Nanocurcumin: a novel antifilarial agent with DNA topoisomerase II inhibitory activity.

http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(5): 395–407 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.869823

ORIGINAL ARTICLE

Nanocurcumin: a novel antifilarial agent with DNA topoisomerase II inhibitory activity Mohammad Ali1, Mohammad Afzal2, Sekh Abdul Nasim3, and Istaq Ahmad4

Journal of Drug Targeting Downloaded from informahealthcare.com by Ondokuz Mayis Univ. on 11/08/14 For personal use only.

1

Nanomedicine Lab, Hamdard Nanobiotechnology Center for Advanced Research, Faculty of Engineering & Interdisciplinary Sciences, Hamdard University, New Delhi, India, 2Dabur Research and Development Centre, Dabur India Limited, Ghaziabad, India, 3Environmental Biotechnology Laboratory, Department of Botany, Hamdard University, New Delhi, India, and 4Department of Biotechnology, Jamia Millia Islamia, New Delhi, India Abstract

Keywords

Objective: The aim of this study is to evaluate the antifilarial, antiwolbachial and DNA topoisomerase II inhibitory activity of nanocurcumin (nano-CUR). Methods: Nano-CUR formulations (F1–F6) were prepared using free radical polymerization and were characterized by particle size, morphology, encapsulation efficiency and in vitro release kinetics. Antifilarial potential was evaluated in vivo against Brugian filariasis in an experimental rodent model, Mastomys coucha, by selecting the formulation that maximized parasite elimination characteristics. Wolbachial status was determined by PCR and a relaxation assay was used to estimate DNA topoisomerase II inhibitory activity. Results: Nano-CUR (F3) having a 60 nm diameter and 89.78% entrapment efficiency showed the most favorable characteristics for the elimination of filarial parasites. In vivo pharmacokinetic and organ distribution studies demonstrate significantly greater C(max) (86.6 2.56 ng ml1), AUC0–1 (796 89.8 ng d ml1), MRT (19.5 7.82 days) and bioavailability of CUR (70.02%) in the organs from which the adult parasites were recovered. The optimized nano-CUR (F3) (5 5 mg/kg, orally) significantly augmented the microfilariciadal and adulticidal action of CUR over free CUR (5 50 mg/kg, orally) or Diethylcarbamizine (50 mg/kg, orally) against the Brugia malayi Mastomys coucha rodent model. The PCR results showed complete elimination of wolbachia from the recovered female parasites. Interestingly, nano-CUR was also found to be a novel inhibitor of filarial worm DNA topoisomerase II, Setaria Cervi in vitro. Conclusion: This study recognizes the beforehand antimicrofilarial, antimacrofilarial, antiwolbachial activity of nano-CUR (F3) over free forms and additionally its strong inhibitory action against the major target filarial parasite enzyme DNA topoisomerase II in vitro.

Brugia malayi, diethylcarbamizine, DNA topoisomerase II, lymphatic filariasis, nanocurcumin, wolbachia

Introduction Lymphatic filariasis (LF) is a neglected, mosquito-borne tropical disease. It is endemic in 81 countries putting 1.2 billion people at risk globally with an estimated 120 million infected [1]. The available standard drugs, Ivermectin (IVM) and Diethylcarbamizine (DEC), reduce microfilaria (Mf) density of tolerants and are not very effective against the adult worms [2]. The World Health Organization through its Global Program to Eliminate Lymphatic Filariasis shares this concern since the drugs administered in mass drug administration programs exhibit adverse effects in many cases [3,4]. Therefore, in past decades, significant efforts have been made to improve antifilarial chemotherapy by identifying a number of compounds that offer promising antifilarial activity [5,6]. Drug development programs have also used novel therapeutic Address for correspondence: Mohammad Ali, Nanomedicine Lab, Hamdard Nanobiotechnology Center for Advanced Research, Faculty of Engineering & Interdisciplinary Sciences, Hamdard University (Jamia Hamdard), New Delhi-110062, India. Tel: +91-8826416916. Fax: +9111-26059663. E-mail: [email protected]

History Received 21 July 2013 Revised 19 October 2013 Accepted 24 November 2013 Published online 30 January 2014

targets from the myriad of parasitic enzymes (e.g. DNA topoisomerase II) and metabolic pathways to combat this deliberating parasitic disease [7]. Despite these efforts, the treatment and hope for eradication of LF still remains a formidable task. The unavailability of vaccines as well as the increased risk of worms becoming drug-resistant point up the urgent need to find a new drug that is effective against LF. A large number of natural products, plant compounds and extracts have been evaluated as potential antifilarial agents against different filarial nematodes, both in vitro and in vivo [8–11]. Although these compounds have exhibited encouraging filariciadal action against the target parasitic species, the majority have failed, largely due to toxicity, pharmacokinetic issues and the complex location of the disease. Therefore, we need to improve the existing antifilarial drug development programs through alternative strategies such as the use of nanodrug delivery systems (nanoDDSs) that take into account the pharmacokinetics and bioavailability of antifilarial agents. Curcumin (CUR) [1,7-bis(4-hydroxy-3-methoxyphenyl)1,6-heptadiene-3,5-dione], a yellow pigment, is the principal curcuminoid of the popular Indian spice turmeric. This yellow

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Table 1. Composition, mean particle size and encapsulation efficiency of nano-CUR formulations.

S.no


1 2 3 4 5 6

Formulation code nano-CUR nano-CUR nano-CUR nano-CUR nano-CUR nano-CUR

(F1) (F2) (F3) (F4) (F5) (F6)

NIPAAM (mg)

VP (ml) (1% w/v)

AA or PEG (ml) (1% w/v)

Mean particle size (nm)

Encapsulation efficiency

95 90 90 80 80 80

5 5 5 10 10 10

0 5 (PEG) 5 (AA) 10 (PEG) 10 (AA) 5 (PEG) þ 5(AA)

110 0.034 65 0.01 60 0.012 133 0.071 174 0.098 378 0.640

82.49 0.467 80.27 0.279 89.78 0.389 85.29 0.031 87.67 0.091 91.307 0.973

polyphenol compound possesses a wide range of therapeutic advantages and has been shown to have antioxidant, antiinflammatory, anticarcinogenic and chemopreventive properties, both in vivo and in vitro, on many cancer cell lines [12–15]. Some investigations have confirmed that CUR possesses potent anti-parasitic activity against several human parasitic diseases; however, many of these studies concentrate on malaria and leishmaniasis. There are only a few reports available regarding the antihelminthic activity of CUR. These reports have demonstrated its schistosomicidal activity and glutathione S-transferase (GST) inhibitory action in a bovine filarial worm [16–21]. Also, no serious effort has been undertaken to use this therapeutic polyphenol as an antifilarial agent. Recently, a molecular-based study by Nayak et al. [22] provided attention-grabbing evidence of CUR-induced apoptosis in the filarial worm Setaria cervi. In other reports, it has also been shown to act as an effective nematicidal inhibitor in vitro against GlutathioneS-Transferase of Meloidogyne incognita [23,24] CUR has been viewed by health scientists as a poor therapeutic agent due to its low aqueous solubility and bioavailability resulting in numerous approaches, including nanoDDSs, to circumvent these pitfalls [25–29]. Recently, nanoparticle-encapsulated CUR or nano-CUR, a water soluble form of CUR having rapid absorption, enhanced plasma concentration and improved bioavailability has been reported to improve the anti-leishmanial and anti-malarial activity of CUR, demonstrating that nano-CUR can be a potent antiparasitic agent against these diseases [30–33]. Though the medical benefits of CUR have not been fully explored for filariasis elimination, enhancing its bioavailability to target filarial parasites in the lymphatic system may nonetheless bring this promising natural compound to the forefront as a strong antifilarial agent for treatment of LF. During the last few years, nanoformulations of antifilarial drugs have been shown to significantly enhance the antifilarial activity of encapsulated drugs, suggesting drug delivery approaches as alternative strategies for the improvement of antifilarial chemotherapy [34–37]. In this study, we have employed N-isopropylacrylamide polymeric nanoparticles prepared by crosslinking with N-vinyl-2-pyrrolidone and acrylic acid or polyethylene glycol through free radical polymerization to improve the antifilarial effectiveness of CUR against the human lymphatic parasite Brugia malayi. Nano-CUR formulations were characterized for their particle size, morphology, encapsulation efficiency and in vitro release kinetics. The antifilarial potential was evaluated in vivo against Brugian filariasis in an experimental rodent model, Mastomys coucha, by selecting the formulation that possessed the greatest number of characteristics for

parasite elimination. The clearance of wolbachia from the recovered worms was detected by PCR.

Materials and methods N-isopropylacrylamide (NIPAAM), N-vinyl-2-pyrrolidone (VP) and 3-(4,5-dimethythiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) were purchased from Akcros Chemicals Ltd (Manchester, UK). Curcumin (CUR), Acrylic acid (AA), polyethylene glycol (PEG), N,N-methylene bis-acrylamide (MBA), potassium di sulphate, ammonium per sulphate (APS) and ferrous ammonium sulphate (FAS) were purchased from Sigma-Aldrich (St. Louis, MO). All the solvents used were of HPLC grade and obtained from Merck (Worli, Mumbai). Milli-Q grade water was used in all the experiments. Preparation of NIPAAM/VP/AA or PEG co-polymeric nanoparticles Polymeric nanoparticles of NIPAAM, VP and AA or PEG were synthesized through free radical polymerization as described by Bisht et al. [38] with slight modifications. Hexane was used to recrystallize NIPAAM, where as VP, AA and PEG was distilled before formulation development. These monomers were dissolved in 10 ml of double distilled water at different concentrations (Table 1). Thereafter, 40 ml of MBA (0.05 g/ml) was added to the each aqueous solution of monomers as a cross linking agent and dissolved oxygen was removed by passing nitrogen gas for 20 min. The polymerization was performed at 30 C for 24 h in nitrogen atmosphere by adding 25 ml of FAS (0.5% w/v) and 30 ml APS. After 24 h, whole the polymeric aqueous solutions were dialyzed for 16 h using a spectrapore membrane dialysis bag (12 kDa cut off) to remove any residual monomers. The dialyzed solutions were then lyophilized immediately to obtain a dry powder for further use. Loading of CUR A post-polymerization method was used to load CUR in the polymeric nanoparticles [38]. Free CUR (Fr-CUR) was dissolved in chloroform at a concentration of 10 mg/ml. For loading of CUR, the chloroform-CUR solution was added to the 10 ml of water in which 50 mg of the lyophilized polymeric nanoparticles formulations (F1–F6) powder were dispersed separately in 40 ml beaker. The solutions were vortexed for 20 min and sonicated in a water bath sonicator for 10 min to achieve physical entrapment of CUR into the hydrophobic core of polymeric nanoparticles. The drug loaded nanoparticles were then lyophilized to dry powder for further use.

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Physiochemical characterization of nano-CUR formulations


Morphology, particle size and size distribution Morphology of the nano-CUR formulations (F1–F6) were observed by scanning electron micrographs (SEM) taken with a scanning electron microscope (Hitachi S-300 N, Erkrath, Germany). The samples were gold coated (at about 100 A) on metal stabs with the aid of double-sided adhesive tape in KSE24M high vacuum evaporator. Selected regions that were scanned depicting distinct morphological feature were photographed. The particles size and size distribution of nano-CUR formulations were determined by Dynamic light scattering (DLS) using a particle size 90 ZS Malvern Instruments. The intensity of scattered light was detected at 90 to an incident beam. The freeze dried powder was dispersed in aqueous buffer and measurements were done, after the aqueous micelles solution was filtered with a Millipore micro filter having an average pore size of 0.2 mm. All the data analysis was performed in automatic mode. Measured size was presented as the average value of 20 runs, with triplicate measurements within each run. Entrapment efficiency To determine entrapment efficiency (%EE), CUR loaded NIPAAM/VP/AA or PEG nanoformulations were separated from unentrapped or Fr-CUR by centrifugation at 5000 rpm (Remi C24, Remi, India) for 25 min at room temperature. The amount of the Fr-CUR in supernatant was measured by High performance liquid chromatographic (HPLC) method as described in the ‘‘HPLC analysis of CUR’’ section. The quantity of CUR loaded in the nanoparticles was calculated as the difference between the total amount of CUR added to the formulations medium and the Fr-CUR, i.e. non-entrapped CUR in the supernatants. The % EE was calculated by following formula: ð½Quantity of CUR added into the system ½Quantity of Fr CUR in the supernatantÞ EECUR ð%Þ ¼ ½Quantity of CUR added into the system 100 In vitro release kinetics of CUR from polymeric nanoparticles formulations The in vitro release studies of CUR from polymeric nanoCUR formulations were performed using dialysis sacs. The nano-CUR formulations were taken in a cellulose membrane dialysis tube (MW: 12 000 kDa) and the dissolution studies were performed by rotating at 250 rpm in 500 ml of phosphate buffer saline (PBS) at pH 7.4, separately. At regular intervals of time, 5 ml of the sample was withdrawn and replaced with 5 ml of fresh media to maintain sink conditions. Withdrawn samples were filtered through syringe filter (0.22 mm) and analyzed for CUR by HPLC method as described in the ‘‘HPLC analysis of CUR’’ section. Pharmacokinetics and organ distribution study of nano-CUR Drug concentration profile achieved in the blood stream after its administration is an important factor determining the

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therapeutic efficacy of the drug. Pharmacokinetics and organ distribution studies of Fr-CUR and nano-CUR (F1–F6) were carried out on Swiss albino mice (weight 200–250 g), obtained from the Animal house facility of Jamia Hamdard. These animals were housed in polypropylene cages, with free access to model laboratory food pellets (Bengal gram soaked in water) under standard conditions (temperature: 25 2 C; relative humidity: 55 5%). Prior the start of the experiment, the animals were starved for 12 h and divided into eight groups having five rats in each group. The rats received FrCUR and nano-CUR (F1–F6) at 25 mg/kg body weight, orally. One group also received sterile phosphate buffered saline solution (pH 7.2) and treated as control. Blood samples were collected from the post orbital venous plexues at 5 min, 15 min 30 min, 1 h, 2 h, 4 h, 8 h, 12 h post-injection and placed in heparinised test tubes. Plasma was immediately separated from each sample by centrifugation at 3000 rpm for 10 min and analysis of CUR concentration was determined by HPLC as described in the ‘‘HPLC analysis of CUR’’ section. After 12 h, the rats were sacrificed following which the lymph nodes, lungs, heart, testes, colon and small intestine were isolated. These organs were homogenized by Micro Tissue Homogeniser (Remi Ltd., Mumbai, India) along with a suitable amount of phosphate buffer (pH 7.4), 1.5 ml of methanol was added to homogenate and kept for 30 min. After appropriate dilution of supernatants with the mobile phase, CUR content was determined by the HPLC method as described. HPLC analysis of CUR CUR was analyzed on reverse phase HPLC column (Supelco column 516, C18, 5 mm, 250 mm 4.6 mm). The HPLC system (SCL-10 AVP, Shimadzu, Japan) consisted of a binary pump (LC-10 ATVP, Shimadzu, Japan) and a UV-VIS detector (SPD-10 AVP, Shimadzu, Japan). The mobile phase was composed of a mixture of acetonitrile–acetic acid (1%) in a ratio of 40:60 v/v and passed through the column with a flow rate of 1.2 ml/min [39]. The detection wavelength was set at 425 nm. The assay was linear (R2 ¼ 0.999) in the concentration range of 0.1–40 mg/ml with the lowest detection limit at 0.005 mg/ml. The percentage recoveries ranged from 99% to 100.1%. All the samples prior to the analysis were filtered through a 0.22 mm pore size membrane filter. Determination of nano-CUR (F3) nanoparticles efficacy in vivo Host parasite model, infection induction Mastomys coucha males (6–8 weeks old) weighing 200–250 g were obtained from the National Laboratory Animal Center, CDRI, Lucknow and used as an experimental animal model. The animals were housed in hygienic and standard conditions of light (12 h light/12 h dark) and temperature (28 C). These were fed on rodent diet and water ad libitum. The infection was induced by inoculating 110 infective larvae (L3) of Brugia malayi parasites (recovered from mosquito vector Ades aegypii) to Mastomys coucha, subcutaneously. Three months post-L3 larvae injection, the infected animals were selected on the basis of rising microfilaremic load in systemic circulation.

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Sample preparation, dosage and treatment groups The lyophilized nano-CUR (F3) was finely resuspended in distilled water, whereas the Fr-CUR, and DEC were solubilized in distilled water with the help of 0.1% Tween80. The animals were divided into seven groups, having five animals in each group. Of these, two groups received oral administration of five doses of 50 mg/kg body weight Fr-CUR and 5 mg/kg body weight nano-CUR (F3) at every third day (i.e. on day 1, 4, 7 and 13). Two other groups treated as control received phosphate buffer saline and nanoparticles without CUR (Blank). Two more groups received five doses of Fr-CUR and nano-CUR (F3) at every third day, each following administration of 50 mg/kg DEC, orally. An additional group of DEC received free DEC at 50 mg/kg orally, to serve as a DEC control was also included. All the experiments were done in duplicate. Evaluation of parasitological parameters Microfilaricidal (MIC) and macrofilaricidal (MAC) efficacies were evaluated as described with some modifications [40]. For determining MIC efficacy, an aliquot of 10 ml of blood sample was taken from the tail vein of animals on days 0, 8, 15, 30 and at every fortnight till days 105. Blood smears were made on clean glass slides just before the initiation of treatment (i.e. on 0 day) and after infection. The slides were observed under a compound microscope to see the percent change in microfilaremic density. Variation in Mf count in comparison to pretreatment level was expressed as percent change in microfilaremia. After MIC observation (on days 105), treated and control Mastomys coucha were euthanized and various organs were isolated (lungs, heart, testes and lymph nodes), teased gently in phosphate buffered saline (PBS, pH 7.2) to recover the adult worms. All the surviving females were teased individually in a drop of saline and the condition of the embryonic stages in the uteri was examined, microscopically. Any abnormality or death/deformation detected in the uterine contents was considered as sterilization effect of the treated samples on females and the percent females’ sterilization were assessed [41]. Detection of wolbachia by polymerase chain reaction (PCR) method The elimination of wolbachia endobacteria from treated worms was performed by PCR as described [42]. In brief, the genomic DNA was isolated from 15 female worms recovered from treated and untreated groups, separately. Primers specific for Brugia malayi wolbachia: Bsymbf (50-ACGAGTTATAGTATAACT-30) and Bsymbr (50-CCTTCGAATAGGAATAAT-30) were used. The PCR conditions includes 3 mM MgCl2 with temperature cycling conditions of 952 C for 7 min followed by 35 cycles of 948 C for 30 s, 458 C for 30 s, 728 C for 30 s, followed by 728 C for 7 min. The PCR product was visualized on 1% agarose gel. Statistical analysis Data were calculated using one-way analysis of variance (ANOVA) and Student’s t-test (Mann–Whitney test) as

J Drug Target, 2014; 22(5): 395–407

appropriate. Individual comparisons following ANOVA were made using a posttest (Bonferroni) with the help of statistical software PRISM 3.0. Results of pharmacokinetic study, Mf density and adulticidal activity have been presented as mean SEM. The values with p50.05 were considered to be significant.

Results Preparation and characterization of CUR polymeric nanoparticles The fate of a drug or therapeutic agent after administration in vivo depends primarily on the physicochemical properties of the drug and on its chemical structure, therefore physicochemical characterization of drug loaded nanoparticles system becomes essential [43]. Particle size is an important parameter for the treatment of lymphatic filariasis as it can directly affect the physical stability, cellular uptake, biodistribution and drug release kinetics from the nanoparticles [44,45]. Therefore, we have fabricated a number of CUR nanoparticles using different types of monomer including NIPPAM, VP and AA or PEG of various concentrations and the basic characteristics of the products involved in the formulation studies are presented in Table 1. Polymeric nanoparticles were synthesized by random copolymerization of the vinyl end groups present in the amphiphilic monomers. The copolymer formed showed an amphiphilic character with a hydrophobic inner core and a hydrophilic outer shell, the latter being composed of water-soluble moieties like amides and carboxylates that project from the monomeric units. This is one of the most widely used methods for the synthesis of NIPPAM based polymers structures [46,47]. Under SEM as shown in Figure 1, all the nanoparticles had a fine spherical shape with slight rough surfaces. The mean size averaged by particle size analyzers of all samples were determined and are listed in Table 1. Figure 2 illustrates the particles size distribution. It can be observed that the size of polymeric formulations (F1–F6) was in ranged from 110 0.034 to 378 0.640 nm. It was observed that the size of CUR nanoparticles was smaller when prepared with acrylic acid (samples F3 and F5), than those of the nano-CUR fabricated using PEG (samples F2 and F4). It was also reported that the size of the nano-CUR formulations (F2–F6) increased as the concentration of VP, AA or PEG was increased. The size of optimized nano-CUR (F3) was 69 nm as reported in SEM analysis, which is comparable to the size obtained from DLS measurements (60 nm) (Figure 2). Similarly, micellar aggregates of curcumin formulation prepared by cross-linking and random copolymerization of NIPAAM with VP and PEG-A resulted in formation of particles size range of 50–500 nm [48]. Other studies such as self-assembling of methoxy poly(ethylene glycol)–palmitate curcumin nanocarrier and micelles of poly(ethylene oxide)-b-poly(ecaprolactone) were also reported to have a similar particle size range [49,50]. The entrapment efficiency (EECUR%) of nano-CUR formulations (F1–F6) was calculated as described in the ‘‘Entrapment efficiency’’ section and is summarized in Table 1, that varied from 82.49 0.467 to 91.307 0.973%.

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Figure 1. Scanning electron photomicrograph of nano-CUR formulations (F1–F6).

The release kinetics of CUR from the NIPAAM nanoparticles was studied for 48 h in buffer. Drug release from nanoparticles usually occurs in a biphasic manner, with an initial burst phase followed by a diffusion-controlled slower drug release phase. In our studies, we also reported that all the nano-CUR (F1–F6) showed an initial burst phase corresponding to about 29–38% within 6 h. The rapid initial release is attributed to the drug which is adsorbed or weakly bound to the surface area of the nanoparticles, than to the drug incorporated inside nanoparticles [51]. A sustained CUR release to a total of about 60.23 0.56 to 48.23 0.64% was found for the nano-CUR (F1–F6) over the entire period of study, as depicted in the graph and shown in Figure 3. However, a slight drop off released was also observed subsequent to 24 h in NIPAAM/VP/PEG nano-CUR formulations (F2, F4 and F6) and 42 h in NIPAAM/VP/AA nanoCUR formulations (F3 and F5). The relatively smooth surface supported the assumption that the release of drug from nanoparticles might be caused by both diffusion and matrix erosion. Similar study has reported a release of 40% CUR in 24 h with co-polymeric micellar aggregates of NIPAAM, VP and PEG [38]. Pharmacokinetics of nano-CUR formulations In clinical trials, several investigators have observed that despite taking gram doses of CUR, plasma levels remained stumpy [52,53]. A major weak point usually identified is poor bioavailability. It is the main hurdle to overcome in clinical trials to demonstrate the therapeutic efficacy of

this promising agent. Bisht et al. [38] has synthesized NIPAAM/VP/PEG acrylate encapsulated nano-CUR and reported that the efficacy of nano-CUR was similar to that of conventional CUR in vitro and in vivo. However, the authors neither determined the in vivo effect of nano-CUR nor its biodistribution or pharmacokinetics, thus failing to show any potential increase in efficacy of nano-CUR over Fr-CUR in vivo. However, we observed improved pharmacokinetics and enhanced gastrointestinal (GI) absorption of nano-CUR (F1–F6) compared to Fr-CUR solution, each of which received a single 25 mg/kg oral dose. As shown in Table 2, the Cmax, AUC and MRT values reflected an enhanced peak plasma concentration and mean residence time of nano-CUR formulations (F1–F6) that were significantly greater than those for Fr-CUR, which had lower values of Cmax (10.3 2.45 ng ml1), AUC (114 31.4 ng d ml1) and MRT (2.23 4.23 days). Among the various formulations, the plasma level of CUR is the lowest in nano-CUR (F6) and the highest in nano-CUR (F2). However, no significant difference in the plasma level of CUR between nano-CUR (F2) and nano-CUR (F1, F3–F5) was observed, indicating that these formulations have no significant effect on the pharmacokinetics of CUR and serve similar functions. The highest Cmax (109.6 1.87 ng ml1), AUC (943 12.3 ng d ml1) and MRT (22.5 1.45 days) values of nano-CUR (F2) indicate that the formulation NIPAAM/VP/PEG (F2) delivered the greatest in vivo effective plasma level of CUR and significantly increased its oral bioavailability after oral administration.

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Figure 2. Dynamic light scattering of nano-CUR formulations: (a) F1, (b) F2, (c) F3, (d) F4, (e) F5 and (f) F6.

The organ distribution study shows a significantly increased concentration of CUR in isolated organs (lungs, heart, testes and lymph nodes) of the nano-CUR (F1–F6) treated animals, as shown in Figure 4. In contrast, after oral administration of plain CUR, much less CUR bioavailability was found in the isolated organs (12 2.4%) and the

maximum amount remained unabsorbed in the small intestine (25 8.1%) or was found intact in the upper part of the GIT (40 2.3%). Among the various formulations (F1–F6), the results of nano-CUR (F3) showed the highest accumulation of CUR in isolated organs with the maximum amount in lymph nodes (30 3.4%), following by lungs (15 1.8%), heart

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1

2

3

4

5

6

(bp)

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(13 8.9%), testes (10 4.7%), colon (6 7.2%) and the small intestine (3 6.6%).

1,534

Therapeutic efficacy of optimized nano-CUR (F3) against blood microfilaremia

1,167

834


689

Figure 3. Detection of wolbachia by PCR using adult female B. malayi recovered from Mastomys after treatment with Fr-CUR and nano-CUR (F3) alone and in DEC combinations. Lane 1 – DNA ladder, Lane 2 – adult B. malayi from DEC treated animals, Lane 3 – adult B. malayi from Fr-CUR treated animals, Lane 4 – adult B. malayi from Fr-CUR þ DEC treated animals, Lane 5 – adult B. malayi from nano-CUR (F3) treated animals and Lane 6 – adult B. malayi from nano-CUR (F3) þ DEC treated animals.

The encapsulation of CUR in NIPAAM/VP/AA nanoparticles significantly increases the MIC efficacy of CUR in Mastomys, as shown in Table 3. A 50.8% decrease (p50.001) in circulating Mf load was observed in the group of animals treated with optimized nano-CUR (F3) on day 8, which subsequently led to total clearance of Mf from blood on day 75 post-initiation of therapy. Mf had not reappeared in the blood by the end of the observation period in this group of animals. However, the free form CUR (Fr-CUR) by the same route did not impart any significant restraint in the Mf densities, which enlarged drastically and crossed the pretreatment level after day 45. Nevertheless, there was a slow rate of increase in the Mf level in this group of animals as compared to the rate in the untreated control. In addition, five oral doses

Table 2. Pharmacokinetic parameters of Fr-CUR and nano-CUR formulations (F1–F6) in Swiss albino mice after administration of 25 mg kg1, orally. Kinetic parameters Tmax (min) Cmax (ng/ml) AUC0–1 (ng d/ml) AUC 0–8 (ng d/ml) MRT (days) T (h)

Fr-CUR solution Nano-CUR (F1) Nano-CUR (F2) Nano-CUR (F3) Nano-CUR (F4) Nano-CUR (F5) Nano-CUR (F6) 69 9.89 10.3 2.45 114 31.4 156 2.45 2.23 4.23 1.02 1.23

23 9.89 95.6 1.87 832 12.3 400 0.89 20.5 1.45 16.65 0.89

19 12.3 109.6 1.23 943 42.3 476 6.38 22.5 5.89 17.59 7.33

21 12.3 86.6 2.56 796 89.8 420 3.99 20.5 3.78 17.65 4.34

23 12.3 90.6 6.81 734 34.1 381 8.67 19.5 7.82 15.65 4.89

24 12.3 76.6 9.25 678 83.8 397 9.23 17.5 6.93 15.65 2.45

25 12.3 69.6 5.67 639 42.6 356 4.98 16.5 7.81 14.65 9.56

Values are in mean SEM. AUC, Area under the concentration–time curve; Cmax, peak concentration; MRT, Mean residence time; Tmax, Time to peak concentration; IVM, IVM solution; IVM NIPAAM NPs, IVM loaded NIPAAM nanoparticles.

Figure 4. CUR content in isolated organs of Swiss albino mice after 12 h oral administration of CUR and nano-CUR formulations (F1–F6).

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of optimized nano-CUR (F3) at 5 mg/kg plus DEC at 50 mg/ kg exhibited superior (p50.05) anti-microfilarial efficacy. After the beginning of this regimen, the Mf started decreasing rapidly below the pretreatment level from day 8 (–84.8%), which ultimately led to the total disappearance of Mf from peripheral blood on day 45, and persisted until termination of experiment. The decreased Mf density in these animals was statistically significant (p50.01) compared to other treated groups or controls at every stage of Mf detection. In contrast, Mastomys treated orally with Fr-CUR plus DEC (Fr-CUR þ DEC) revealed mild parasite suppression on day 15 after initiation of treatment, and the microfilaremia data pattern in these animals was almost the same as that of the DEC-treated animals (the DEC control). Mastomys received blank nanoparticles and were treated as controls.

They revealed no effect on Mf density, and therefore are displayed as pooled data (Table 3). Therapeutic efficacy of optimized nano-CUR (F3) against adult Brugia malayi parasites including effects on intrauterine contents After 105 days of observation of MIC activity, the animals were sacrificed and the worms recovered were evaluated for MAC activity. As demonstrated in Figure 5, the oral nanoCUR (F3) treatment at a dose of 5 mg/kg exhibited significant adulticidal efficacy against filarial parasites as compared to the untreated control (p50.01), causing 48.23% worm mortality. A marked reduction was observed in the recovery of live parasites with 56% of the surviving females sterilized,

Table 3. Microfilaricidal efficacy of Fr-CUR and nano-CUR (F3) in Brugia malayi M. coucha model in vivo. Percent change (mean SD) in microfilaria/10 ml blood at different days over pretreatment level Treatment groups

Days 8

Days 15

Days 30

Days 45

Days 60

Days 75

Fr-CUR (50 mg/kg, oral) –19.5 4.5 –23.2 3.5 –13.2 3.5 –9.6 2.4 þ53.2 2.4 þ173.1 4.2 Nano-CUR (F3), –50.3 3.9 –59.7 5.7 –67.4 1.9 –79.3 1.5 –99.4 4.2 –100.3 4.1 (10 mg/kg, oral) Fr-CUR (50 mg/kg, oral) þ –65.8 3.4 –74.8 5.7 –56.4 1.7 –24.3 9.3 –13.3 9.3 þ 25.4 2.4 DEC (50 mg/kg, oral) –84.8 3.4 –98.8 5.7 –76.4 1.7 –100.2 1.3 –100.5 4.2 –100.1 7.2 Nano-CUR (F3), (10 mg/kg, oral) þ DEC (50 mg/kg, oral) DEC (50 mg/kg, oral) –61.8 3.4 –48.8 5.7 –23.4 1.7 –4.9 9.3 þ13.3 5.9 þ 37.4 2.4 Nanoparticles (Blank) þ28.2 5.6 þ58.4 3.7 þ71.7 9.6 þ84.3 2.9 þ96.4 1.6 þ129.4 3.4 Control þ23.2 9.8 þ 54.4 8.4 þ 78.6 3.9 þ 88.3 1.9 þ 90.9 6.9 þ 144.4 2.2

Days 90

Days 105

þ261.1 7.4 –100.6 2.3

þ367.3 2.9 –100.9 3.6

þ 123.5 2.3 þ 189.2 3.6 –100.3 9.9

–100.9 2.7

þ 107.5 2.3 þ 212.2 36 þ198.4 6.7 þ243 2.9 þ 205.4 8.6 þ 268.4 8.4

Figure 5. Effect of Fr-CUR, nano-CUR (F3), alone and in DEC combination on recovery, mortality and female sterilization of adult Brugia malayi worms.

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DOI: 10.3109/1061186X.2013.869823

having degenerated uterine contents with no developing embryos or live microfilaria. However, the regimen of Fr-CUR using the same dose and route showed low detrimental effect on adult parasites (26.34% worm mortality and 29.13% female sterilization). DEC in a single dose when given a day after completion of nano-CUR therapy significantly augmented (p50.001) the worm mortality (78.43%); however, no further improvement in female worm sterility was reported. In contrast, the free form of CUR þ DEC resulted in marginal adulticidal action (44.09%) and did not show any effect on embryogenesis (Figure 5). The intrauterine contents of these female parasites also showed only the presence of degenerated eggs and no further developing embryos or microfilaria were seen. Free DEC resulted in 40.5% worm death and 58.9% female sterilization. Untreated control or blank nanoparticles did not have any significant effect on female sterilization or worm mortality. PCR for clearance of wolbachia from adult female parasites Wolbachia, an endobacterium, subsists in symbiotic association with adult worms in the lymphatic system, and has recently been identified as an important drug target for the LF treatment option. The antiwolbachial activity of CUR was ascertained in the recovered filarial worms using specific primers and PCR. The complete clearance of wolbachia was observed from the parasites obtained from animals treated with orally administered nano-CUR (F3) at 5 mg/kg, as shown in Figure 3. In contrast, the regime of Fr-CUR and DEC at a dose of 50 mg/kg orally, either alone or in combination, failed to eliminate wolbachia from the recovered parasites. The wolbachial status was also not detected in nano-CUR (F3) þ DEC treated animals. Blank nanoparticles and the

403

untreated control groups did not have any effect on the wolbachial activity of the recovered parasites. DNA topoisomerase-II inhibitory activity In past decades, CUR has also been shown to target several molecules, including growth factors, transcription factors, cytokines, and enzymes involved in the etiology of diverse diseases [12]. In this study, the inhibitory activity of Fr-CUR and nano-CUR (F1–F6) was evaluated for DNA topo II against the filarial parasite Setaria cervi in vitro. The evaluation included the standard antifilarial drug (DEC) and a topoisomerase II inhibitor (Novobiocin). The inhibitory effect on the topo II enzyme was evaluated by gel electrophoresis (Figures 6 and 7). Nano-CUR (F2) exhibited maximum (88.56%) inhibition at 50 mg/ml, whereas Fr-CUR exhibited only 15% DNA topo II inhibitory activity at this concentration. The other nano-CUR formulations (F1, F2, F4–F6) also showed significant DNA topo II inhibition compared to that of the control; however, the activity decreased with increased size of the nano-CUR formulations. Standard filaricides, DEC and Novobiocin were included in the study as reference topoisomerase II inhibitor compounds and had 52.56 and 80.09% inhibition at 50 mg/ml, respectively, against a DNA topo II enzyme of filarial nematode Setaria cervi.

Discussion During the past decades, researchers have expressed renewed interest in plant products as offering a potential remedy for the treatment of LF [8,11]. In this study, antifilarial, antiwolbachial and DNA topo II inhibitory activity of CUR (a yellow polyphenol), an ingredient of the medicinal plant Curcuma longa, were evaluated. Currently, this amazing yellow polyphenol is undergoing human clinical trials for a

Figure 6. Topoisomerase II inhibitory activity of Fr-CUR and nano-CUR (F3) at various concentrations against filarial worm Setaria cervi.

M. Ali

J Drug Target, 2014; 22(5): 395–407

number of conditions [54,55]. However, poor solubility and bioavailability limits its practical utility in medical applications and may also render this interesting polyphenol a poor antifilarial agent. Therefore, we have attempted to enhance the antifilarial potential of CUR by improving its pharmacokinetics and bioavailability using a NIPAAM polymeric nanoDDS. The particles size and %EE have been modulated by varying the ratio of co-monomers, thereby providing a platform to choose the optimal formulations (those with the greatest number of characteristics) for filarial parasite elimination. During the optimization process, it was seen that with an increased AA or PEG concentration, the size and %EE of nano-CUR formulations was also increased; however, the entrapment of CUR did not show a linear relationship with particle size (Table 1). Apart from this, all the nano-CUR formulations exhibited very high encapsulation due to superior solubility in the solvents used and their inclusion in the hollow hydrophobic core of NIPAAM/VP/AA or PEG nanoparticles. Since the encapsulation was high and CUR (which is slightly soluble in water) has a low tendency to diffuse out from the core of nanoparticles, the in vitro release experiments were monitored for up to 48 h (Figure 8). 1

2

3

4

5

6

7

8

9

Figure 7. Effect of Fr-CUR and nano-CUR (F1-F6) at 50 mg/ml on topo II mediated supercoiled pBR 322 relaxation. Lane 1 – Supercoiled pBR 322 alone, Lane 2 – pBR 322 DNA þ enzyme protein, Lane 3 – pBR 322 DNA þ enzyme protein þ Fr-CUR and Lane 4–9 – pBR 322 DNA þ enzyme protein þ nano-CUR (F1–F6).

F1

F2

F3

All of the nano-CUR (F1–F6) showed sustained release up to 24 h, though their release started to gradually decrease after 42 h with NIPAAM/VP/AA nano-CUR formulations (F3 and F5). This trend demonstrates that entrapment led to continuous slow release of CUR; therefore, the optimized nano-CUR (F3) was administered at an interval of 72 h at 5 mg/kg in Mastomys coucha. The Cmax, AUC and MRT values were highest in nano-CUR (F2) followed by (F3), as shown in Table 3. The differences in pharmacokinetics of the nanoCUR formulations suggest that PEG can enhance GI absorption of CUR compared to that of AA (see the composition in the preparation section); however, the difference was not statistically significant. The results of the organ distribution study indicated that the maximum concentration of CUR was found in the isolated organs of nano-CUR (F3) treated animals. Recently, various critical parameters for the development of polymeric nanodrug delivery systems for the treatment of LF have been described, showing that 20–70 nm diameter particles provide the most suitable range for enhanced antifilarial drug targeting of the parasites and effective killing of Mf and adult filarial worms [44]. Therefore, these nanoparticles should have the ability to concentrate the maximum amount of drug in the circulatory system or the organs where the adult parasites reside [44]. It is evident from the data that nano-CUR formulations (F2) and (F3), having minimum particle size (of 65 and 60 nm, respectively), high %EE and the best pharmacokinetics and organ distribution profiles revealed the most promising features required for LF treatment. The final selection of the most active optimized formulation for in vivo analysis was critically evaluated by using two well-characterized in vitro systems (motility and MTT assays) that employ rodent filariids as a target and that are used as prescreens [56]. The nano-CUR (F3) was found to be the most active against both forms of the parasites, the adult and Mf, and had the highest selectivity index (data not shown). Hence, based upon in vitro investigations, nano-CUR (F3) was considered to be the superior antifilarial candidate F4

F5

F6

30

36

70 60 50 Release (%)


404

40 30 20 10 0

0

6

12

18

24

42

48

Time (h)

Figure 8. In vitro release profile of CUR from polymeric nano-CUR formulations (F1–F6).

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DOI: 10.3109/1061186X.2013.869823

for in vivo follow up analysis in mastomys coucha infections with the L3 stage of Brugia malayi. The selected nano-CUR (F3) demonstrated encouraging results as only five doses at 5 mg/kg orally significantly augmented the antifilarial potential of CUR and successfully eliminated wolbachia bacteria from the recovered worms. The antimicrofilarial data unequivocally showed effective suppression of parasitic load up to the end of the experiment, and 67.4% MIC efficacy was achieved as early as day 30 posttreatment. Conversely, Fr-CUR exhibited only 19.5% reduction in Mf density on initiation of treatment (by day 8), suppressing parasite load only up to day 45 (Table 3). In vivo pharmacokinetics data of nano-CUR (F3) indicating significantly greater (p50.001) Cmax (86.6 2.56 ng ml1), AUC0–1 (796 89.8 ng d ml1) and MRT (19.5 7.82 days) values compared to those for Fr-CUR, as shown in Table 2, demonstrate considerable increased bioavailability and long-term retention of CUR in Mf circulating blood, substantiating enhanced MIC activity of nano-CUR (F3) and extended Mf suppression from the peripheral blood until the end of the experiment. DEC used as a standard filaricide is a strong microfilaricidal drug. It imparted an additive effect on the MIC efficacy of nano-CUR (F3), although the MIC activity of the Fr-CUR and DEC combination is seen to be DEC-mediated only. The synergistic effect of the DEC combination was also observed in the adulticidal activity of nano-CUR; however, the female sterilization effect appeared to be entirely due to nano-CUR alone. Thus, there was no synergistic effect on embryogenesis (Figure 5). Bajpai et al. [57] also observed that co-administration of DEC and IVM provided a marginal increase in MAC activity, enclosing nix embryo-static activity for IVM that alone caused 95% female sterilization. Nano-CUR (F3) without DEC exhibited significant MAC efficacy (48.23% worm mortality and 56% surviving females sterilized) against the adult filarial parasites compared to the Fr-CUR (26.34% worm mortality and 29.13% female sterilization), which can be better explained by the in vivo organ distribution study results. It was observed that 70.02% of the administered nano-CUR (F3) was accumulated in isolated organs from which the adult parasites were recovered, thus demonstrating improved bioavailability of CUR to the parasites, whereas the concentration of orally administered plain CUR was found to a much lesser extent in these organs (Figure 4). Therefore, the in vivo pharmacokinetics and organ distribution study, performed to analyze the targeting potential of nano-CUR to the filarial parasites, clearly suggests that the squat MIC and MAC activities of CUR are due to its poor absorption through the GIT (as reflected by longer Tmax of 69 9.89 min). The maximum proportion remained unabsorbed (in the small intestine and colon), leading to very low bioavailability to the parasites. The proposed scheme for improvement in the antifilarial potential of nano-CUR due to improved pharmacokinetics and organ bioavailability is shown in Figure 9. The results of PCR confirmed that only five doses of nano-CUR (F3) at 5 mg/kg orally resulted in the complete clearance of wolbachia from the parasites, whereas Fr-CUR and DEC did not show antibacterial activity at 50 mg/kg (Figure 3, Lane 4). The disappearance of wolbachia from worms recovered from the nano-CUR þ DEC treated

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animals was due to the antiwolbachial property of nano-CUR only (Figure 3, Lane 6). Thus, the end results show nano-CUR as a potential anti-wolbachial agent other than DEC or CUR. In this study, CUR was also found effective against DNA topo II enzyme in vitro. It has been reported that compounds having 4-methoxy-phenyl substituent or bearing only phenyl with no substitution showed significant DNA topo II inhibition (50–60%) [58]. Therefore, the structural activity of CUR, 3-methoxy phenyl, or only the phenyl group might be responsible for the DNA topo II inhibition activity of this yellow polyphenol. Interestingly, the enzyme inhibition activity was improved in the nano-form of CUR (highest in nano-CUR (F2): 88.56%), which is greater than the inhibitory activity of the standard topo II inhibitors (Novobiocin-80%) and standard filaricide, DEC (52.56%) (Figure 6). The higher activity of nano-CUR (F2) than that of nano-CUR (F3) could be due to the fact that AA is more hydrophilic than PEG, as the topo II inhibition increases with an increase in hydrophobicity of the test compound. The data indicate that nano-CUR (F2) can be used as the lead DNA topo II inhibitor against filarial infection based on extensive investigations in vivo from a pharmacological point of view. However, the related understanding of molecular interactions of CUR with the filarial targets (wolbachia and DNA topo II), along with recent advances in targeted drug delivery approaches, may facilitate in vitro and in vivo level studies, and would further engender renewed interest in therapeutic use of this interesting compound for the treatment of LF. The results obtained in this study also indicate that the nano-form of CUR beforehand had exhibited greater antimicrofilarial, antimacrofilarial and antiwolbachial activity than that of Fr-CUR or the standard filaricide DEC (Figure 9). In addition, its strong inhibitory effect against the major target, the filarial parasite enzyme DNA topo II in vitro, amalgamated with its natural origin makes this pleiotropic molecule a novel and worthy antifilarial agent for the treatment of human LF.

Conclusions LF is a disease about which there has been much discussion as it poses a worldwide health problem. The drugs presently available are not effective against the adult stage of the parasites and are also at a billion-fold risk of the parasites becoming resistant to the drugs. These circumstances have led the filaraisis research community to search for safe lead molecules with adulticidal activity. In view of this fact, we have assessed and improved the antifilarial properties of CUR using nanoDDSs. Experimental results demonstrated a radical swell in therapeutic antifilarial qualities of CUR when it was modified to nano-form using NIPAAM/VP/AA nanoparticles and employed against the human lymphatic filarial parasite Brugia malayi. Studies reveal that the developed formulation is safe, easy to prepare, and provides significantly improved pharmacokinetics and organ distribution of CUR. Further, its strong inhibitory action against a major target filarial parasite enzyme DNA topo II in vitro suggests nano-CUR as a novel antifilarial drug lead for the treatment of human LF.

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J Drug Target, 2014; 22(5): 395–407

Improved bioavailability in target organs

Improved pharmacokinetics

Tmax

NanoCurcumin

Cmax

GI track Lymph nodes


AUC

Nanosizing Increase topoII inhibition

MRT

Enhanced Mf load

Heart

Enhanced adulticiadal activity

Long Mf suppression

Improved MIC activity

Lungs

Enhanced antiwolbachial activity

Improved DNA topo II activity

Testes

Improved MAC activity

A novel antifilarial agent Figure 9. The proposed mechanism of improvement in anti-microfilarial, anti-macrofilarial and anti-wolbachial activity of CUR, leads nano-CUR as a novel antifilarial agent.

Declaration of interest The authors report no declarations of interest. The authors are thankful to Indian Council of Medical Research (ICMR), New Delhi, India, for providing financial support in carrying out the research.

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