Pharmacokinetics and in vivo chemosuppressive activity studies on cryptolepine hydrochloride and cryptolepine hydrochloride-loaded gelatine nanoformulation designed for parenteral administration for the treatment of malaria

Acta Tropica 127 (2013) 165–173

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Pharmacokinetics and in vivo chemosuppressive activity studies on cryptolepine hydrochloride and cryptolepine hydrochloride-loaded gelatine nanoformulation designed for parenteral administration for the treatment of malaria N. Kuntworbe a , M. Ofori b , P. Addo b , M. Tingle c , R. Al-Kassas a,∗ a

School of Pharmacy, The University of Auckland, Auckland, New Zealand Noguchi Memorial Institute of Medical Research, The University of Ghana, Legon, Ghana c Department of Pharmacology, The University of Auckland, New Zealand b

a r t i c l e

i n f o

Article history: Received 18 September 2012 Received in revised form 9 April 2013 Accepted 17 April 2013 Available online 30 April 2013 Keywords: Cryptolepine hydrochloride Nanoparticles Pharmacokinetics Chemosuppression

a b s t r a c t The main objective of this investigation was to establish the pharmacokinetics profile and in vivo chemosuppressive activities of cryptolepine hydrochloride-loaded gelatine nanoparticles (CHN) designed for parenteral administration for the treatment of malaria in comparison to the drug free in solution (CHS). Single-dose pharmacokinetics was investigated in Wistar rats by administering CHN or CHS (equivalent to 10 mg/kg of drug) by IV bolus injection via the lateral tail vein. The drug concentration in plasma was monitored over a 24-h period following administration. Chemosuppressive activity was investigated in Wistar rats challenged with P berghei parasites. Animals were given a daily dose of either CHN or CHS, equivalent to 2.5–100 mg/kg by intraperitoneal injection. The level of parasitaemia was determined by light microscopy by examining Giemsa-stained thin blood smears prepared from the tail end on day four of infection. It was found that CHN attained a higher (4.5-folds) area under the curve (AUC (0–24)) compared to CHS. CHS however produced a higher volume of distribution (4-folds). Distribution and elimination rates were higher with CHS which resulted in a lower (11.7 h) elimination half-life compared to that of CHN (21.85 h). The superior pharmacokinetic profile of CHN translated into superior chemosuppressive activity at all dose levels relative to CHS. As a conclusion, loading cryptolepine hydrochloride into gelatine nanoparticles improved both pharmacokinetics and in vivo antiplasmodial activity of the compound with the highest chemosuppression (97.89 ± 3.10) produced by 100 mg/kg of CHN. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Malaria remains a major health challenge in several countries. Recent WHO malaria report covering 106 countries indicated that up to 216 million cases of malaria were recorded in 2010, resulting in 655,000 deaths (WHO, 2011), despite numerous interventions including vector control, and deployment of insecticide-treated nets among others. Chemotherapy remains the main tool against malaria, although the continual emergence of resistance to most recent antimalarial drugs (Eisenstein, 2012), gives cause for concern and the need for newer drugs as well as improved formulations of existing drugs to enhance their efficacy. Cryptolepine hydrochloride (Fig. 1) is an indoloquinoline compound reported to have a number of pharmacological activities including; antihyperglycemic

∗ Corresponding author. Tel.: +64 9 373 7599. E-mail address: [email protected] (R. Al-Kassas). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.04.010

(Bierer et al., 1998), anti-inflammatory (Bamgbose and Noamesi, 1981; Olajide et al., 2009), antihypertensive (Oyekan, 1995), antibacterial (Boakye-Yiadom and Heman-Ackah, 1979), antifungal (Sawer et al., 2005) and antiplasmodial (Arzel et al., 2001; Cimanga et al., 1997; Kirby et al., 1995; Onyeibor et al., 2005; Wright et al., 2001) activities. The antiplasmodial activity of the compound has attracted particular attention as the search for new antimalarial drugs continues. The action of cryptolepine is due, at least in part to a chloroquinelike mode of action (Wright et al., 2001). Effective accumulation of the quinoline compounds to which cryptolepine belonged has been attributed to the presence of basic nitrogenous groups in their molecules (Egan et al., 2000; Hawley et al., 1996). Thus any formulation strategy which will enhance this property may likely improve activity of these compounds. This observation led to the proposition of the hypothesis that loading cryptolepine into gelatine which has amino groups could further enhance the ability of cryptolepine to accumulate into the parasite food vacuole. Thus in an earlier

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Wright et al., 2001). Even though the compound has been reported to have antimalarial activity, it is important to establish the antimalarial profile of the nanoformulation in comparison to the pure drug compound by the intended route of administration.

2. Materials and methods 2.1. Materials and reagents

Fig. 1. Chemical structure of cryptolepine hydrochloride.

study (Kuntworbe and Al-Kassas, 2012), cryptolepine hydrochloride was loaded onto gelatine nanoparticles as a means of testing this hypothesis. There are few reports on the pharmacokinetics of cryptolepine. A whole-body autoradiographic study on the distribution of 3 Hcryptolepine in mice was reported by Noamesi et al. (1991). They detected a rapid distribution and localisation of the compound to various tissues in the body except the central nervous system within 4 h following a single IV injection. In another study, Salako et al. (1985) investigated the distribution of 131 I-labelled cryptolepine in rats following a single IV administration. They also detected rapid disappearance of the compound from plasma but no evidence of any localisation in tissues. They inferred that the main clearance pathway of the compound could be via the hepatobiliary tract. Up to 8% of the administered dose was also detected in the stomach 1 h after injection: They postulated a possible enterohepatic reflux. In yet another study (McCurrie et al., 2009), cryptolepine was detected in serum within 30 min following oral administration of 10 mg/kg of the hydrochloride salt to rats. The Cmax was 0.42 ± 0.08 ␮M. Contrary to the earlier studies; cryptolepine persisted in blood up to 10 h following administration. That observation could be due to prolonged absorption from the gastrointestinal tract as well as enterohepatic circulation as suggested by Salako et al. (1985). Although the above studies provided very useful information on the pharmacokinetics of the compound, it is important that when the compound is incorporated into a dosage form, the in vivo profile be reinvestigated. Apart from this, two of the previous studies were based on whole animal distribution of radio-labelled cryptolepine, after i.v. administration, while the other study involving oral bioavailability did not investigate tissue or organ distribution. Other vital pharmacokinetic parameters such as the extrapolated area under the curve (AUC∞), clearance (CL) and volume of distribution (Vd) among others remain unknown. Though there are few formulation studies (Singh et al., 1996) on cryptolepine, there are no reports of pharmacokinetics of those formulations. This part of the study was aimed at investigating the pharmacokinetics profile including tissue distribution of CHN in comparison to CHS. Among the requirements for the development of bioactive compounds and dosage forms are evaluations of effectiveness, toxicity profile, and stability as well as other physicochemical properties such as solubility, ionisation, and permeability among others. Evidence of biological activity and biodisposition are the primary requirements for further investigation of any bioactive compound. The continued attention being given to cryptolepine stems from evidence of the folkloric use of Cryptolepis sanguinolenta, the primary source of cryptolepine, for the management of malaria (Cimanga et al., 1997). There have been several reports of the antimalarial activity of cryptolepine; both in vivo and in vitro (Cimanga et al., 1997; Kirby et al., 1995; Onyeibor et al., 2005;

Cryptolepine hydrochloride (>98%) was isolated from Cryptolepis sanguinolenta (Kuntworbe et al., 2012). Cryptolepine hydrochloride-loaded gelatine nanoparticles were prepared as reported earlier (Kuntworbe and Al-Kassas, 2012). Giemsa stain, Plasmodium berghei cell line NK65 and sterile centrifuge tubes were kindly donated by the Department of Immunology, Noguchi Memorial Institute for Medical Research. Immersion oil produced by Leica Microsystems was used for examining slides. Sterile needles 26G × 1/2 Terumo® equipped with 1 ml sterile syringes were used for administration of drugs. Frosted microscope slides 25.4 mm × 76.2 mm (ADD surgical medicals, Middlesex, England) were used for preparing blood smears. Vacutainer® tubes 1.8 ml capacity (BD Beliver industrial estate, Plymouth, UK), were used for blood collection. 2.2. Methods 2.2.1. Detection and quantification of cryptolepine hydrochloride in blood and in tissues A validated HPLC method developed in an earlier work (Kuntworbe et al., 2012) for the detection and quantification of cryptolepine hydrochloride in aqueous samples was used with modifications to detect and quantify cryptolepine hydrochloride in blood and tissues. The HPLC system consisted of an Agilent Series 1100 liquid chromatography system equipped with a quaternary pump (part G1311A) and, a degasser (part G1322A). The chromatographic column used was Gemini 5 ␮ C18 110 A˚ 250, 250 mm × 2.00 mm equipped with a column guard and a cartridge (Gemini C18, 4 mm × 2.0 mm). Both analytical and guard columns were purchased from Phenomenex (Auckland, NZ). Data acquisition was by ChemStationTM software version A.10.02 Build 1757 (Agilent Technologies, Cheshire, UK). HPLC mobile phase consisted of water containing 1% formic acid (mobile phase A) and acetonitrile containing 1% formic acid (mobile phase B) used in a gradient method. Injection volume was set at 10 ␮L and flow rate was maintained at 0.2 ml min−1 . 9-Aminoacridine hydrochloride was used as an internal standard (IS). 2.2.2. Determination of response factor (RF) Stock solution of cryptolepine hydrochloride (analyte) standard was prepared by dissolving 2 mg of the compound in 5 ml of mobile phase (A) to produce 400 ␮g/ml solution. A 600 ␮g/ml solution of 9-aminoacridine hydrochloride (internal standard) was similarly prepared by dissolving 3 mg of the compound in the same solvent system. Working standard solutions were prepared by appropriate dilution of stock standard solutions just before use. Solutions containing mixtures of pure cryptolepine hydrochloride (80 ␮g/ml or 40 ␮g/ml) and 9-aminoacridine hydrochloride (60 ␮g/ml or 300 ␮g/ml) were prepared from stock standard solutions and analysed by six replicate injections. The areas under the peaks and corresponding concentrations were used to compute the response factor (RF) as shown in Eq. (1): AUCIS [IS] = RF AUCal [al]

(1)

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2.2.3. Preparation of plasma and tissue samples Fresh blood of Wistar rats without drug history was taken into buffered sodium citrate (0.109 M) blood collection tubes (BD Vacutainer® UK) and stored at −20 ◦ C and used within 48 h of collection. Prior to use, the blood was thawed at 37 ◦ C in a horizontally shaking water bath (GLS Aqua18, Cambridge, Ltd., England) set to oscillate at 50 cycles per minute, for 15 min. Aliquots of thawed blood (100 ␮l) were spiked with equal volume (100 ␮l) of the working standard solution of cryptolepine hydrochloride. Contents were mixed by vortex (Labnet International, Inc., Edison, NJ, USA) for 10 s. Chilled methanol (300 ␮l) was added to the samples, vortexed and set aside for 10 min, followed by centrifugation (Eppendorf MiniSpin centrifuge) at 13,000 rpm for 5 min. Supernatant (100 ␮l) was withdrawn and spiked with equal volume of a working standard solution of the internal standard (to give an effective IS concentration of 60 ␮g/ml) and filtered (0.22 ␮M syringe filters) into Agilent HPLC vials equipped with 250 ␮l capacity vial inserts and analysed using the HPLC conditions described earlier. Tissue samples (1 g) were homogenised in 10 ml phosphate buffer using a glass tissue homogeniser and treated as described for plasma. Post extraction spiking of the samples with internal standard was used because an accurate knowledge of the concentration of the internal standard in each sample was critical for the RF to remain valid. In other words, all the parameters in Eq. (1) except the concentration of the analyte must be accurately known. 2.2.4. Determination of recovery, precision and accuracy from plasma and tissue samples Recovery, accuracy and precision were determined as described in the earlier work (Kuntworbe et al., 2012), but over a wider concentration range (0.15 ␮g/ml-40 ␮g/ml for plasma samples) to reflect the intended application of the method to in vivo studies. A fixed effective concentration (60 ␮g/ml) of internal standard was used throughout. 2.2.5. Freeze-thawed stability of cryptolepine hydrochloride in plasma and in tissues Plasma and tissue samples were recovered from storage and thawed. Samples were then spiked with cryptolepine hydrochloride to give known concentrations (0.15, 10 and 40 ␮g/ml) of cryptolepine hydrochloride and stored at -20 ◦ C. Samples were thawed within 1 h of freezing and on day 7 of continuous freezing, processed and analysed. The recovered concentrations were expressed as percentages of the initial spiked concentrations. 2.2.6. Pharmacokinetics of cryptolepine hydrochloride and cryptolepine hydrochloride-loaded gelatine nanoparticles This study was conducted under approval number AEC R772 granted by the Animal Ethic Committee of the University of Auckland. The study was conducted at the Vernon Jansen Unit at the Faculty of Medical and Health Sciences, University of Auckland. The study was conducted in 36 healthy male Wistar rats weighing 200 ± 10 g which were obtained from an inbred stock from the Vernon Jansen Unit, University of Auckland, New Zealand. Animals were specific pathogen-free and had no drug history. Three animals were housed per cage, and were allowed food and water ad libitum and 12 h day and night cycles during the experimental period. Prior to drug administration, animals were weighed to determine the dose required. Rats were gently restrained in a rodent restrain device appropriate for their sizes, and were given a single dose of 10 mg/kg of either CHN or CHS by a slow intravenous bolus injection via the lateral tail vein over 30 s, after tail veins were dilated under a warm lamp. Three animals were euthanised using CO2 at predetermined times (0, 1, 2, 4, 6, and 24 h) following drug administration. Blood was taken via cardiac puncture into heparinised tubes and stored at −20 ◦ C until analysed. The brain,

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heart, lungs, kidney, spleen and liver were harvested for determination of drug concentrations. Harvested organs were rinsed with phosphate buffered saline and blotted clean with a soft tissue paper to remove external blood. Weighed tissues (1 g) were homogenised in 2 ml phosphate buffer (pH 7.4) and stored at -20 ◦ C until analysed. Plasma drug levels at the various time-points were used to estimate the pharmacokinetic parameters for CHN and CHS using PK solution® version 2.0 (Summit Research Services, Montrose, Co 81401, www.summitPK.com). The software relies on noncompartmental methods of analysis for estimation of pharmacokinetic parameters employing two techniques; trapezoidal rule and curve stripping. The trapezoidal approach is based on the estimation of the area associated with the curve described by the concentration–time profile. The curve stripping method on the other hand resolves the curves into series of exponential terms corresponding to the absorption (oral route), distribution and elimination phases with the assumption that the dispositions phases follow first order rate processes. 2.2.7. Four day in vivo chemosuppressive study The study was conducted on 65 specific parasite free male Wistar rats weighing between 200 ± 10 g and with no drug history. The animals were obtained from an inbred stock from the animal house of the Animal Experimentation Unit of the Noguchi Memorial Institute for Medical Research, Legon, Ghana where this study was conducted. The animals were divided randomly into three groups made up of two experimental groups (A and B) containing 30 animals each, and a positive control group C, containing five animals. Groups A and B animals were further divided into six sub-groups of five animals each such as A1 , A2 , A3 and A6 , for group (A) animals and B1 , B2 , B3 , and B6 for group (B) animals. Each sub-group was housed in the same cage throughout the study period. Housing and grouping were made 48 h prior to commencement of the experiment to allow the animals to acclimatise. Animals were allowed food and water ad libitum and 12 h day and night cycles throughout the study period. A vial of cryopreserved P. berghei parasites stored in liquid nitrogen (-180 ◦ C) was defrosted at 37 ◦ C in a gently shaking water bath to thaw for 2 minutes The samples were then centrifuged at 1500 rpm for 10 minutes The exterior of vials containing parasites were disinfected with 70% alcohol and handled aseptically under a biological safety cabinet. The supernatant was removed and the parasites were washed twice with thawing mix (3.5% NaCl solution) and centrifuged at 1500 rpm for 10 min on each occasion. Washed cells were then suspended in sufficient complete parasite medium (CPM) to give parasite density of approximately 107 cells per ml. The initial parasite density was determined based on the approach by Greenwood and Armstrong (1991). Briefly, a thick film was prepared with 5 ␮l (5 × 10−3 ml) of parasite suspension and stained with Giemsa stain. The number of parasites per microscope field (10× eye piece and ×100 objective) was multiplied by the factor 5 × 105 , assuming a blood volume of 2 × 10−6 ml per field (Greenwood and Armstrong, 1991). Two primary donor rats were inoculated with 200 ␮l of parasite suspension. Thin blood smears were prepared from each animal on daily basis until about 25% parasitaemia was established. This was determined by counting the number of infected RBCs in 50 fields and dividing by the total number of RBCs counted. Two secondary donor rats were each inoculated with 200 ␮l of plasma collected from a primary donor rat. Thin smears were prepared and examined as before until 25% parasitaemia was established. A 4-day chemosuppressive activity of CHN and CHS was carried out based on the approach by Peters (1975). A day before the commencement of the study, the animals were weighed. Each animal in the experimental groups and positive control group received 200 ␮l of infected blood from a secondary donor rat via intraperitoneal

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Table 1 Determination of response factor for cryptolepine hydrochloride (n = 6 per combination). Concentration of cryptolepine hydrochloride/internal standard (␮g/ml)

AUC

RF

Cryptolepine HCl (40 ␮g/ml) 9-Aminoacridine HCl (300 ␮g/ml)

2020.0667 ± 21.1457 1729.9833 ± 17.5871

0.1142 ± 0.0001

Cryptolepine HCl (80 ␮g/ml) 9-Aminoacridine HCl (60 ␮g/ml)

4158.2500 ± 5.4968 352.7333 ± 8.2696

0.1120 ± 0.0001 0.1131 ± 0.0015

Average RF

3. Results

tional to the ratio of their respective concentrations. The constant of proportionality, referred to as the response factor (RF) in this study, was determined by analysing a solution containing known concentrations of relatively pure samples of internal standard (9-aminoacridine hydrochloride) and analyte (cryptolepine hydrochloride) as shown in Eq. (1). In this study, two different combinations of cryptolepine hydrochloride and 9-aminoacridine hydrochloride concentrations were used to determine the overall average factor (Table 1). The standard deviation (0.0015) confirms the constancy and reliability of the factor. Chromatograph (Fig. 2) of the mixture of pure 9-aminoacridine and cryptolepine showed well resolved peaks with retention times of 8.6 ± 0.2 min and 9.6 ± 0.4 min, respectively. Chromatograph (Fig. 3) of the two compounds in plasma extract showed little or no interference with peaks resulting from plasma debris. The method demonstrated high recovery (Table 2). Precision and accuracy depicted by the %recovery and %RSD respectively were all below 15%. The compound also demonstrated good freeze–thaw stability in blood over a 1-week storage period. This was done by comparing the percentage recovery from freshly spiked samples to the percentage recovery of the same sample after a 1-week period (Table 3). Results (Table 4) from tissue analysis were similar to those of plasma in terms of accuracy and precision. Cryptolepine hydrochloride also exhibited good freeze–thaw stability in tissues (Table 5), but marginally less compared to the results obtained with plasma.

3.1. Detection and quantification of cryptolepine hydrochloride in blood

3.2. Pharmacokinetic studies

Analysis of plasma samples involving the use of internal standard is based on observations that the ratio of the respective areas under the peak of internal standard and analyte is propor-

The main objective of the pharmacokinetic study was to compare the plasma profile and tissue distribution of CHN to that of CHS. This was done by monitoring the concentration of cryptolepine

injection. Infected rats were treated with intraperitoneal injection of CHN or CHS in sterile phosphate buffer saline at daily doses equivalent to 2.5, 5, 15, 40, 60 or 100 mg/kg of drug on days 0, 1, 2, and 3. Group C animals were given phosphate buffer saline (200 ␮l) and served as parasite positive/drug negative control group. On day 4, thin blood smears on frosted slides were prepared from the tail end of each animal and allowed to dry in air. These dried smears were fixed with methanol and allowed to dry in a slide rack and then stained with freshly prepared and filtered (0.45 ␮m filter membrane) Giemsa solution over 10 min. All slides were examined under the microscope (Leica DMR light microscope, Leica Microsystems, Germany), using 100× objective under oil immersion. The total numbers of RBCs as well as those infected with parasites were counted from 50 different fields. The percentage parasitaemia was determined as the infected RBCs expressed as a percentage of the total RBCs counted. The percentage chemosuppression was calculated using Eq. (2): Cs = 100% ×

Pcg − Ptg Pcg

(2)

9.742

where Cs is the chemosuppression; Pcg is percentage parasitaemia in the controlled group, and Ptg is the percentage parasitaemia in the test group. Statistical analyses of results were carried out by nonparametric t-test (P < 0.05), using GraphPad Prisms (version 5.02) statistical software. All animals were euthanised by CO2 euthanasia.

DAD1 H, Sig=280,16 Ref=360,100 (PLASANAL1 2011-09-12 11-21-21\003-0302.D)

mAU

400

8.876

300

200

100

0 2

4

6

8

10

12

14

16

min

Fig. 2. Chromatograph of a mixture of pure 9-aminoacridine hydrochloride (IS) and cryptolepine hydrochloride (analyte) in water containing 0.1% (v/v) formic acid (mobile phase B).

169

9.212

N. Kuntworbe et al. / Acta Tropica 127 (2013) 165–173

DAD1 H, Sig=280,16 Ref=360,100 (PLASMA2 2011-09-21 11-40-20\015-1503.D)

mAU 800 700

8.622

600 500 400 300

14.611

7.807

3.277

1.401

100

2.060 2.306

200

0 2

4

6

8

10

12

14

16

min

Fig. 3. Chromatograph of 9-aminoacridine and cryptolepine in extracted plasma sample. Table 2 Recovery of cryptolepine hydrochloride from blood samples: accuracy and precision of the method. Spiked concentration (␮g/ml)

Recovered concentration (␮g/ml)

Intra-day (n = 3)

0.15 0.20 0.31 0.63 1.25 2.50 5.00 10.00 20.00 40.00

0.15 0.20 0.34 0.63 1.28 2.46 4.87 9.35 19.60 37.64

± ± ± ± ± ± ± ± ± ±

0.01 0.00 0.00 0.01 0.01 0.04 0.06 0.43 1.80 0.58

100.43 100.83 108.30 101.04 102.44 98.37 97.40 93.53 97.98 94.09

± ± ± ± ± ± ± ± ± ±

5.93 1.70 0.66 1.12 0.92 1.45 1.23 4.33 9.01 1.43

5.90 1.68 0.61 1.11 0.89 1.47 1.27 4.63 9.19 1.52

Inter-day (n = 9)

0.15 0.20 0.31 0.63 1.25 2.50 5.00 10.00 20.00 40.00

0.14 0.21 0.34 0.63 1.27 2.48 4.82 9.44 19.06 37.56

± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.01 0.01 0.02 0.02 0.04 0.10 0.47 1.28

95.87 103.09 108.72 100.46 101.57 99.11 96.49 94.41 95.29 93.90

± ± ± ± ± ± ± ± ± ±

4.94 1.96 0.62 0.55 0.76 0.65 0.78 0.97 2.33 3.19

5.16 1.90 0.57 0.54 0.75 0.65 0.81 0.103 2.45 3.40

Table 3 Freeze–thaw stability of cryptolepine hydrochloride in blood (n = 3). Time

Spiked concentration (␮g/ml)

% recovery

Within 1 h

0.15 10.00 40.00

95.55 ± 3.85 99.73 ± 1.75 96.34 ± 2.00

On day 7

0.15 10.00 40.00

94.67 ± 4.12 98.65 ± 3.20 97.88 ± 5.11

%RSD

Plasma drug concentrations of cryptolepine hydrochloride were higher for CHN than CHS at all time points. The main pharmacokinetic parameters (Table 6) showed that the observed area under the curve (AUC (0–24)) for CHN was significantly (P < 0.001) higher (4.5-fold) than CHS whiles the extrapolated area (AUC∞ (area), Plasma drug concentration(µg/ml)

hydrochloride in plasma over a 24-h period following a single slow i.v. bolus injection via the lateral tail vein at doses of 10 mg/kg of CHN or CHS. Rapid administration of CHN resulted in sudden death of two animals. There were no other observable signs of discomfort among remaining animals over the course of the study. The pharmacokinetic results were obtained directly using the PK solution® software based on noncompartmental open model system with appropriately built in equations. The graphical results (Fig. 4) indicated biphasic systems depicted by a rapid distribution phase in the case of CHS and a relatively slower one in the case of CHN, followed by a very gradual elimination phase in both cases.

% recovery

100 80 60 40 20 0 0

10

20

30

Time (hours) Fig. 4. Average plasma concentration–time curves for cryptolepine hydrochloride following administration of aqueous solution and nanoparticles, Symbols: 䊉, aqueous cryptolepine hydrochloride injection (CHS); ,cryptolepine hydrochloride nanoparticles injection (CHN).

Liver drug concentration (µg/ml)

4.99 5.02 11.16 101.07 ± 5.04 99.04 ± 4.98 98.40 ± 10.98

10

5

0 10

4.42 4.47 5.02

20

30

Time (hours) Fig. 5. Liver drug distribution of cryptolepine hydrochloride aqueous injection (CHS) and cryptolepine hydrochloride nanoparticles (CHN) Symbols: 䊉, aqueous cryptolepine hydrochloride injection; , cryptolepine hydrochloride nanoparticles injection.

AUC∞ (expo)) of CHN was at least 7 times higher. There was also a higher (about 4-fold) volume of distribution (Vd (area), Vd (expo), Vd (area)/kg) and rate of distribution (D rate), which was 2-fold higher with CHS compared to CHN. At steady state, the volume of distribution (Vss (area), Vss (expo)) of CHS was only 2-folds higher than that of CHN. A rapid decline of cryptolepine level was observed especially with CHS (D half-life of 0.37 h). The longer halflife (21.85 ± 6.37 h) encountered with the CHN injection translated into a higher mean residence time (MRT (area), MRT (expo)) of about 3-fold compared to the CHS injection. These observations were partly due to the higher clearance (CL (area), CL (expo), CL (area)/kg) of cryptolepine hydrochloride following administration of CHS compared to CHN. Cryptolepine was not detected in the brain at any time point with CHN and CHS injections. A comparison of the distribution profile of the two dosage forms in other tissues showed that CHS injection attained a marginally higher liver and a much higher kidney drug levels than the CHN injection. However, after about 10 h, the liver drug level became higher for CHN injection and was depicted by a crossing over of the two lines (Fig. 5). The trend observed with the kidney distribution (Fig. 6) could be due to increased presentation of the compound for renal excretion when the drug was administered as CHS. Tissue distributions observed in the spleen (Fig. 7), heart (Fig. 8), and lungs (Fig. 9) revealed higher drug levels with the CHN especially in the heart and lungs compared to CHS. Comparison of general tissue distribution using the extrapolated areas under the curve (Table 7) had shown higher figures for CHN than CHS injection except in the kidney where the reverse was the case. The observed trend was due to a much slower tissue clearance of nanoparticles – bearing drug in the liver, spleen, heart and lungs. 40

Kidney drug concentration (µg/ml)

99.57 ± 4.40 95.58 ± 4.27 94.46 ± 4.74 6.00 2.66 6.42 97.06 ± 5.78 98.36 ± 2.61 99.46 ± 6.38 6.46 4.77 3.80 99.10 ± 6.40 99.05 ± 4.72 99.46 ± 3.78 3.57 1.43 3.47 2.92 2.84 7.72 0.15 10 40

93.63 ± 2.73 96.92 ± 2.75 95.36 ± 7.36

94.43 ± 3.37 97.91 ± 1.40 95.42 ± 3.31

% recovery

100.47 ± 2.17 97.02 ± 3.72 101.72 ± 3.40 5.34 6.90 3.89

% RSD % recovery

94.84 ± 5.06 97.82 ± 6.75 98.81 ± 3.84 4.40 3.44 1.26

%RSD % recovery

95.17 ± 4.40 96.11 ± 3.31 97.22 ± 1.23 1.41 2.73 2.26

%RSD % recovery

98.39 ± 1.39 100.20 ± 2.74 97.92 ± 2.21 2.48 3.67 2.36 2.71 3.14 3.73 94.70 ± 2.56 96.23 ± 3.02 97.02 ± 3.62

%RSD % recovery % RSD % recovery

0.15 10 40

Spleen

95.72 ± 2.38 95.44 ± 3.51 96.95 ± 2.29

Brain Lungs Heart Kidney

15

0

Liver

30

20

10

Inter-day (n = 9)

0

Intra-day (n = 3)

Spiked concentration (␮g/ml)

Table 4 Recovery of cryptolepine from tissue samples: accuracy and precision of the method.

2.16 3.84 3.35

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%RSD

170

0

10

20

30

Time (hours) Fig. 6. Kidney drug distribution of cryptolepine hydrochloride aqueous injection (CHS) and cryptolepine hydrochloride nanoparticles (CHN) Symbols: 䊉, aqueous cryptolepine hydrochloride injection; , cryptolepine hydrochloride nanoparticles injection.

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Table 5 Freeze–thaw stability of cryptolepine hydrochloride in tissue samples (n = 3). Freeze time

Spiked concentration (␮g/ml)

% recovery

Liver

Spleen

Kidney

Heart

Lungs

Brain

Within 1 h

0.15 10 40

94.04 ± 5.97 98.18 ± 2.37 97.89 ± 1.94

99.06 ± 4.94 95.59 ± 4.53 96.62 ± 1.64

99.72 ± 4.20 96.7 ± 7.53 97.69 ± 1.77

92.07 ± 3.91 97.74 ± 2.50 99.35 ± 6.98

91.21 ± 2.57 95.33 ± 3.62 92.79 ± 2.51

100.90 ± 4.49 98.66 ± 4.20 98.47 ± 1.53

On day 7

0.15 10 40

92.12 ± 5.33 92.84 ± 4.97 91.50 ± 2.42

92.17 ± 1.50 92.53 ± 2.90 91.40 ± 3.47

92.76 ± 4.52 89.21 ± 9.45 93.03 ± 5.05

91.39 ± 6.51 90.47 ± 5.49 92.28 ± 5.54

91.54 ± 2.33 92.48 ± 4.64 91.98 ± 3.05

98.61 ± 3.99 92.27 ± 4.41 93.92 ± 4.30

Table 6 Main pharmacokinetic parameters for the aqueous solution (CHS) and nanoparticles (CHN. Parameter

Units

E intersect E slope E rate E half-life D slope D rate D half-life C initial AUC (0–24) AUC∞ (area) AUC∞ (expo) AUMC∞ (area) AUMC∞ (expo) MRT (area) MRT (expo) Vcv Vd (area) Vd (expo) Vd (area)/kg Vss (area) Vss (expo) CL (area) CL (expo) CL (area)/kg

␮g/ml 1/h 1/h 1/h 1/h 1/h h ␮g/ml ␮g h/ml ␮g h/ml ␮g h/ml ␮g h h/ml ␮g h h/ml h h ml ml ml ml/kg ml ml ml/h ml/h ml/(h kg−1 )

CHS

CHN 2.34 −0.03 0.06 11.70 −0.88 2.034 0.37 107.13 88.30 102.87 92.37 925.17 818.57 8.57 17.43 20.83 326.63 362.27 1633.13 161.77 165.67 19.83 22.61 99.15

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.56 0.01 0.02 3.96 0.31 0.72 0.12 47.20 9.30 17.37 24.98 618.14 705.93 4.45 5.90 7.32 78.09 38.01 390.47 59.16 63.39 3.48 5.30 17.39

21.39 −0.01 0.03 21.85 −0.30 1.06 0.67 77.60 400.47 740.20 739.90 23,408.57 23,073.20 29.47 32.50 25.87 85.80 85.87 429.00 79.37 78.40 2.90 2.90 14.16

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.95††† 0.00† 0.01† 6.37† 0.26† 0.22† 0.15† 6.68* 42.65††† 223.95†† 223.43†† 14,523.50† 13,710.90† 11.01† 9.00† 2.15* 6.58†† 6.77††† 32.87†† 6.09† 4.88† 0.98†† 0.98†† 5.10††

Values recorded in this table are mean values ± standard deviation (n = 3). Abbreviations: E, elimination; D, distribution; C, concentration; AUC (0–t), area under the curve from time (0) to time (t); AUC∞, area under the curve extrapolated to infinity; AUMC∞, area under the moment curve extrapolated to infinity; MRT, mean residence time; Vcv, volume of central compartment; Vd, volume of distribution; Vss, volume of distribution at steady state; CL, clearance. “(area)” and “(expo)” denote parameters obtained with the trapezoidal and exponential calculations, respectively. * P values of CHN in relation to CHS: P > 0.05. † P values of CHN in relation to CHS: P < 0.05. †† P values of CHN in relation to CHS: P < 0.01. ††† P values of CHN in relation to CHS: P < 0.001.

3.3. Four day in vivo suppressive study

4. Discussion

In this study, the chemosuppressive activities of CHS and CHN were investigated by the 4-day study as outlined above. The results (Table 8) showed both CHS and CHN to be moderately chemosuppressive at lower doses, with activity being dose dependent. The chemosuppressive activities were calculated using Eq. (2).

4.1. Pharmacokinetics and tissue distribution Chemotherapy has been the main approach to the fight against malaria, with the objective to clear disease-causing parasites already in blood and to prevent development of parasites to disease

Table 7 Tissue distribution of cryptolepine hydrochloride following administration of aqueous (CHS) and nanoparticles (CHN) injection. Tissue

AUC∞ (area) (␮g h/ml) Aqueous injection

Liver Spleen Kidney Heart Lungs

149.00 171.60 278.43 88.27 71.60

± ± ± ± ±

46.10 42.75 61.44 4.18 29.98

CL (area)/kg (ml/(h kg)) Nanoparticles injection 274.5 329.80 268.57 530.90 547.53

± ± ± ± ±

24.81† 197.40* 143.32* 255.26† 126.18††

Abbreviations: AUC (area) and CL (area)/kg have the same meanings as defined under Table 6. * P values of CHN in relation to CHS: P > 0.05. † P values of CHN in relation to CHS: P < 0.05. †† P values of CHN in relation to CHS: P < 0.01. ††† P values of CHN in relation to CHS: P < 0.001.

Aqueous injection 72.46 60.93 37.10 113.47 155.22

± ± ± ± ±

26.11 16.01 8.08 5.32 56.71

Nanoparticles injection 36.64 39.07 46.56 23.89 19.03

± ± ± ± ±

3.46† 23.32* 27.52* 15.8††† 4.99†

N. Kuntworbe et al. / Acta Tropica 127 (2013) 165–173

Spleen drug concentration(µg/ml)

172

Table 8 Mean percentage chemosuppression obtained from the thin blood smear from the tails.

10 8

Dose (mg/kg)

CHS 4

2.5 5 15 40 60 100

2 0 0

10

20

30

Time (hours) Fig. 7. Spleen drug distribution of cryptolepine hydrochloride aqueous injection (CHS) and cryptolepine hydrochloride nanoparticles (CHN) Symbols: 䊉, aqueous cryptolepine hydrochloride injection; , cryptolepine hydrochloride nanoparticles injection.

heart drug concentration(µg/ml)

25

20

15

10

5

0 0

10

20

30

Time (hours) Fig. 8. Heart drug distribution of cryptolepine hydrochloride aqueous injection (CHS) and cryptolepine hydrochloride nanoparticles (CHN) Symbols: 䊉, aqueous cryptolepine hydrochloride injection; , cryptolepine hydrochloride nanoparticles injection.

bearing state. Bioactives of plant origin were the first to be deployed against this devastating parasitic disease, including quinine from cinchona bark, artemisinin from Artemisia annua, and cryptolepine from Cryptolepis sanguinolenta. Decoctions containing these natural products were used for many years prior to isolation and identification of their active components. Cryptolepine is still undergoing intensive studies as there are still missing links in the body of knowledge on the compound. Though formulations containing cryptolepine are in circulation, there are few studies on the pharmacokinetics of the compound. This inadequate information makes it difficult to appreciate the dosage regimens of these products. In this study, the pharmacokinetics of cryptolepine hydrochlorideloaded gelatine nanoparticles formulation was compared to that of an aqueous solution of the compound and to determine how the 30

Lungs drug concentration(µg/ml)

% chemosuppression

6

20

10

0 0

10

20

11.20 17.93 27.47 55.94 71.22 78.36

CHN ± ± ± ± ± ±

5.63 7.01 6.44 11.01 8.84 5.58

24.81 30.76 62.92 88.05 94.46 97.89

± ± ± ± ± ±

5.89†† 10.01† 11.00††† 7.32††† 4.19††† 3.10†††

The chemosuppression is the difference in the mean parasitaemia between the controlled group and treated group expressed as a percentage of the controlled group (n = 5 per sub-experimental group and for group C). The % parasitaemia in the controlled group was 35.89 ± 3.18. † P values of CHN in relation to CHS: P < 0.05. †† P values of CHN in relation to CHS: P < 0.01. ††† P values of CHN in relation to CHS: P < 0.001.

pharmacokinetics profile affect the antimalarial activity of the compound in vivo. The nanoparticles were designed to take advantage of the presence of basic nitrogenous groups on the carrier polymer which may improve upon the delivery of cryptolepine into the parasite food vacuole. Basic nitrogenous groups have been identified as important for the accumulation of the quinolines into the parasite food vacuole (Egan et al., 2000; Hawley et al., 1996). The attributes of the cryptolepine hydrochloride-loaded gelatine nanoparticles observed in this study include notably higher initial drug level, slower tissue distribution, reduced clearance and higher plasma residence time, all desired characteristic of a good antimalarial formulation (Santos-Magalhães and Mosqueira, 2010). Higher plasma residence time is particularly important for the clearance of erythrocytic stage parasites. The persistence of the compound in blood with elimination half-life (E half-life) of 11.7 h for CHS injection and 21.85 h for CHN injection was however unexpected with respect to the aqueous injection, as the longest plasma residence time so far reported for the compound was 10 h (McCurrie et al., 2009) which was observed after oral administration. The rapid decline of plasma level of cryptolepine observed with CHS was in line with earlier investigations (Noamesi et al., 1991; Salako et al., 1985) which detected rapid distribution of the compound to tissues following administration of a radio-labelled cryptolepine. Though there was significant tissue drug level with both CHS and CHN, the rate of tissue distribution (D rate) of CHN was half that of CHS and may potentially reduce acute tissue effect relative to CHS. The observed trend in liver distribution suggest that though there was a more rapid delivery of the compound to the liver with the CHS injection, the rate of liver clearance was also higher. It is known that conventional nanoparticles are recognised by the mononuclear phagocytes system (MPS), mainly kupffer cells of tissues of the liver, lungs, spleen and that of other organs which could partially account for the long term accumulation of cryptolepine in those tissues when the drug was administered in the nanoparticulate form. The reduced clearance may also be due to retention of the drug-bearing nanoparticles by the MPS. Cryptolepine was not detected in brain tissue at any time-point, an indication that the compound does not cross the blood–brain barrier. The significant levels of drug attained in other tissues with CHN meant the current formulation may have to be modified further, such as through surface modification of the nanoparticles to further reduce systemic distribution.

30

Time (hours) Fig. 9. Lungs drug distribution of cryptolepine hydrochloride aqueous injection (CHS) and cryptolepine hydrochloride nanoparticles (CHN) Symbols: 䊉, aqueous cryptolepine hydrochloride injection; , cryptolepine hydrochloride nanoparticles injection.

4.2. Four day in vivo suppressive study There are a number of reports on the antimalarial activity of cryptolepine and its hydrochloride salt both in vitro and in vivo. While there is a general agreement on its in vitro activity, there

N. Kuntworbe et al. / Acta Tropica 127 (2013) 165–173

seems to be variations in the reports of its in vivo schizonticidal activity. Kirby et al. (1995) reported the compound (free base) to be very potent in vitro with an IC50 of 0.134 ␮M but they did not detect any in vivo activity against the rodent parasite P. berghei even for doses as high as 112.63 mg/kg/day × 4. Later studies with the hydrochloride salt suggested the compound has significant in vivo activity even with doses as low as 12.5 mg/kg/day × 4 (Cimanga et al., 1997; Grellier et al., 1996). From the pharmacokinetic data in the present study, it is clear that the plasma residence time of the CHN was significantly (P < 0.05) longer than the plain drug. This increased contact time between nanoparticles and erythrocytes coupled with potentially improved delivery of the compound across parasite membranes into the food vacuole may have accounted for the improved efficacy observed with CHN at all dose levels as shown in Table 8. This observation is also in line with numerous studies which have established the fact that nanoformulations of antimalarial bioactives have higher efficacy than conventional formulations of such compounds (Haas et al., 2009; Santos-Magalhães and Mosqueira, 2010). Cryptolepine is a member of the quinoline antimalarial compounds which act mainly within the acidic food vacuole of the plasmodium parasite. Effective accumulation of these compounds has been attributed to the presence of basic nitrogenous groups in their molecules (Egan et al., 2000; Hawley et al., 1996). Thus any formulation strategy which will enhance this property may likely improve activity of these compounds. This observation led to the proposition of the hypothesis that loading cryptolepine into gelatine which has amino groups could further enhance the ability of cryptolepine to accumulate into the parasite food vacuole. Thus the significant improvement of the suppressive activity of CHN compared to CHS strongly supports this hypothesis. Another important observation from this study was the fact that no animal deaths were recorded during the experimental period except illness (reduced mobility and feeding habit as would be expected from the disease progression) among the controlled group. This contrasts with earlier research in which intraperitoneal administration of cryptolepine to mice resulted in toxicity after the second dose (Wright et al., 2001). Thus different species could show different degree of tolerance to the compound. For instance, there has not been any reported incidence of human toxicity though the compound has been used for several decades in humans. Thus different species could show different degree of tolerance to the compound.

5. Conclusion The pharmacokinetics and in vivo schizonticidal activity of cryptolepine hydrochloride and its loaded gelatine nanoparticles have shown the compound to be generally tolerated in vivo causing no observable acute toxicity except when administration was done rapidly with the nanoparticles. The long plasma half-life of cryptolepine hydrochloride (21-h) obtained with the nanoparticles injection could allow a once a day administration of the compound in the nanoparticulate dosage form. The reduced distribution rate encountered with the CHN injection is important for both activity and safety of the formulation as an antimalarial agent. On a dose to dose comparison, CHN produced greater chemosuppressive activity compared to CHS. The observed trend could be linked to the pharmacokinetic profiles of the two dosage forms which have shown that CHN delivered superior plasma drug level, reduced distribution rate, reduced clearance and extended half-life, all of which have the potential to enhance delivery of the active compound to parasites in blood.

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References Arzel, E., Rocca, P., Grellier, P., Labaeid, M., Frappier, F., Gueritte, F., Gaspard, C., Marsais, F., Godard, A., Queguiner, G., 2001. New synthesis of benzo-␦-carbolines, cryptolepines, and their salts: in vitro cytotoxic, antiplasmodial, and antitrypanosomal activities of ␦-carbolines, benzo-␦-carbolines, and cryptolepines. J. Med. Chem. 44, 949–960. Bamgbose, S.O., Noamesi, B.K., 1981. Studies on cryptolepine. II. Inhibition of carrageenan induced oedema by cryptolepine. Planta Med. 41 (392), 396. Bierer, D.E., Fort, D.M., Mendez, C.D., Luo, J., Imbach, P.A., Dubenko, L.G., Jolad, S.D., Gerber, R.E., Litvak, J., Lu, Q., Zhang, P., Reed, M.J., Waldeck, N., Bruening, R.C., Noamesi, B.K., Hector, R.F., Carlson, T.J., King, S.R., 1998. Ethnobotanical-directed discovery of the antihyperglycemic properties of cryptolepine: its isolation from Cryptolepis sanguinolenta, synthesis, and in vitro and in vivo activities. J. Med. Chem. 41, 894–901. Boakye-Yiadom, K., Heman-Ackah, S.M., 1979. Cryptolepine hydrochloride effect on Staphylococcus aureus. J. Pharm. Sci. 68, 1510–1514. Cimanga, K., De Bruyne, T., Pieters, L., Vlietinck, A.J., Turger, C.A., 1997. In vitro and in vivo antiplasmodial activity of cryptolepine and related alkaloids from Cryptolepissanguinolenta. J. Nat. Prod. 60, 688–691. Egan, T.J., Hunter, R., Kaschula, C.H., Marques, H.M., Misplon, A., Walden, J., 2000. Structure–function relationships in aminoquinolines: effect of amino and chloro groups on quinoline–hematin complex formation, inhibition of ␤-hematin formation, and antiplasmodial activity. J. Med. Chem. 43, 283–291. Eisenstein, M., 2012. Drug development: holding out for reinforcements. Nature 484, S16–S18. Greenwood, B., Armstrong, J., 1991. Comparison of two simple methods for determining malaria parasite density. Trans. R. Soc. Trop. Med. Hyg. 85, 186–188. Grellier, P., Ramiaramanana, L., Millerioux, V., Deharo, E., Schrével, J., Frappier, F., Trigalo, F., Bodo, B., Pousset, J., 1996. Antimalarial activity of cryptolepine and isocryptolepine, alkaloids isolated from Cryptolepis sanguinolenta. Phytother. Res. 10, 317–321. Haas, S.E., Bettoni, C.C., de Oliveira, L.K., Guterres, S.S., Dalla Costa, T., 2009. Nanoencapsulation increases quinine antimalarial efficacy against Plasmodium berghei in vivo. Int. J. Antimicrob. Agents 34, 156–161. Hawley, S.R., Bray, P.G., O’Neill, P.M., Park, B.K., Ward, S.A., 1996. The role of drug accumulation in 4-aminoquinoline antimalarial potency: The influence of structural substitution and physicochemical properties. Biochem. Pharmacol. 52, 723–733. Kirby, G.C., Paine, A., Warhurst, D.C., Moamese, B.K., Philipson, J.D., 1995. In vitro and in vivo antimalarial activity of cryptolepine, a plant-derived indoloquinoline. Phytother. Res. 9, 359–363. Kuntworbe, N., Al-Kassas, R., 2012. Design and in vitro haemolytic evaluation of cryptolepine hydrochloride-loaded gelatine nanoparticles as a novel approach for the treatment of malaria. AAPS PharmSciTech 13, 1–14. Kuntworbe, N., Martini, N., Brimble, M., Alany, G.R., Al-Kassas, R., 2012. Isolation and development of an HPLC method for the detection and quantification of cryptolepine and its application in the determination of the total cryptolepine content in the root powder of Cryptolepis sanguinolenta. Pharmacol. Pharm. 3, 263–270. McCurrie, J., Albalawi, S., Wright, C., Kuntworbe, N., 2009. Investigation of the absorption and vascular effects of the indoloquinoline alkaloid, cryptolepine. In: British Pharmaceutical Conference, J. Pharm. Pharmacol., Manchester Central, pp. A67–A68. Noamesi, B.K., Larsson, B.S., Laryea, D.L., Ullberg, S., 1991. Whole-body autoradiographic study on the distribution of 3H-cryptolepine in mice. Arch. Int. Pharmacodyn. Ther. 313, 5–14. Olajide, O.A., Ajayi, A.M., Wright, C.W., 2009. Anti-inflammatory properties ofcryptolepine. Phytother. Res. 23, 1421–1425. Onyeibor, O., Croft, S.L., Dodson, H.I., Feiz-Haddad, M., Kendrick, H., Millington, N.J., Parapini, S., Phillips, R.M., Seville, S., Shnyder, S.D., Taramelli, D., Wright, C.W., 2005. Synthesis of some cryptolepine analogues, assessment of their antimalarial and cytotoxic activities, and consideration of their antimalarial mode of action. J. Med. Chem. 48, 2701–2709. Oyekan, A.O., 1995. Cryptolepine-induced vasodilation in the isolated perfused kidney of the rat: role of G-proteins, K+ and Ca2+ channels. Eur. J. Pharmacol. 285, 1–9. Peters, W., 1975. The chemotherapy of rodent malaria. XXII. The value of drugresistant strains of P. berghei in screening for blood schizontocidal activity. Ann. Trop. Med. Parasitol. 69, 155–171. Salako, Q., Ablordeppey, S.Y., Dwuma-Badu, D., Thornback, J.R., 1985. Radioiodination and preliminary in vivo investigation of the alkaloid cryptolepine. Int. J. Appl. Radiat. Isot. 36, 1003–1004. Santos-Magalhães, N.S., Mosqueira, V.C., 2010. Nanotechnology applied to the treatment of malaria. Adv. Drug Deliv. Rev. 62, 560–575. Sawer, I.K., Berry, M.I., Ford, J.L., 2005. The killing effect of cryptolepine on Staphylococcus aureus. Lett. Appl. Microbiol. 40, 24–29. Singh, M., Singh, M.P., Ablordeppey, S., 1996. In vitro studies with liposomal cryptolepine. Drug Dev. Ind. Pharm. 22, 377–381. WHO, 2011. World Malaria Report. Wright, C.W., Addae-Kyereme, J., Breen, A.G., Brown, J.E., Cox, M.F., Croft, S.L., Gokcek, K.Y., Kendrick, H., Phillips, R.M., Pollet, P.L., 2001. Synthesis and evaluation of cryptolepine analogues for their potential as new antimalarial agents. J. Med. Chem. 44, 3187–3194.