanta N, Prasanta C. Kinetic modeling on drug release from controlled drug delivery systems. Acta Poloniac Pharmaceutica-Drug Research 2010;67:217–223. 24. Babalola CP, Kanu DN, Okafor GO, et al. Toxicological effect of sub-therapeutic, therapeutic and overdose regimens of halofantrine hydrochloride on male albino rats. Pharmacologia 2013;4: 180–185. 25. Saganuwan AS, Onyeyili PA, Ameh EG, Etuk EU. In vivo antiplasmodial activity by aqueous extract of Abrus precatorius in mice. Rev Latinoamer Quı´m 2011;39:32–44. 26. Maina MB, Garba SH, Jacks TW. Histological evaluation of the rats testis following administration of a herbal tea mixture. J Pharmacol Toxicol 2008;3:464–70. 27. Simmons JE, Yang RS, Berman E. Evaluation of the nephrotoxicity of complex mixtures containing organics and metals: advantages and disadvantages of the use of real-world complex mixtures. Environ Health Perspect 1995;103:67–71. 28. Obi E, Orisakwe OE, Asomugha LA, et al. The hepatotoxic effect of halofantrine in guinea pigs. Indian J Pharm 2004;36:303–305. 29. European Community Council Directive on the ethics of experiments involving laboratory animals (86/609/EEC), November 24, 1986. 30. Higuchi TJ. Mechanism of sustained medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. Pharm Sci 1963;84:1464–1477. 31. Sachs J, Malaney P. The economic and social burden of malaria. Nature 2012;415:680–685. 32. Uronnachi EM, Ogbonna JDN, Kenechukwu FC, et al. Pharmacokinetics and biodistribution of zidovudine loaded in a solidified reverse micellar delivery system. International J Drug Deliv 2013;5:73–80.
RESEARCH ARTICLE
Formulation, in vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Nyu Medical Center on 08/05/15 For personal use only.
John D. N. Ogbonna, Franklin C. Kenechukwu, Chinekwu S. Nwobi, Onochie S. Chibueze, and Anthony A. Attama Drug Delivery Research Unit, Department of Pharmaceutics, University of Nigeria, Nsukka, Nigeria
Abstract
Keywords
Context: Formulation, characterization, in vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles (SLMs). Objective: The objective of the study was to formulate and evaluate halofantrine-loaded SLMs. Materials and methods: Formulations of halofantrine-loaded SLMs were prepared by hot homogenization and thereafter lyophilized and characterized using particle size, pH stability, loading capacity (LC) and encapsulation efficiency (EE). In vitro release of halofantrine (Hf) from the optimized SLMs was performed in SIF and SGF. In vivo study using Peter’s Four day suppressive protocol in mice and the mice thereafter subjected to histological studies in kidney and liver. Results: Results obtained indicated that EE of 76.32% and 61.43% were obtained for the SLMs containing 7% and 3% of Hf respectively. The SLMs loaded with 3% of Hf had the highest yield of 73.33%. Time-dependent pH stability analysis showed little variations in pH ranging from 3.49 ± 0.04 to 4.03 ± 0.05. Discussion: The SLMs showed pH-dependent release profile; in SIF (43.5% of the drug for each of H2 and H3) compared with SGF (13 and 18% for H2 and H3 respectively) after 8 h. The optimized SLMs formulation and HalfanÕ produced a percentage reduction in parasitemia of 72.96% and 85.71% respectively. The histological studies revealed that the SLMs formulations have no harmful effects on the kidney and liver. Conclusion: SLMs formulations might be an alternative for patients with parasitemia as there were no harmful effects on vital organs of the mice.
Hematological parameters, halofantrine, histological studies, parasitemia, solid–lipid microparticles
Introduction 1
Malaria, a major public health problem in endemic regions affects about 500 million people annually and is a leading cause of mortality and morbidity in the developing World. The global impact of malaria parasite, the development of resistance especially to chloroquine by Plasmodium falciparum (P. falciparum) coupled with the emergence of new parasites all translate into incessant scientific challenge of drug discovery and formulation development. Over the past decades many researchers have focused on development of new antimalarials that are able to target important therapeutic processes. Treatment failures have been linked majorly to the development of resistance of the malaria parasite to standard antimalarial agents2,3. Effective treatment of malaria had been a great challenge to medicine and this has had a great impact on man’s health and economy (Sachs and Malaney, 2002). Halofantrine hydrochloride, a 9-phenanthrenemethanol is one of the most active antimalarial drugs against P. falciparum with a long half-life which lessens the need for repeated administration. The antimalarial activity is mediated partly by the parent drug and partly by its N-desbutyl metabolite4. It has a place in treatment of
Address for correspondence: John D. N. Ogbonna, Drug Delivery Research Unit, Department of Pharmaceutics, University of Nigeria, Nsukka 410001, Nigeria. Tel: +2348063674303. E-mail: johnixus@ yahoo.com, [email protected]
History Received 7 May 2014 Revised 7 July 2014 Accepted 22 July 2014 Published online 18 August 2014
multiresistant and chloroquine-resistant strains of P. falciparum when administered orally. However halofantrine has serious cardiotoxicity, prolonging the Q-T interval, which also causes the cardiac side effects of quinine and mefloquine. Owing to these adverse effects, including severe local irritation with strong erythema (probably related to the toxicity of the solvents used in preparation) as well as serious cardiac side effects (Q-T interval prolongation), which worsens in intravenous administration4 its oral use is a better formulation strategy, hence the need for novel formulation such as solid–lipid microparticles (SLMs). The oral route is generally the most widely and preferred route of drug administration because it improves patient compliance, avoids pain and discomfort as well as the possibility of infections associated with injections5. Most lipid drug delivery systems used as drug carriers have high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances and feasibility of variable routes of administration, including oral application and inhalation6. SLMs have the advantages of liposomes (possibility of controlled drug release and drug targeting), in addition to these advantages (use of non organic solvent, non-toxicity of the carrier system, chemical and physical storage stability for both the carrier and the drug, low cost of ingredients, not difficult in preparation and high scale-up potential)7–9. The most important limitation of SLMs is that drugs to be incorporated into SLMs must be lipophilic enough so as to ensure high-entrapment efficiency10 and to overcome this problem, solid reversed micellar solution
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(SRMS)-based carriers have been investigated, and successfully employed to achieve controlled release of a hydrophilic drug, zidovudine11. The aim of the present work was to formulate Hf-loaded SLMs and assess the activity of the formulations both in vitro and in vivo in Plasmodium berghei infected mice and compare their activity with a commercial sample, HalfanÕ tablets.
Materials and methods
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Materials The materials used were HalfanÕ tablets (GlaxoSmithKline, Lagos, Nigeria), PhospholiponÕ 90H (Phospholipid GmbH, Ko¨ln, Germany), sorbic acid, sorbitol, Polysorbate 80 (Merck, Darmstadt, Germany), distilled water (Lion Water, Nigeria), goat fat (obtained from a batch processed in our Laboratory). All other reagents and solvents were of analytical grade and were used without further purification. Parasites P. berghei NK-65, a strain free of contamination with Eperythrozoon coccoides and sensitive to chloroquine, was used for antimalarial evaluation in vivo. This strain is known to induce high mortality in mice, providing a good model to estimate antimalarial efficacy in reducing parasitemia, and is sensitive to all currently used antimalarial drugs. It was obtained from Nigerian Institute of Medical Research (NIMR), Yaba, Lagos. Animals Animal experiments were carried out according to the Principles of Laboratory Animal Care and legislation in force in Nigeria. Eperythrozoon-free Swiss albino mice (CD1) weighing 20 to 25 g were obtained from Department of Pharmacology and Toxicology, University of Nigeria. Extraction of goat fat The lipid (goat fat) used in the formulation was first extracted from goat fat (Capra hircus) using wet rendering method12 after which the lipid matrix was prepared using the fusion technique.
(to 100%w/w). The lipid matrix consisted of goat fat and PhospholiponÕ 90H. For each batch, the lipid matrix was placed in a stainless steel bowl and heated at 60 C until it had completely melted. The remaining excipients were weighed out appropriately and mixed with the corresponding quantity of water at 70 C. The excipients mixture with water at 70 C was poured into the lipid matrix-drug mixture and homogenized at 5000 rpm for 10 min with an Ultra-Turrax homogenizer (IKAÕ T25, Basic Digital, Werke Staufen, Germany). The hot emulsion was then poured into a bottle and allowed to recrystallize at room temperature for 24 h. The same procedure was adopted for the drug-loaded SLMs with varying quantities of the drug (halofantrine in concentrations of 3%, 5%, and 7%) as shown in Table 1 but the drug was poured into the melted matrix and mixed. The SLMs obtained after cooling at room temperature were lyophilized using a freeze-dryer (Amsco/Finn-Aqua Lyovac GTZ, Germany)8. Briefly, lyophilisates of the SLMs are obtained by freezing the formulations at a pressure of 2.7 Pa and temperature of 30 C; sublimation and drying at 15–25 C and all these operations took 6–12 h. SLMs containing no drug (unloaded SLMs) which served as negative control were also formulated. Characterization of the formulated SLMs Particle size analysis Particle size analysis was carried out on the SLMs after formulation. A 5 mg of the SLMs from each batch was dispersed in distilled water and smeared on a microscope slide using a glass rod. The mixture was covered with a cover slip and viewed using a polarized photomicroscope (HundÕ , Weltzlar, Germany), attached with a Motic image analyzer which is an Automated imaging system at a magnification of 100. Triplicate readings were taken. Determination of the loading capacity of the formulated SLMs Loading capacity (LC) expresses the ratio between the entrapped active pharmaceutical ingredient (API) and total weight of the lipids15. The loading capacity of the formulated SLMs was determined using the formula below:
Preparation of lipid matrix
Loading capacity ¼ 13
The lipid matrix was prepared according to Friedrich et al., ; Attama et al.,14. Briefly, a 70 g quantity of the prepared goat fat was weighed and melted in a beaker placed in a water bath at a temperature of 60 C, then 30 g of PhospholiponÕ 90H was added to the melted goat fat and stirred using a magnetic stirrer and hot plate (Jenway 400, EU), until an even mix was obtained. The molten lipid matrix was then placed in a cold water bath for 30 min at room temperature until solidification to obtain the solidified reverse micellar solution (SRMS).
Determination of the percentage yield of the SLMs After lyophilization, the water-free SLMs from all the batches were weighed. The yield of SLMs (% w/w) was calculated according to the following formula12.
Preparation of SLMs
W1 100 ðW2 þ W3Þ
H0 H1 H2 H3
ð2Þ
where, W1 ¼ weight of SLMs formulated (g), W2 ¼ weight of drug added (g), W3 ¼ weighed of the lipid matrix + polysorbate 80 + sorbitol + sorbic acid (g).
Table 1. Formula and composition of halofantrine SLMs. Batches
ð1Þ
where, W1 ¼ weight of lipid added in the formulation and W ¼ amount of API entrapped by the lipid.
% Recovery ¼ The solid lipid microparticles were prepared to contain: lipid matrix (17% w/w), halofantrine (0, 3, 5, 7%w/w), Polysorbate 80 (1.5%)), sorbic acid (0.05%), sorbitol (4%w/w) and water
W 100 W1
Halofantrine (%)
Lipid matrix (%)
Polysorbate 80 (%)
Sorbitol (%)
Sorbic acid (%)
Distilled water q.s (%)
0 3 5 7
17 17 17 17
1.5 1.5 1.5 1.5
4 4 4 4
0.05 0.05 0.05 0.05
100 100 100 100
In vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles
DOI: 10.3109/10837450.2014.949270
Determination of encapsulation efficiency of the SLMs
Evaluation of anti-malaria activity
A 50 mg of the SLMs from a batch was placed in a beaker containing 100 ml of distilled water. The dispersion was shaken properly and then filtered. The filtrate was then analyzed spectrophotometrically at a wavelength of 340 nm using a UV-VIS spectrophotometer (Unico 2102 PC UV/Vis Spectrophotometer, East Norwalk, CT). The encapsulation efficiency EE% was calculated using the formula16.
Preparation of the animals
Encapsulation efficiency ð%Þ ¼
actual drug content 100 theoritical drug content ð3Þ
3
The animal experimental protocols were in accordance with the guidelines for conducing animal experiments stipulated by our Institution’s Animal Ethics Committee and in compliance with the Federation of European Laboratory Animal Science Association and the European Community Council Directive of November 24, 1986 (86/609/EEC). Twelve healthy, non-pregnant adult Swiss albino mice were selected and divided into three groups of four mice per group. The mice were allowed water and food ad libitum and allowed to acclimatize for seven days.
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In vivo studies Time dependent pH stability studies of the formulations The formulations were subjected to pH analysis according to the method of Chime et al.,17 with slight modification; using a pH meter (Jenway 3510, EU) on days 0, 7, 60 and 90 to check the effect of storage with time on the pH stability of SLMs. The particles were stored as lyophilized powder and resuspended at the programme time point. Briefly, pH of 20 ml dispersion of SLMs from each batch was determined and triplicates readings taken. In vitro drug release studies The dissolution medium consisted of 200 ml of freshly prepared simulated gastric fluid (SGF, pH ¼ 1.2) 37 ± 1 C. The dialysis membrane (MWCO 6000–8000 Spectrum Labs, The Netherlands) was pre-treated by immersing it in the dissolution medium for 24 h, prior to the commencement of each release procedure. In each case about 500 mg of the formulation containing 5%, 7% of Hf and the commercial sample HalfanÕ was enclosed in a dialysis membrane containing 3 ml of the dissolution medium securely tied with a thermo-resistant thread and immersed in the dissolution medium containing 100 ml under agitation by a stirrer at 100 rpm. At predetermined time intervals, 5 ml aliquots of the dissolution medium (SGF, pH ¼ 1.2) was withdrawn and immediately replaced with 5 ml of fresh SGF and analyzed spectrophotometrically (Unico 2102 PC UV/Vis Spectrophotometer, East Norwalk, CT) at 340 nm. The amount of drug released at each time interval was determined using the standard Beer’s plot for halofantrine at 340 nm. The same procedure was repeated using formulations containing 5%, 7% of halofantrine and the commercial sample HalfanÕ with simulated intestinal fluid (SIF, pH ¼ 7.4)) as the dissolution medium.
The parasite, a chloroquine-sensitive strain of Plasmodium berghei NK 65 which was maintained in mice was obtained from the Nigerian Institute of Medical Research (NIMR), Yaba, Lagos. The 4-day test was performed as described by Peters et al.20. Each mouse was inoculated intraperitoneally (i.p) with 0.2 mL of infected blood containing about 10 000 000 Plasmodium berghei parasitized erythrocytes. The animals were left for four days; on day 4 after parasitic inoculation, parasitemia levels were measured and average parasitemia calculated for each group. Group E received SLMs containing Hf; group F received commercial sample (Halfan) and group G received no treatment at all and the treatment doses were based on body weight. The baseline packed cell volume (PCV), haemoglobin content (Hb), white blood cell content (WBC) and red blood cell content (RBC) were taken, these parameters were also determined before treatment after parasite inoculation and post treatment. The parasite count was done 4 days after infection and also post treatment from thin blood smears of the tail blood of mice, fixed with methanol and stained with Giemsa’s stain. The efficacy of the developed formulation was determined by monitoring the mean percentage parasitemia suppression activity against time as well as the animal survival period. Percentage parasitemia was calculated based on the parasite count pre-treatment and post-treatment using the formula; % parasitemia ðAv pretreatmentAv posttreatmentÞ parasitemia 100 ¼ Average pretreatment parasitemia
ð6Þ
Histological studies In vitro release kinetics The dissolution data for the SLMs were analyzed to determine the in vitro release kinetics using two kinetic models of zero order equation and Higuchi square root equation models. The zero order rate Equation describes the systems where the drug release rate is independent of its concentration18. C ¼ K0 t
ð4Þ
where, K0 is zero-order rate constant expressed in units of concentration/time and t is the time. According to Higuchi relationship, the amount of drug released per unit surface area is proportional to the square root of time. This equation explains diffusion release rate as indicated below: Qt ¼ KH t
1=2
ð5Þ
where, KH is Higuchi rate constant, Qt has same meaning as defined earlier19.
The mice were sacrificed seven days post treatment and the liver and kidney of a mouse from each group subjected to histological studies. Tissue sections of the liver and kidney of mouse from each group (E–G) were fixed in 10% normal saline and dehydrated in ascending grades of ethanol. Thereafter, the tissues were cleared in chloroform overnight, infiltrated and embedded in molten paraffin wax. The blocks were later trimmed and sectioned at 5–6 mm. The sections were deparaffinized in xylene, rinsed with water and subsequently stained with Haematoxylin and Eosin (H and E) and fixed for viewing which was done with a moticam (D-MOTICAM 580, U.S) fitted to the polarized photomicroscope. Statistical analysis Statistical analysis was done using SPSS version 14.0 (SPSS Inc., Chicago, IL). All values were expressed as mean ± SD. Data were analyzed by one-way ANOVA. Differences between means were assessed by a two-tailed Student’s t-test. p50.05 was considered statistically significant.
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Table 2. Particle size, yield, loading capacity, Encapsulation efficiency and pH stability studies.
Batches H0 H1 H2 H3
MPS*,y
% Yield*,y
12.41 ± 0.40 16.85 ± 0.10 22.75 ± 0.10 30.35 ± 0.33
87.74 ± 1.10 73.33 ± 0.70 60.48 ± 0.56 61.56 ± 0.51
EE (%)*,y
pH Day 0
stability Day 14
Study*,y Day 60
Day 90
61.43 ± 0.01 70.30 ± 0.30 76.32 ± 0.02
5.30 ± 0.02 3.60 ± 0.04 3.70 ± 0.01 3.70 ± 0.05
5.40 ± 0.02 3.49 ± 0.04 3.60 ± 0.01 3.65 ± 0.05
4.83 ± 0.05 3.77 ± 0.10 4.02 ± 0.07 4.03 ± 0.10
4.87 ± 0.03 3.74 ± 0.01 4.03 ± 0.05 3.70 ± 0.10
LC (%)*,y 10.84 ± 0.17 20.68 ± 0.41 31.43 ± 0.39
MPS: Mean particle size, LC: loading capacity, EE: encapsulation efficiency, *Mean_SD. yn ¼ 3.
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Results SLMs are a suitable carrier for Hf because the free base is very soluble in oil (4180 mg/ml) and has a favorable oil-water partition coefficient (log p ¼ 6.5)21. A high drug concentration was obtained (Table 2), and the drug was encapsulated with a yield of 61.56–73.33%; EE of 61.43–76.32%; mean diameter of the particle sizes were 16.85–30.35 mm. Percentage recovery of SLMs The results of percentage recovery of the SLMs presented in Table 2 showed that percentage recovery values of unloaded SLMs (H0) was 87.74%, while the recovery values for Hf-loaded SLMs ranged from 60.48 to 73.33%.
Figure 1. Release profile of the SLMs formulations and commercial sample in SIF.
Mean particle diameter of SLMs From the values of mean particle diameter presented in Table 2, it can be seen that halofantrine-loaded SLMs prepared with the lipid matrix, (H1–H3) exhibited the highest mean particle diameter and the SLMs were mostly irregular in shape. Encapsulation efficiency (EE%) and loading capacity (LC) Table 2 shows the EE% and the LC of various batches of formulated Hf-loaded SLMs. EE% ranged from 61.43% for batch H1 to 76.32% for batch H3 SLMs. Lipid matrices containing 7% of Hf exhibited the highest encapsulation efficiency of 76.32% for SLMs formulated with SRMS, which was followed by lipid matrix containing 5% drug loading. The lipid matrix with 3% drug loading had the least encapsulation efficiency, however LC increased with increase in drug loading as shown in Table 2. Batch H3, with 7% halofantrine, exhibited the highest loading capacity of 31.43 g of halofantrine/100 lipid. Time-dependent pH stability studies of the formulations In Table 2, the SLMs containing no drug (H0) had a relatively variable pH of 4.87–5.30. However, the pH of the SLMs formulated with 3% halofantrine (H1) exhibited a relatively stable pH; H2 exhibited a significant increase in pH from approximately 3.70 to 4.03 at 3 months (p50.05) while the pH of batch H3 exhibited variable values within the 3 months of the stability study. In vitro drug release studies The results of in vitro release presented in Figures 1 and 2 showed the release profile in SIF and SGF respectively of halofantrineloaded SLMs and Halfan. In vitro kinetics release The result of the in vitro release of the different batches was fitted into zero order, first order, Higuchi and Peppas models to determine the kinetics of release as shown in Table 3. These dependent methods are based on differential mathematical
Figure 2. Release profile of the SLMs formulations and commercial sample in SGF. Table 3. Kinetic models for the release studies. Zero Batch H2 in SIF H2 in SGF H3 in SIF H3 in SGF Halfan in SIF Halfan in SGF
2
–0.750 –0.760 –0.360 0.514 –1.270 –0.460
r
Order
Higuchi
K0
r
2
Kh
7.392 2.155 6.408 2.541 3.019 0.266
–4.580 –5.460 –1.230 0.874 –7.280 –1.400
18.850 5.481 16.610 6.245 7.822 0.674
Key: r2 ¼ regression coefficient, K0 ¼ Zero order Kh ¼ Higuchi rate constant.
rate constant,
functions which describe the dissolution profile. Determining the regression coefficients assessed the fitness of the data into various kinetic models of the batches. The rate constants for the respective models were calculated from their slopes22. The statistical analysis showed a high significance difference (p50.05) between the halofantrine-loaded SLMs and the Halfan tablet. In vivo studies The hematological parameters of the mice (packed cell volume, hemoglobin content, red blood cell count, white blood cell count)
In vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles
DOI: 10.3109/10837450.2014.949270
5
Table 4. Heamatological parameters. Baseline
pretreatment
posttreatment
Group
PCV
Hb
RBC
WBC
PCV
Hb
RBC
WBC
PCV
Hb
RBC
WBC
E F G
38.0 34.0 36.0
10.8 14.2 14.5
9.9 9.3 8.7
10875 9150 8700
32.0 30.8 28.0
9.3 8.3 10.4
7.3 5.7 6.6
11075 9825 10725
37.5 31.5 21.33
13.7 14 11.5
8.6 8.8 5.5
7950 8650 7250
PCV (packed cell volume), Hb (heamoglobin content), RBC (red blood cell count), WBC (white blood cell count).
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Table 5. Percentage parasitemia reduction.
Groups E F G
AWA (g)
4 days post-infection treatment
Pre-TP
Post-TP
PP (%)
27.50 ± 0.06 30.70 ± 0.09 31.40 ± 0.07
3 doses of 0.4 ml of 0.5 mg/ml 3 doses of 0.1 ml of 2.5 mg/ml No treatment
27.00 ± 0.2 31.50 ± 0.5 54.40 ± 0.5
7.33 ± 0.14 4.50 ± 0.04 46.31 ± 0.62
72.96 ± 1.95 85.71 ± 2.11 14.89 ± 0.12
Group E received SLM containing halofantrine, group F received commercial sample of halofantrine and group G received no treatment; AWA is average animal weight (g), Pre-TP is Pre-treatment parasitemia, Post-TP is Posttreatment parasitemia, PP (%) is Percentage parasitemia (%).
and the effect of drug treatment (percentage parasitemia reduction) are presented in Tables 4 and 5, respectively. Peter’s Four day suppressive protocol studies were carried out in order to investigate the in vivo properties of the formulations and compare with the in vitro drug release and the reference drug. The result of anti-malaria properties of Hf-loaded SLMs presented in Tables 4 and 5 showed that halofantrine-loaded SLMs based on SRMS had a high parasitemia reduction. Histological studies The effect of the formulations in the liver and kidney of the mice are shown in Figures 3 and 4. The information from the study will give insight on the toxic effect of the halofantrine-loaded SLMs and the commercial tablet formulations (HalfanÕ ) on the vital organs of the body studied.
Discussion The percentage of SLMs recovered from the formulation showed that the unloaded SLMs had higher percentage recovery than the loaded SLMs while 5% Hf-loaded SLMs had the least percentage recovery of 60.48% (Table 2). This implies that the formulation technique adopted was reliable since more than 50% of the formulations were recovered. The SLMs containing 7% halofantrine (H3) had the highest particle size of 30.35 mm while the batch containing 3% (H1) had the least particle size of 16.85 mm which suggests that increased drug loading resulted in larger particle sizes of the SLMs Table 2. The SLMs showed stable, discrete, polydispersed, poorly flowable, mostly irregularly shaped Hf HCl-loaded SLMs with size range 16.85 ± 0.10 to 30.35 ± 0.33 mm were successfully prepared with the lipid matrices. The sizes of the SLMs were all within the micrometer range, indicating that the production was able to achieve the intended end point for oral administration as the size influences the rate of drug release and subsequent pharmacokinetics. This is because the rate of absorption, the speed of onset of effect and the duration of therapeutic response can all be determined by particle size for most route of administration; therefore the deliberate manipulation of particle size leads to a measure of control of activity and side effects of the formulations21. The time-dependent pH stability test was carried out to determine the pH stability of the SLMs formulations when stored
at room temperature at different time intervals. The H0 batch which contains drug unloaded SLMs formulation varied in pH from a value of 5.30 ± 0.02 on the day of formulation to 4.87 ± 0.03 after 90 days. The H1 batch which contains 3% of Hf varied from pH of 3.60 ± 0.04 on the day of formulation to 3.74 ± 0.01 after 90 days. The H2 batch which contains 5% of Hf varied from pH of 3.70 ± 0.01 on the day of formulation to 4.03 ± 0.05 after 90 days. The H3 batch which contains 7% of Hf varied from pH of 3.70 ± 0.05 on the day of formulation to 3.70 ± 0.10 after 90 days. The pH change upon storage for a period of 3 months as presented in Table 2, was insignificant hence there was little drug and excipient degradation during the storage period. Change in pH of a liquid drug formulation could be a function of degradation of the drug or the excipients. A prior stable drug may be affected by degradation of excipients with storage through generation of an unfavorable pH (increase or decrease) or reactive species for the drug14. However, the slight decline in the pH values in the formulations was not attributed to drug degradation since there was also a fall in the pH of the unloaded SLMs14. The pH decrease may be due to release of fatty acids from the lipid matrix. However, the little decrease in pH of the Hf-loaded SLMs indicated that the preparation would need a buffer to keep the pH more stable. The difference in mean particle size between the unloaded SLM (H0) and loaded SLMs (H1, H2 and H3) was quite significant; and the same applies for the percentage yield. The result of encapsulation efficiency and the loading capacity presented in Table 2 showed that the encapsulation efficiency and the loading capacity increased with increasing drug loading, SLMs containing 7% halofantrine gave the highest encapsulation efficiency and the loading capacity of 76.32% and 31.43% respectively, while the batch containing 3% gave the least encapsulation efficiency and the loading capacity of 61.43% and 10.84% respectively. The in vitro drug release study showed greater drug release from SLMs than from the commercial tablet formulation (HalfanÕ ) which could be attributed to the granular nature of the tablet as it has to dissolve before releasing the drug. There was also greater drug release in SIF than SGF (Figures 1 and 2). The zero order model is based on drug dissolution from dosage forms that do not disaggregate and release the drug slowly such as from matrix tablets with low soluble drugs in coated forms, osmotic systems etc23. None of the halofantrine loaded SLMs or
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Figure 3. Photomicrographs of liver sections of mice in groups E, F, G and H that is SLMs formulation, HalfanÕ , no treatment group and uninfected animals respectively. H and E 400.
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E
F
G
Figure 4. Photomicrographs of kidney sections of mice in groups E, F, G and H that is SLMs formulation, HalfanÕ , no treatment group and uninfected animals respectively. H and E 400.
H
E
F
G
H
the commercial tablet formulation (HalfanÕ ) showed zero order release kinetics. The first example of a mathematical model aimed to describe the drug release from a matrix system was proposed by Higuchi in 196114,24. It is based on a hypothesis that (i) initial drug concentration in the matrix is much higher than drug solubility; (ii) drug diffusion takes place only in one dimension; (iii) drug particles are much smaller than system thickness; (iv) matrix swelling and dissolution are negligible (v) drug diffusivity is constant; and (vi) perfect sink conditions are always attained in
the release environment23. The drug release kinetics showed that Higuchi plot of amount of drug release against square root of time for none of the batches of halofantrine-loaded SLMs were linear (r2 6¼ 0.9) except H3 which contains 7% of halofantrine-loaded SLMs in SGF as a medium. However, plot of log Q against log t according to Higuchi gave n value of 0.5 in most of the batches, indicating that diffusion-controlled process was predominant in these batches. All the mice infected with Plasmodium berghei exhibited the following signs: dullness, rough hair coat, paleness of the visible
DOI: 10.3109/10837450.2014.949270
In vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles
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Figure 5. Micrographs of the blood work of mice in group E (Bar represents 40 mm).
Figure 6. Micrographs of the blood work of mice in group F (Bar represents 40 mm).
Figure 7. Micrographs of the blood work of mice in group G (Bar represents 40 mm).
mucous membranes, weakness, diarrhea and thirst in accordance to the exhibited sign in the work of Saganuwan et al25. The in vivo studies showed that there were variations in the hematological parameters (PCV, Hb, RBC and WBC) due to the various effect of different treatments administered to the different groups as illustrated in Table 3. The data in Table 4 showed that group A which received SLMs containing halofantrine had 72.96% parasite clearance; group B which received commercial formulation of halofantrine had 85.71% parasite clearance while group C which received no treatment had 14.89% parasite clearance. The percentage parasitemia reduction properties of the SLMs formulation were comparable to that of the reference formulation. The in vivo pharmacodynamic properties of halofantrine-loaded SLMs were comparable to the in vitro release of halofantrine from SLMs. The formulated SLMs and commercial sample treatments yielded a more significant reduction in parasitemia compared to the negative control (group G). It has been reported that a change in relative or absolute weight of an organ after drug administration is an indication of the toxic effects of that drug26,27. The ability of
a drug to produce liver damage in vivo often results from the interaction of the uptake, biotransformation and elimination of potentially toxic compounds24,28. The observation from the histological studies conducted on the liver of the mice from various groups, showed varying degrees of hepatitis on the liver of the mice in groups E, F and G. Also, studies on the kidneys of various animals from various groups revealed different cases of mild tubular dilatation and very mild tubular degeneration for the mice in group G. Photomicrograph of liver section of mice from experimental groups E, F, G showing varying degrees of periportal mononuclear infiltration of cells-periportal hepatitis (arrow) while the group H shows normal portal area and hepatocytes (H and E 400). Photomicrograph of kidney section of mice from experimental groups E, F and H showing apparently normal glomerulus (GM) and renal tubules (white arrow) but mild tubular degeneration in G (black arrow) (H and E 400). The results of this study showed that short-term administration of halofantrine did not cause any significant changes in the body regarding the relative organs’ morphology of the treated mice.
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This implies that short-term oral administration of this drug had no negative effects on somatic growth. The micrographs of the fixed blood of mice are shown in Figures 5–7 where the parasitic infection are shown in black dots in the red blood cells in groups E–G.
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Conclusion It can be concluded that SLMs are good candidates for oral administration of halofantrine. The parasitemia reduction on the halofantrine-loaded SLMs was comparable with the reduction from the commercial sample, Halfan. The histological studies carried out also established the safety of the SLMs formulations as the results showed no evidence of disastrous side effect on the organs of mice used.
Declaration of interest This research work was funded by the International Foundation for Science (IFS), Sweden (IFS No. F/4467-1) and Organization for the Prohibition of Chemical Weapons (OPCW), The Hague. Dr. A. A. Attama is highly grateful to the Foundation and the Organization.
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