INTRODUCTION:

Chitosan is a cationic polysaccharide derived from chitin, which is a copolymer of glucosamine and N-acetyl glucosamine units^1,2. It has been extensively studied in the pharmaceutical industry as a carrier for drugs owing to its biocompatibility and biodegradibilty^3-5. Chitosan is polycationic in acidic media (pK_a 6.5) and can form a gel matrix with counter-ions such as sodium tripolyphosphate (TPP)^6-8. TPP is a non-toxic and multivalent anion that can form either inert or intra-molecular link with positively charged amino groups of chitosan ^9-11. This characteristic can be employed to prepare cross-linked chitosan beads. The interaction of chitosan with TPP leads to formation of biocompatible cross-linked chitosan beads, which can be efficiently employed in drug delivery. The cross-linking density, crystallinity, and hydrophilicity of cross-linked chitosan can allow modulation of drug release and extend its range of potential applications in drug delivery.

Methotrexate (MTX), a folic acid analogue, anticancer agent, immunosuppressive agent, selected as a model drug. MTX was chosen, because it is a cell-cycle phase specific drug where by prolonged exposure of drug to the cancer cells, is necessary for optimal efficacy¹¹. Methotrexate competitively and reversibly inhibits dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis.

The affinity of methotrexate for DHFR is about one thousand-fold that of folate for DHFR. Dihydrofolate reductase catalyzed the conversion of dihydrofolate to the active tetrahydrofolate. Folic acid is needed for the de novo synthesis of the nucleoside thymidine, required for DNA synthesis. Also, folate is needed for purine base synthesis, so all purine synthesis will be inhibited. Methotrexate, therefore, inhibits the synthesis of DNA, RNA, thymidylates, and proteins. Methotrexate acts specifically during DNA and RNA synthesis, and thus it is cytotoxic during the S-phase of the cell cycle. Logically, it therefore has a greater toxic effect on rapidly dividing cells (such as malignant and myeloid cells, and GI and oral mucosa), which replicate their DNA more frequently, and thus inhibits the growth and proliferation of these non-cancerous cells as well as causing the side effects listed above. Methotrexate is a weak dicarboxylic acid with pKa 4.8 and 5.5, and thus it is mostly ionized at physiologic pH. When it is given orally has a short elimination half life and can cause gastrointestinal irritation, diarrhoea and ulcerative stomatis. Oral absorption is saturatable and thus dose-dependent, with doses less than 40 mg/M2 having 42% bioavailability and doses greater than 40 mg/M2 only 18%. Mean oral bioavailability is 33% (13-76% range), and there is no clear benefit to subdividing an oral dose. Mean intramuscular bioavailability is 76%. Methotrexate is metabolized by intestinal bacteria to the inactive metabolite 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) and accounts for less than 5% loss of the oral dose^12-15

Table1 Formulation summary as per 3² factorial design

Batch no.	X₁	X₂	Mean particle size (µm)		DC %	t₅₀ min
CH1	-1	-1	770		58.56	218
CH2	0	-1	751		42.30	240
CH3	1	-1	742		35.70	300
CH4	-1	0	670		49.70	183
CH5	0	0	634		40.16	200
CH6	1	0	664		33.46	230
CH7	-1	1	544		39.48	120
CH8	0	1	556		36.43	153
CH9	1	1	543		30.61	170
Coded value	Actual values
Coded value	X₁			X₂
-1	1			0.5
0	2			1.0
1	3			1.5

X₁ = Concentration of TPP (%), X₂ = Concentration of Span 80 (%)

Thus, the objectives of present study are to prepare MTX containing sustained release chitosan beads by using 3² factorial design which eliminate the inherent drawbacks experienced with oral administration. The concentration of TPP (X₁) and the concentration of Span 80 (X₂) were selected as independent variables. The DC and t₅₀ were selected as dependent variables. These various concentrations of TPP (1%, 2%, 3%) and Span 80 (0.5%, 1%, 1.5%) were used to investigate the influence of these varied concentration on release and physical characterization of beads.

MATERIALS AND METHODS:

Materials:

Chitosan and MTX were procured from Central Institute of Fisheries, Cochin and Sun Pharmaceuticals, Mumbai respectively. Tripolyphosphate (TPP), Sorbitan mono-oleate (Span 80), Glacial acetic acid, and other reagents were procured from SD fine Chemicals Ltd. Mumbai.

Methods of preparation:

Beads were prepared by Ionotropic gelation method with some modifications^16-18. The MTX (50 mg) was dispersed in 10 mL of 1 % wt/vol chitosan solution previously prepared in aqueous acetic acid (Disperse phase). The beads were formed by dropping the bubble-free dispersion through 18 gauge disposable syringe (10 mL) into a gently agitated (Magnetic stirrer Remi equipment, Mumbai, Model ms 500) 1 % wt/vol aqueous TPP solution containing 0.5 % vol/vol Span 80 (Continuous phase). The chitosan beads were separated after 15 minutes by filtration and rinsed with distilled water. Beads were oven dried at 60⁰C for 6 h and dried beads were stored for their characterization.

CHARACTERIZATION OF BEADS:

Factorial design:

Well established statistical tools such as factorial designs are very essential to understand the complexity of pharmaceutical formulations. The number of experiments required for these studies are dependent on the number of independent variables selected by the formulator. For simplicity, it was decided to perform a two variable study as three experimental levels to achieve the set objectives efficiently. The concentration of TPP (X₁) and the concentration of Span 80 (X₂) were selected as independent variables. The DC and t₅₀ were selected as dependent variables. These various concentrations of TPP (1%, 2%, 3%) and Span 80 (0.5%, 1%, 1.5%) were used to investigate the influence of these varied concentration on release and physical characterization of beads. Various compositions of formulations as per the 3² factorial design were shown in Table 1. The statistical analysis of the factorial batches was performed by multiple regression analysis using Microsoft Excel followed by ANOVA. The results are depicted in Table 2.

Figure 1 Response surface plot of effect of X₁ and X₂ on DC

Drug content (DC):

The MTX-Chitosan beads were tested for their DC. The beads containing drug were extracted with 0.1N HCl solution for 36 h. The content was filtered and absorbance of the filtrate was measured at 303 nm, using 0.1N HCl as the blank by spectrophotometer (UV 1601 Shimandzu Corp., Japan Model) and DC were determined.

In-vitro release study:¹⁹

The release properties of beads were studied in 0.1N HCl using USP XXI rotating paddle apparatus (Scientific USP stds, Model DA). The dissolution medium 500 mL, was maintained at (37 ± 0.5⁰C), and stirring speed was set at 100 rpm. Beads (50 mg) were added to the dissolution medium and aliquots of 1mL were taken and replaced with fresh medium at predetermined time intervals. The concentration of MTX in each filtered sample was determined by UV spectrophotometer at wave length of 303 nm using corresponding blank.

Size distribution of beads:

The size distribution of beads into various size fractions was carried out using mechanical sieve shaker. The mean particle sizes of the beads were determined by sieving method.^20,21Beads were separated into different size fractions by sieving for 10 minutes using mechanical sieve shaker (Cuprit Electrical Co. India) containing standard sieves having apertures of 1000,

T50

X1 = A: TPP

X2 = B: span 80

Figure 2 Response surface plot of effect of X₁ and X₂ on t₅₀

710, 500, 355, 250 and 180 µm (Indian Pharmacopoeia, 1996). The particle size distribution of the beads for all the formulations was determined and mean particle size of beads was calculated by using the following formula.

Similarity factor:

The following criteria were established for the desired drug release profile; (a) 20 %< Y60<40%; (b) 50 %< Y360<70%; (c) 65 %< Y480<80 %²². Based on these criteria, a desired drug release profile was established and compare with best batch (CH1) as shown in the figure1. For the purpose of comparison, the similarity factor between the two formulations was determined using the data obtained from the drug release studies. The data were analyzed by the formula²³ shown in Equation (1)

f₂ = 50 log { [1+(1/N)Σ(Ri - Ti)²]^-0.5 × 100} (1)

Where,

N- Number of time points

Ri and Ti – Dissolution of reference and test product at time i

If f₂>50, it is consider that two products share similar drug release behavior.

RESULTS AND DISCUSSION:

A statistical model [Eq. (2)] incorporating interactive and polynomial terms was used to evaluate the selected responses.

Y=b₀+b₁X₁+b₂X₂+b₁₁X₁²+ b₂₂X₂²+b₁b₂X₁X₂ (2)

where Y is the dependent variable, b ₀ is the arithmetic mean response of the 9 runs, and b _i is the estimated coefficient for the factor X _i. The main effects (X ₁ and X ₂) represent the average result of changing 1 factor at a time from its low to high value. The interaction terms (X ₁ X ₂) show how the response changes when 2 factors are simultaneously changed. The polynomial terms (X ₁ ² and X ₂ ²) are included to investigate nonlinearity.

Figure 3 Comparison of dissolution profile of batch CH1 and desired drug release profile.

The fitted equation relating the response (DC) to the transformed factors is shown in Eq. (3)

DC = 40.02-7.995X₁-5.0X₂+1.621X₁²-0.593X₂²+3.49X₁X₂ (3)

(R=0.9784)

The DC value for the nine batches showed a wide variation i.e. the response ranges from a minimum of 30.61 to a maximum of 58.56 %. These data indicates that the DC value is dependent on the factors.

The significant level of coefficients of b₁² and b₂²were found to be 0.350461 and 0.713508 respectively and hence they were removed from regression Eq. (3) to generate the reduced model. The coefficients b₁, b₂and b₁b₂ showed significant values of less than 0.05 and hence they were retained in the reduced model. [Eq. (4)]

DC = 40.02-7.995X₁-5.0X₂+3.49X₁X₂ (4)

It was concluded that the low levels of X₁ (TPP concentration) and X₂ (Span 80 concentration) appears to favor the preparation of beads with better DC. The Eq. (4) is presented in the form of a response surface plot in Fig. 1 to visualize the impact of changing independent variables on DC.

It was found that as the concentration of Span 80 and TPP increases the DC was decreased. Here the span 80 is a representative of non ionic dispersing agent which was used to stabilize the system. It has the HLB value 4.3 and is expected to have a high dispersity by reducing the surface tension at the interface. Thus, greater amount of Span 80 might be included in the beads which resulted in the decreased DC.

The decreases DC value with higher concentration of TPP attributed to solubility of MTX in the TPP solution and loss of the drug from disperse phase to continuous phase. An increase in concentration of TPP in continuous phase leads to increased solubility of MTX in the TPP solution and lower the DC in the beads. Results showed similar decreased in the DC, as found by R. Bodmeier [17].

t₅₀ = 200.44+29.83 X₁-52.5X₂+5.83X₁²-4.166X₂²-8.0X₁X₂ (5)

(R=0.9839)

The t₅₀ value for the nine batches showed a wide variation i.e. the response ranges from a minimum of 120 to a maximum of 300 min. The data clearly indicates that the t₅₀value is strongly dependent on the factors.

The significant level of coefficients of b₁², b₂² and b₁b₂were found to be 0.508153, 0.629694 and 0.242176 respectively and hence they were removed from regression Eq. (5) to generate the reduced model. The coefficients b₁and b₂showed significant values of less than 0.05 and hence they were retained in the reduced model. [Eq. (6)]

t₅₀ = 200.44+29.83 X₁-52.5X₂ (6)

It was concluded that the high level of X₁ (TPP concentration) and low level of X₂ (span 80 concentration) appears to favor the preparation of controlled release beads of MTX. The Eq. (6) is presented in the form of a response surface plot in Fig. 2 to visualize the impact of changing independent variables on t₅₀.

It was found that as the concentration of TPP increases the drug release in terms of t₅₀value was decreased. This was due to the fact that as the concentration of TPP increases the cross linking between TPP and chitosan was also increased which reduces the degree of swelling with subsequent decreased in rate of drug release. The same tendency was seen in this study. The MTX release in terms of t₅₀was decreased with increasing concentration of TPP in 0.1N HCl.

It was also found that as concentration of Span 80 increases the drug release in terms of t₅₀ value was decreased. This may be attributed to the presence of more free drug on the surface of beads with increasing concentration of span 80 and also the presence of Span 80 prevented the aggregation of beads, provided greater the surface area for dissolution resulted in faster release of the drug.

The beads of batch CH1 were found comparatively smooth, spherical in shape and free flowing. The size analysis study revealed that the beads mean particle sizes ranging from 500 to1000 (see Table 1). Most of the particles were smaller than 600 µm, and only a few particles were larger than 700 µm, with none larger than 1000 µm.²⁴

As reported the swelling of chitosan beads was dependent on the dissolution medium. More specifically it was reported that when chitosan beads wetted by the acidic dissolution medium they swelled extensively, and formed a hydrogel matrix before they dissolved completely. They did not swell or dissolve in simulated intestinal fluid. Thus in present investigation the dissolution study was performed in 0.1N HCl dissolution medium. The dissolution data of beads of batch CH1, a potential candidate for 12 h in vitro release (Fig. 3).

Similarity factor:

The principal purposes of dissolution testing are 3-fold: (1) for quality control, to ensure the uniformity of product from batch to batch; (2) to help to predict bioavailability for formulation development; and (3) as a measure of change when formulation changes are made to existing formulation. The so called f₂ method can be used to compare 2 dissolution profiles. Similarity factors analysis between prepared formulation (CH1) and predicted dissolution profile showed an f₂ factor (f₂= 90.90072) greater than 50. Thus, the f₂ factor confirms that the release of drug from prepared formulation was similar to that of desired drug release profile.

CONCLUSION:

MTx containing chitosan beads can be prepared successfully by an ionotropic gelation technique using 3² full factorial design. The DC value and t₅₀ value showed the response ranges from a 30.61 to 58.56 % and 120 to 300 min, respectively. The beads of best batch (CH1) were found comparatively smooth, spherical in shape and free flowing. The size analysis study revealed that the beads mean particle sizes ranging from 500 to1000. The results of similarity factor, f_2,confirmed that the release of drug from prepared formulation was similar to that of desired drug release profile. The results of multiple linear regression analysis concluded that for obtaining controlled drug release with high drug content, the beads should be prepared using relatively lower levels of Tripolyphosphate and Span 80.

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Received on 16.08.2009 Modified on 23.08.2009

Research J. Pharm. and Tech.2 (3): July-Sept. 2009,;Page 592-596

Parameters	df	SS	MS	F	Significant F
For DC
Regression	5	588.8141	117.7628	27.2537	0.010527
Residual	3	12.96295	4.320984
Total	8	601.7771
For t₅₀
Regression	5	22236.44	4447.289	36.67587	0.006834
Residual	3	363.7778	121.2593
Total	8	22600.22

Kotadiya RM1,*, Patel VA2 and Patel HV1

Kotadiya RM^1,*, Patel VA² and Patel HV¹