Indian Journal of Experimental Biology Vol. 39, September 2001, pp 902-905

Sustained release implants of chloroquine phosphate for possible use in chemoprophylaxis of malaria

Beena Saparia, Ajay Solanki & R S R Murthy*

Pharmacy department, Faculty of Technology and Engineering,M.S. University of Baroda, Vadodara 39000 I, India

Received 16 January 2001; revised 7 March 2001

Implants of chloroquine phosphate (CQP) using biodegradable polymer, gelatin (G) and cross-linked gelatin (CLG) were prepared and evaluated to assess their physicochemical properties and in vitro release profile. The mechanism and ki­netics of release were studied to correlate the release phenomenon with the formulation parameters. Out of many batches of the implants investigated, the implant prepared with 20% gelatin at 2: 1 drug polymer ratio, 10% crosslinking agent and 2% plasticizer (Batch J) was found to provide optimum release behavior conforming to the requirements of a long term implant for a week. In vivo studies conducted on albino rats showed consistent therapeutic blood level over a period of 7 days. Mean residence time (MRT) of the drug released in the body, calculated as the ratio of the area under the first moment curve (AUMC) to area under concentration time curve (AUC) was 72 hr for implant against 2.42 hr for subcutaneous injection.

Malaria is still a major health problem worldwide es­pecially in the third world countries in spite of the introduction of effective insecticides and antimalarial drugs. Enormous increase in the cases of chloroquine resistance has made the management of the disease extremely difficult and so the demand for a simple and effective prophylactic method is always felt. Ac­cordingly, travellers to endemic area are recom­mended for prophylactic measures to avoid infection I. World Health Organization2 recommends a minimum of 3 months protective measure against malaria by using a suitable injectable prophylactic medication. Many efforts are made to prolong the duration of ac­tion of many antimalarial drugs by using low soluble derivatives, prodrugs, non-biodegradable silicon rub­ber implants, biodegradable dihydropyran implants etc3.5.

For the present study, chloroquine · phosphate (CQP) was chosen as it is the most widely prescribed antimalarial drug in the world. In spite of high inci­dences of drug resistance in Plasmodium Jalciparum, it still remains as the drug of choice for malaria. It is inexpensive, widely available and well tolerated under controlled dose and so it can be well adopted as drug for mass prophylaxis program provided a suitable and safe long acting delivery system is brought out. The study is essentially oriented to develop an implant formulation of CQP using biodegradable polymer, gelatin.

*Correspondent author : Fax-91-0295-423898: E-mail-murthyrsr@satyam.net.in

Materials and methods Chloroquine phosphate (CQP) was obtained as a

gift sample from IPCA Labs Pvt. Ltd., Mumbai. Gelatin of U.S.P. grade with isoelectric point of 4.8 was procured from Burgoyne Burbridges & Co., Mumbai, India. Formaldehyde was obtained from BDH, Mumbai and polyethylene glycol-400 was ob­tained from Fine Chemicals Div., Hyderabad, India. All other reagents and solvents used were of analyti­cal grade.

Gelatin molds were prepared6 with some modifica­tions from the original method. Weighed quantity of CQP was dissolved in sufficient quantity of distilled water and to it weighed quantity of gelatin was added. The mixture was heated at 55° C to obtain a clear so­lution. Required quantity of polyethylene glycol-400 and formaldehyde was added, gently stirred and poured immediately in to polystyrene cylindrical mold of 6mm inner diameter. It was allowed to set at room temperature and then kept in refrigerator for 24 hr. The molds obtained were slowly extruded out and cut transversely in to implants of 10 mm length. All batches of implants were tested for free formalde­hyde by the method' involving the reaction of formal­dehyde with phenyl hydrazine to form red color.

Characterization oj implants - The implants were characterized for swelling and gelling properties by exposing them to water at 37° C for 5 days and the increase in dimension and weight were determined at regular intervals. Gel strength of hydrated gelatin im­plants was determined by using penetrometer. Maxi-

SAPARIA et al.: IMPLANTS OF CHLOROQUINE PHOSPHATE 903

mum weight required for the needle to penetrate the physical gel was taken as the indicator of strength. The implants were also examined microscopically (lOx) to study the surface continuity.

CQP content in implants was determined by meas­uring absorbance at 343 nm using Hitachi, 2000 UVNis spectrophotometer8

. Approximately 5 mm length implant was accurately weighed and placed in ' 50 mL volumetric flask. Added 40 mL of 3N HCI, heated slightly to dissolve the implant and the volume was made up with 3 N HCl. The solution was suitably diluted with 3N HCI and absorbence was measured at 343 nm against a blank prepared using the implant containing no drug. Amount of CQP was calculated using the calibration curve constructed in the concen­tration range of 1 to 20 j..lg/mL (Regression equation Y = 0.5636 - 0.0323 X ).

In vitro drug release study--ln vitro release of CQP from implants was determined9 over a period of • 7 days in distilled water at 37°C using USP XXIII dissolution test apparatus with 900 mL of the dissolu­tion fluid. The implant was weighed and placed in the dissolution media and stirred (Peddle) at 100 rpm. Samples (10 mL) were withdrawn at 1,2,4,6, 12,24 hr on the first day and every 24 hr for the rest of the days. The samples were analyzed for CQP content by the method described above.

In vivo studies-In vivo study of CQP implants was conducted on rats of either sex in two groups of six · rats each weighing 250-300 g. Rats of first batch were injected with injection (S.C) of CQP(4.5 mg) and the other group of rats were implanted with CQP implant (equivalent to 9 mg of the drug) subcutaneously in the right axilla. Blood samples were withdrawn by punc­turing the sino-orbital plexus of the rat eye with a fine

capillary at different time intervals for 7 days. Blood (0.2 mL) was mixed with trichloroacetic acid to make the volume up to 1 mL. Sodium hydroxide (0.1 N ; 2mL) was added and extracted successively with three fractions of 1mL, 1mL and 0.5 mL of dichlo­romethane. These fractions were pooled and re­extracted with 1mL, 1mL and 0.5 mL of O.OIN HCl. Concentration of CQP was determined by the method described above. Results and Discussion

Several batches of implants were prepared by varying parameters like polymer concentration (10 to 30%), drug to polymer ratio (1: 1 to 3: 1), concentra­tion of cross linking agent (4 to 20%) and of the plas­ticizer (2 to 10%). The concentration of the cross linking agent (formaldehyde) was optimized based on the residual free formaldehyde content in the implant. Batches prepared with 15% or more formaldehyde content gave positive test for free formaldehyde and hence the concentration of the cross linking agent was restricted to 10% w/w. Swelling studies showed an average of 38% increase in volume and 31 % in weight at equilibrium. Gel strength of optimized for­mulation at hydrated state was between 68 to 75 g. Microscopic examination of implants showed smooth but porous surface with all batches except with batches prepared with high drug to polymer ratio (3: 1) where the surface was rough with deposits of drug crystals scattered at the surface.

Details regarding different batches of implants along with their average weight and drug content are given in Table 1. Batches A, B and C prepared with 10, 20 and 30 % gelatinr.concentration respectively showed significant difference in consistency and gel strength. Batch A implant was too slimy to handle

Table I--Physical characteristics and in vitro dissolution profile of CQP implants

Batch Gelatin Percentage of Weight Drug Per cent Gel t3O% tro% No conc.(%) CLA PEG-400 (mg) content Swelling Strength (hr) (hr)

(d : g Ratio) (mg)

A 10 (1:1) 10 448 98.7 32/30 56 6.4 28.0 B 20 (1:1) 10 504 95.4 34/29 69 16.0 67.4 C 30 (1:1) 10 559 102.4 29/26 86 24.0 96.0 D 20 (I : 1) 10 2 498 99.4 32/29 71 16.0 67.4 E 20 (1 :1) 10 4 501 98.7 30/27 57 6.4 28.0 F 20 (I: 1) 10 10 506 97.9 32/28 54 5.8 24.0 G 20 (1:1) 4 2 497 103.2 35/32 66 13.0 49.0 H 20 (1 :1) 10 2 503 101.3 31/29 71 16.0 67.4 I 20 (1:1) 20 2 508 98.4 28/25 75 24.0 96.0 J 20(2:1) 10 2 512 205.5 32/28 65 24.0 72.0 K 20 (3:1) 10 2 519 304,2 31128 67 16.0 50.0

A, B K are the batches of CQP implants prepared with gelatin concentration, drug to polymer ratio, percentage of cross-linking agent (CLA) and PEG 400 as given above in the table. D-drug; and g-gelatin

904 INDIAN J EXP BIOL, SEPTEMBER 2001

while the implant of batch C was too rigid. It was ob­served that the batch containing 30% gelatin had rough surface and hence 20% gelatin concentration was found optimum. To avoid brittleness in implants on storage, polyethylene glycol-400 was used as plas­ticizer. The batches D, E and F prepared with 2, 4 and 10% of PEG-400 respectively showed negligible dif­ference in consistency, and surface smoothness but · significant increase in drug release rate with batch F having 10% PEG-400 (Fig. i). Hence the concentra­tion of PEG-400 was fixed at 2% w/w. Batches J and K were prepared with higher drug to polymer ratio of 2: 1 and 3: 1 respectively in order to study the influ­ence of drug loading on the drug release profile. Comparing the in vitro dissolution behavior of these batches with batches containing 1: 1 drug . to polymer ratio, no significant difference in t30 values but, slight decrease in tw value was observed (Fig. 1). This is beneficial for implants for long term drug delivery, where pay load capacity of drug can be increased without significantly increasing formulation dimen­sion and also the release rate of the loaded drug . These findings are in agreement with the report of Goto et al. 10 where, in vitro release of sulfonamide from gelatin microparticles has been reported to be independent of drug content up to 67 % by weight.

All batches of implants except batch A, E and F showed linear relationship (R2 values between 0.9948 and 0.9975) when % release was plotted with root of time indicating the matrix diffusion behavior. Regres­sion of the root time plot of batches AlE and F showed low R2 values of 0.977 and 0.9858 respec­tively. In addition these batches showed positive Y­intercept values indicating burst effect in the initial release phase followed by decrease in the release rate as the time proceeds. This release . behavior with batches AlE and F was obvious as they were prepared either with low polymer concentration or high con­centration of plasticizer, as a result, the implant may undergo erosion during the period of study. In order to confirm this, Hopfenbergll analysis for cylindrical devices was used by plotting (l_MtlMoc)112 vs. time with the dissolution data of these batches of implants.

Table2- Pharmacokinetic parameters of CQP implants and

Parameter Cmax

Tp AUC AUMC MRT

s.c.injection in albino rats Implant Subcutaneous injection

l5.0j.LglmL ± 3.0 34.8 j.Lg/mL ± 5.0 j.LglmL j.Lg/mL 4.0 hr 108.0

7776.0 72.0 hr

1.0 hr 23.8 57.6

2.4 hr

Linear plots were obtained (Fig.2) with R2 values of 0.9923 and 0.9941 for batches AlE and F res·pectively indicating the possibility of erosion.

Based on the above results, batch J was selected for

140

1 FIX'

120 2 Al£1.)

3 C(O)

4 KI+'

QI 5 8/DIH (0) .. 6 J ,.)

" 7 CIliA) QI

! 80

CI

2 c :.e 0 40

o+-----------,-----------,-----~

o 5 10 15

Root of time (hO•5

)

Fig.l-Per cent drug release vs. root time plot of dissolution data for various batches of CQP implants. F (X); CII (A); AlE (. );G(O);K (+); BlDtH (O);J (e).

0.8

S_ 0.6

~ 0." =F 0.2

0

~.2~------____________________ ~

Time (hr)

Fig.2-Plot of (1- MtIM)112 vs. time of the dissolution data of the batch A and E (~) and the batch F (D).

40~---------------------,

::i E 30 -Cl .:. c..i 20 c 0 u

" 10 0 0 iii

0 0 50 100 150 200

Time (hr)

Fig.3-Blood concentration vs. time plot following implantation of subcutaneous implant (A) of CQP (9 mg) in albino rats com­pared with that of subcutaneous injection (0) of CQP sterile solu­tion (4.5 mg).

SAPARIA et al.: IMPLANTS OF CHLOROQUINE PHOSPHATE 905

in vivo studies in albino rats. Implant being designed for subcutaneous region, subcutaneous injection of CQP was taken for comparative study.Blood concen­tration vs. time data generated during in vivo studies in albino rats is plotted in Figure 3 and the pharmaco­kinetic parameters calculated from these data are tabulated in Table 2. Blood concentration of CQP in rats following subcutaneous injection reached maxi- . mum level (34.8 Jlg ImL) in one hour and the drug concentration could be estimated upto 6 hr. The im­plant showed the Cmax of 15 Jlg ImL after 4 hr post implantation and the concentration maintained above 4.0 Jlg/mL for 168 hr (7 days). Comparative bioavail­ability of implants to sc injection calculated using AUC to dose ratio of implant (108) to that of sc injec­tion (23.8) was found to be 4.54. The mean residence time (MRT) of the drug in the body calculated as the ratio of the area under the first moment curve (AVMC) to the area under the curve (AVC) was found to be 72 hr for the implant as against 2.40 hr for the subcutaneous injection. The blood level concen­tration of CQP observed following subcutaneous im­plantation (>4 JlglrnL) in albino rats was well above the minimum concentration of 30 nglmL, as sug­gested by Hardman and Limbard 12 for antimalarial activity with any type of plasmodium parasite. These results signify the importance of implant delivery system for long term delivery of CQP particularly for prophylaxis.

Acknowledgement Authors wish to acknowledge the financial assis­

tance from University Grants Commission, New Delhi to the first and the second author during the course of the study.

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