3.1. Geral
O presente trabalho visa o desenvolvimento de formulações microencapsuladas de sulfato de quinina, para a sua utilização na terapia antimalárica.
3.2. Específicos
♦ Obter e caracterizar físico-químicamente microesferas de PLGA contendo sulfato de quinina;
♦ Realizar um estudo preliminar de estabilidade das formulações
♦ Avaliar a cinética de liberação in vitro de sulfato de quinina microencapsulado;
4. ARTIGO I
ATIVIDADE ANTIMALÁRICA IN VIVO DE MICROESFERAS DE PLGA
CONTENDO SULFATO DE QUININA
IN VIVO ANTIMALARIAL ACTIVITY OF QUININE
SULPHATE-LOADED PLGA MICROSPHERES
Lúcio Figueira Pimentel1,2, Vanessa Carla Furtado Mosqueira3, Nereide Stela Santos-Magalhães1,4*
1
Universidade Federal de Pernambuco (UFPE), Laboratório de Imunopatologia Keizo Asami (LIKA), Recife, PE, Brazil
2
Departamento de Ciências Farmacêuticas (UFPE)
3
Universidade Federal de Ouro Preto (UFOP), Departamento de Farmácia, Ouro Preto, MG, Brazil
4
Departamento de Bioquímica (UFPE)
Keywords: Malaria; Plasmodium falciparum; Quinine sulphate; PLGA microspheres; Antimalarial
activity
* Corresponding author
Dr. Nereide Stela Santos Magalhães
Universidade Federal de Pernambuco (UFPE)
Grupo de Sistemas de Liberação Controlada de Medicamentos e Vacinas Laboratório de Imunopatologia Keizo Asami (LIKA)
Av. Prof. Moraes Rego, 1235, Cidade Universitária, 50670-901, Recife, PE, Brazil
Tel: +55 81 21268484; Fax: +55 81 21268485 E- mail: nssm@ufpe.br
Abstract
Quinine sulphate is the drug of choice for the treatment of the most cases of chloroquine resistant falciparum malaria. However, the quinine has a low therapeutic index and its use at large dose is usually associated with severe side effects, such as cinchonism. In order to circumvent this drawback, quinine sulphate- loaded microspheres were prepared and the antimalarial activity was investigated on mice infected with Plasmodium berghei. Quinine sulphate- loaded microspheres prepared using the double emulsion technique presented spherical shaped particles ranging from 1.53 to 7.65 µm. The drug encapsulation efficiency attained 50.34 ± 0.62%. The in vitro release profile of quinine sulphate from microspheres showed a bimodal behavior. A burst effect of 42% was initial observed, followed by a gradual drug release achieving 72% within 144 h. Microspheres administered in mice by subcutaneous route were at least as efficient as the free quinine sulphate administered by s.c. injection at 20 mg/kg for four days. Nevertheless, no efficacy improvement was achieved with microencapsulation of quinine sulphate. A parenteral microsphere-based formulation of quinine sulphate is however feasible, which can ensue a potential strategy to achieve a controlled therapeutic effect in the treatment of malaria.
1. Introduction
The malaria is a worldwide spread disease, which afflict and kill million people in the world. Drug resistance is probably the major drawback to achieve a successful malaria control. Since the emergence of multidrug-resistant falciparum malaria, the search for new antimalarial drugs and more efficient treatments involving drugs combinations are urgently needed [1]. About 40% of the world’s population lives in malaria-endemic areas [2]. Population of high contamination risk is that living under poverty, isolation, and inadequate health services with delay in receiving appropriate treatment [3]. The WHO estimates the prevalence of this disease in order of 300 million new clinical cases per year and it is predicted that two million people will pass away each year due to malaria [4]. Malaria is even believed as a major obstacle to the development and the economic advancement of countries within the tropical Africa [3].
Plasmodium falciparum malaria causes annually hundred of thousand of death, and a large
proportion of morbidity [5,6]. P. falciparum is the most pathogenic among the four species that infect humans, and is the principal cause of severe disease, since the other species of malaria rarely cause death or persistent sequela [2]. It is encountered throughout the tropics, and has developed resistance to almost all presently available drugs. Resistance has severely limited the use of chloroquine for P. falciparum [7]. Chloroquine-resistance is widespread, however quinine has been used to treat malaria for more than 350 years, and moderate resistance has only recently developed in limited geographic areas [8,9]. Quinine is consequently an effective replacement for chloroquine and remains a first- line drug for the treatment of falciparum malaria, including severe cases [7, 10]. Suggested regimens by WHO are based on quinine administered intravenously or intramuscularly for the treatment of severe malaria in both children and adults [2, 11].
There are several pharmacological factors that may have contributed to the upholding efficacy of the quinine. First, it quite often causes annoying side effects, so people are unlikely to
take the drug unless absolutely necessary. Second, quinine has a relatively short elimination half- life (11 hours) [12]. That requirement for repeated administrations, less probability of subtherapeutic concentrations in the blood stream and little evolutionary selective resistance [8].
Quinine has a small therapeutic index, and its use is often associated with side effects such as fever, confusion, respiratory arrest, and arrhythmias. Even standard doses produce symptoms of “cinchonism,” i.e., tinnitus, giddiness, blurred vision. Hypoglycaemia and hypotension are further more serious side effects of a rapid administration of quinine [13].
These constraints reduce the patient compliance and the usefulness of quinine in P.
falciparum treatment. In this way, a controlled release dosage forms may be valuable to improve the
efficacy of this drug by reducing the frequency of administrations and by decreasing plasma level fluctuations [14]. Controlled delivery systems provide more uniform concentration of drug at the absorption site and are able to maintain plasma concentration within the therapeutic range. Simultaneously, they minimize side effects, and improve patient compliance to treatment. The drugs associated to these formulations could present more suitable administration regimens and habitually are less toxic when compared with the free drug administration [15].
A variety of synthetic biodegradable polymers have long been used to develop controlled drug delivery systems. Biodegradable microspheres prepared from a copolymer of poly(lactic-co- glycolic acid) (PLGA) have been used as oral or injectable drug delivery systems [15]. The adjustable degradation rate of these biodegradable devices provides an efficient means to control and prolong the release of encapsulation of a variety of compounds [16].
Ogawa et al., 1988 [17], developed a water- in-oil- in-water (w/o/w) emulsion technique to encapsulate water soluble active agents with higher efficiency. Since then, several studies report on the encapsulation of hidrophylic drugs into microspheres using this technique [13,18-22].
The water solubility property of quinine sulphate turn it into a actual candidate to be loaded in microspheres prepared by the water-oil-water double emulsion method, which is suitable for
parenteral administration. It is expected that this kind of formulation could able to modify the pharmacokinetics of the entrapped drug, producing sustained drug blood concentration.
An attempt was thus made in this study to develop and characterize a formulation of poly (D,L-lactide)-co-glycolide microspheres containing quinine sulphate as a controlled release system for the treatment of malaria. Their in vitro kinetic profile and their in vivo efficacy were evaluated on mice infected with P. berghei using a four-day standard test.
2. Materials and Methods
2.1. Materials
Poly (D,L- lactide-co-glycolide) acid (PLGA) 50:50 (0.17 dl.g-1), was purchased from Birmingham Polymers Inc. (Alabama, USA); Poly vinyl alcohol (PVA, MW. 30 000-70 000); Polyethylene Glycol (PEG, mw. 4000), D (+) Trehalose dihydrate and standard quinine sulphate were obtained from Sigma-Aldrich Inc. (St. Louis, USA). Quinine sulphate was obtained from Henrifarma (São Paulo, Brazil). HPLC grade acetonitrile, analytical grade solvents and reagents were obtained from Merck (Darmstadt, Germany). Ultrapure water was obtained by a Milli-Q purification System from Millipore (Molshein, France). Plamodium berghei strains were kindly supplied by Dr. Érika Martins Braga, from the Department of Parasitology of the Federal University of Minas Gerais, Brazil.
2.2. Methods
2.2.1. Preparation of empty and quinine sulphate–loaded PLGA microspheres
The microspheres were formulated according to the modified method previously described [23], using the double emulsion solvent evaporation technique. Several batches were developed by
modifying the continuous phase pH, the polymer/drug ratio, and the solvent organic phase volume, as an attempt to derive more stable formulations (Table1).
Briefly, PLGA (450 mg) was dissolved in methylene chloride (8, 10 or 12 ml) and homogenized with several amounts of quinine sulphate (QS) (10, 25 or 50 mg) dissolved in deionized water (five milliliters) with PEG 4000 (400 mg), using a mechanical homogenizer (Ultra- Turrax T25, Ika, Germany) at 8,000 rpm for 60 seconds in an ice bath, resulting in a simple
emulsion (w/o). The result ing emulsion was added to a continuous phase constituted of 50 ml PVA solution (0.5% w/v) at pH (6, 8, 9 or 10) adjusted with diethylamine solution (10% w/v) and emulsified at 8,000 rpm for 30 seconds, resulting in a double emulsion (w/o/w). This emulsion was then stirred with a four-blade propeller (Mechanic stirrer RE 162/P, IKA, Germany) at room’s temperature at 400 rpm for four hours to allow the solvent evaporation followed by the microspheres formation. The microspheres were collected by centrifugatio n (Kubota KN-70 centrifuge, Japan) at 3,000 rpm for five minutes and washed three times with 20 ml of deionized water. Microspheres were finally dispersed with 1.0 % threalose aqueous solution (w/v), which was frozen overnight at –80ºC before lyphilisation (EZ-DRY, FTS System, New York, USA) operated at 200 bars for 16 hours. Unloaded microspheres were prepared under the identical conditions. The PLGA- microspheres were stored at 25ºC ± 2 ºC in vacuum desiccators.
2.2.2. Characterization of quinine sulphate-loaded microspheres
Morphological Analysis
The morphology of microspheres was analyzed by light microscopy (Olympus CH-2, USA) to evaluate the microsphere homogeneity and the particle size. The average particle size was visually determined by counting 200 microspheres. The shape and surface morphology of dried microspheres were examined by scanning electron microscopy (SEM), using an electron
microscope (JSM-T200, JEOL, Japan). A sample of lyophilized microspheres was ressuspended in distilled water to obtain a homogeneous suspension, dry in vacuum desiccators, and placed in a surface of a metallic support, which was coated with colloidal gold using a sputter module in a high- vacuum evaporator (JFC-1100, JOEL, Japan). Microspheres were then examined and photographed under the microscope at 10 kV.
Quinine sulphate assay by HPLC Method
The Pharmacopoeia method [24] was adapted to quantify the quinine sulphate content using a computer-assisted HPLC System (Shimadzu SCL-6B, Japan) composed of a UV/VIS detector and manual injector with a 20 µl loop. The chromatographic run was performed using a µBondapack C18 column (10 µm particle size, 125 Å porous size, 300 mm × 3.9 mm I.D. Waters, USA) and a
mobile phase of HPLC grade acetonitrile/ultrapure water (Milipore ultra pure water system) / 10%
diethylamine solution (w/v)/ 10% orthophosphoric acid solution (w/v) (80:16:2:2), pH adjusted to 2.6 with 10% orthophosphoric acid solution (w/v). Sample aliquots (20 µl) were injected and eluted with the mobile phase at a flow rate of 1.5 ml.min–1. The quinine sulphate peak was verified at a wavelength of 250 nm (0.005 a.u.f.s.) with a retention time of about 4.5 min. The amount of quinine sultafe into microspheres was determined through the standard calibration curve, which was prepared for concentrations varying from 2.5 µg.ml–1 to 50 µg.ml–1. A stocked standard solution (1 mg.ml–1) was prepared with ten milligrams of standard quinine sulphate in 50 ml of mobile phase. Each experiment was performed in triplicate.
Content of quinine sulphate and encapsulation efficiency
Accurately weighed samples of loaded microspheres (5 mg) were dissolved in methylene chloride (10 ml) under ultrasound agitation for 15 minutes with the aim of determining the drug content after the dissolution of microspheres. One milliliter of resulting solution was diluted with
five milliliters of HPLC mobile phase, filtered through a 0.45 µm membrane filter (Millipore) and injected into an HPLC system to obtain the drug concentration.
The entrapment efficiency was evaluated from the ratio of the quinine sulphate content into the microspheres and the theoretical amount of this drug into microspheres, calculated by the quinine sulphate amount used for preparation of microspheres.
Percentage entrapment efficiency (%) =
es microspher into QS of content l Theoretica es microspher into content sulphate Quinine × 100
Preliminary stability studies of quinine sulphate loaded-microspheres
A preliminary stability studies of quinine sulphate encapsulated into lyophilized PLGA microspheres was performed by measuring the lost of quinine sulphate content in the microspheres along the time, when the microspheres were stored at 25ºC. At pre-determined time intervals, samples of microspheres were collected and morphological characteristics were observed. Assay by HPLC method was used to determine the quinine sulphate content in the microspheres as above described.
In vitro kinetic release of quinine sulphate-loaded PLGA microspheres
The in vitro release profile of quinine sulphate from microspheres was determined along with the procedure by Herrmann and Bodmeier, 1998 [14]. Drug- loaded PLGA microspheres (5 mg) were resuspended in five milliliters of 20 mM phosphate buffered saline (PBS, pH 7.4). The tubes were incubated in a horizontal shaker with a water bath (Polytest 20, Bioblock Scientific 86507) at 37ºC ± 1ºC and shaken at 180 rev.min–1 with samples intervals (1, 24, 48, 72, 96, 120 and 144 hours). Samples of two milliliters of the dissolution medium were withdrawn and centrifuged at 2,800 rpm for five minutes in order to separate any microsphere present in the sample. The supernatant was removed and the amount of quinine sulphate was measured by the above described
HPLC method. Assays were performed in triplicates and results were expressed in percentage of the mean values and their corresponding standard deviations.
2.2.3. Antimalarial activity of quinine sulphate-loaded PLGA microspheres
The in vivo antimalarial activity of quinine sulphate- loaded PLGA microspheres was determined by means of the "four-day suppressive test" described by Peters et al., 1975 [25]. Tests were performed against Plasmodium berghei NK-65, which is sensitive to all current antimalarial drugs and is characterized by a high mortality in mice, providing thus a suitable model to estimate efficacy in reducing parasitemia. This strain was frozen stored in liquid nitrogen with glycerolyte. It was also maintained through weekly blood passages in mice infected by intraperitoneal route (i.p.) using the heparin as an anticoagulant in saline solution.
Female, seven week aged, adult Swiss albino mice weighing 27 ± 3 g from “Ouro Preto Federal University animal facility” were inoculated by retro orbital route with 1×106 Plasmodium
berghei infected-red blood cells in 0.2 ml inoculum’s obtained from the donor mouse with rising
parasitaemia. Animals were firstly infected on the day namely zero (D+0), and were randomly divided in four groups of ten animals per cage.
Two hours later, the treatment started with four consecutive daily doses of quinine sulphate microspheres (20 mg/kg per day); or unloaded microspheres; quinine sulphate solution (20 mg/kg per day) as a free drug. Untreated controls received 0.5 ml of saline (0.9%) by s.c. route. All microspheres preparations were subcutaneously (s.c.) administered in 0.5 ml of suspens ion with saline 0.9% and 0.1% Tween 80.
Thin blood smears were made from tail blood from untreated controls and from treated animals on the days (D+4, D+6, D+8 and D+10), fixed with methanol, and stained with Giemsa. Levels of parasitemia (the percentage of infected erythrocytes, ring stages and schizonts) were
microscopically determined (magnification 1,000×) by examining 3,000 cells. The percentage of parasitemia suppression was calculated for each group in comparison with infected non treated control group.
At days 5, 7 and 9, the red blood cell content (RBC) of treated animals was indirectly estimated by hematocrit percentage and compared with the untreated groups. The parasitemia levels, the variation in body weight from D0 to the D10, and the mean sur vival time were used to evaluate the in vivo antimalarial activity.
2.2.4. Statistics
All erythrocyte counts and parasitemia levels are expressed as mean value ± S.D (n= number of surviving mice at a determined time of treatment). The parasitemia data were analyzed by the one-way ANOVA test. Mean survival times were compared using Student's t-test considering a significance level of 5%.
3. Results and discussion
3.1. Preparation of quinine sulphate-loaded microspheres
Pre-formulation studies were performed to guide the choice of the concentrations of constituents for obtaining stable formulation of quinine sulphate- loaded microspheres. The preparations conditions of quinine sulphate-loaded PLGA microspheres are listed in Tables 2 to 4.
The solvent evaporation method is a basic technique to prepare microspheres and involves two major steps, namely the formation of stable droplets of the drug-containing polymer solution and the subsequent removal of solvent from these droplets. Some factors can make hard this manufacturing, such as the polymer concentration, the solvent nature, the phase volume ratio of organic and aqueous phases, the continuous phase pH, the used stabilizer, the time and stirring speed, etc. [15, 26, 27]. Some of these effects were investigated, but further information is required to elucidate the complete processes of microspheres formation.
Table 1
Formulation of quinine sulfate- loaded PLGA microspheres.
Formulations Constituents 1 2 3 4 5 6 7 8 PLGA 50:50 (g) 450 450 450 450 450 450 450 450 Quinine sulphate (g) 25 25 25 25 50 10 25 25 PEG 4000 (mg) 400 400 400 400 400 400 400 400 PVA solution 0.5% (w/v) (ml) 50 50 50 50 50 50 50 50 pH of PVA solution 0.5% 6 9 10 11 10 10 10 10 Methylene choride (ml) 10 10 10 10 10 10 8 12
The effect of the pH of the aqueous phase on the second emulsification process was examined (Table 2). It was observed that the pH of the external aqueous phase plays an important role on the entrapment efficiency of quinine sulphate into PLGA microspheres. The alkaline pH of the external phase raises the retention of drug in the internal aqueous phase during the manufacturing of microspheres by the double emulsion method. This strategy was used in accordance with Bodmeier and McGinity [27], to reduce drug loss in external aqueous phase. The effect of the continuous phase pH in the encapsulation efficiency of the quinine sulphate in PLGA microspheres was similar at pH 9 and 11, but increased at pH 10, achieving the highest encapsulation rate (50.34 ± 0.62).
Table 2
Evaluation of the influence of the aqueous phase pH on the quinine sulphate entrapment efficiency in PLGA microspheres.
pH of the aqueous continuous phase
Quinine sulphate encapsulation rate (%)
6 30.57± 0.60
9 47.01 ± 0.33
10 50.34 ± 0.62
11 44.73 ± 2.86
Data: mean ± S.D. (standard deviation), n=3.
The low encapsulation efficiency could be explained by the hidrophilicity of quinine sulphate, its affinity to the aqueous phase and a much higher tendency to diffuse into water phases. Quinine has two basic nitrogen atoms of pKa approximately 4.3 and 8.5 (the quinoline nitrogen
having the lower basicity). The molecule is thus likely to be at least partially protonated over the whole pH range between two and eight [28]. This effect allows decreasing the solubility of drug in alkaline solutions, and subsequently limiting potential drug loss into the external PVA solution.
Previous investigations described that the loss of the encapsulated hydrophilic active agent during the microspheres formatio n process essentially resulted from its diffusion in the inner water phase to the outer water phase [29]. Furthermore, the entrapment efficiency can be affected by the stability of o/w and w/o/w emulsions, the solvent removal rate, the interactions among drug, polymer and solvent, and particle size [30].
Despite the fact of the quinine sulphate physicochemical properties include photo sensibility and chemical instability, an encapsulation of (10, 25 or 50 mg) quinine sulphate was achieved into microspheres prepared with 450 mg of PLGA and pH 10 of external aqueous phase. At the 1:18 drug-polymer ratio, the best quinine sulphate encapsulation efficiency (50.34 ± 0.62) and more stable microspheres were attained for an initial payload of 25 mg of QS. The encapsulation efficiency of the quinine sulphate into PLGA microspheres for different drug-polymer ratio is presented in Table 4. The encapsulation efficiency decreases at higher quinine sulphate concentrations achieving 41.2 for a 1:9 drug-polymer ratio.
Table 3
Encapsulation efficiency of quinine sulphate into PLGA microspheres (450 mg of polymer)
Quinine sulphate amount (mg) Encapsulation Efficiency (%)
10 43.41 ± 0.70
25 50.34 ± 0.62
50 41.21 ± 0.87
The influence of the solvent volume at a constant aqueous phase volume on the QS-MS encapsulation efficiency was evaluated as shown in Table 3. Methylene chloride was used in the organic phase because of its ease removal, and excellent ability to dissolve polymer and allow the microspheres formation. The drug encapsulation efficiency decreased when eight or twelve milliliters was used. This can probably be accounted for the primary emulsion instability and the diffusion of quinine sulphate amount not emulsified to the external aqueous phase during the solvent evaporation step, explaining the low encapsulation efficiency obtained. Some publications have evaluated the relevance of the primary emulsification stage for increasing entrapment efficiency and for controlling the internal structure of microparticles prepared using the (w/o/w) emulsification solvent evaporation technique [30, 31].
Table 4
Encapsulation efficiency of quinine sulphate into PLGA microspheres (450 mg of polymer)
Solvent volume (ml) Encapsulation Efficiency (%)
8 36.95 ± 0.13
10 50.34 ± 0.62
12 41.40 ± 0.13
Data: mean ± S.D. (standard deviation), n=3.
When eight milliliters of methylene chloride was used, the higher polymer concentration leaded to a less effective emulsifying process. This result is in accordance with those of Schlicher et
al., 1997 [32], who observed a decreasing in the encapsulation efficiency of a water soluble
antimalarial agent, after increasing the volume of the internal phase at a fixed polymer concentration and volume (i.e. increasing the ratio of inner aqueous phase/organic phase in primary