INTRODUCTION:

Dapsone-USP, 4, 4’-diaminodiphenylsulfone (DDS) is a primary treatment for Dermatitis herpetiformis. It is an antibacterial drug for susceptible cases of leprosy. Dapsone is used to control the dermatologic symptoms of Dermatitis herpetiformis. Dapsone is used alone or in combination with other anti-leprosy drugs for leprosy.

Biopharmaceutical classification system (BCS), based on solubility and permeability of drug is divided into four classes.

Dap shows low aqueous solubility and high membrane permeability (class II) ^(1-3)as per the BCS classification given below:

BCS class I	BCS class II
High solubility	Low Solubility
High permeability	High permeability
BCS Class III	BCS Class IV
High solubility	Low solubility
Low permeability	Low permeability

Increasing dissolution rate of poorly water soluble drug is major challenge in dosage form development. Bioavailability of orally administered drug mainly depends on its solubility and permeability. Drug discovery shows that compounds are often high molecular weight and highly lipophilic hence exhibits poor solubility.

Dissolution of various drugs can be improved by ⁽⁴⁾

· Increasing the surface area available for dissolution.

· Optimizing wetting characteristics of compound surface.

· Decreasing boundary layer thickness.

· Ensuring sink conditions for dissolution.

· Improve apparent solubility.

Various solubility enhancement strategies in solid dispersion are fusion (melting), solvent evaporation, lyophilization (freeze drying), melt agglomeration process, extruding method, spray drying technique, use of surfactant, electrostatic spinning method and super critical fluid technology. One approach is formation of solid dispersion of drug with hydrophilic excipients. Ideal type of solid dispersion for increasing dissolution requires glass solution in which amorphous drug has low thermodynamic barrier to dissolve together with maximally reduced particle size. Also presence of hydrophilic excipients may lead to increase wetting leading to super saturation in the diffusion layer.

Glass solution is formed when two or more components are entirely miscible in molten state and cooled to form amorphous one phase system. For glass solution, melt extrusion studies were preferred due to several applications and advantages as given below:

Applications include: ⁵

· Improving dissolution rate and bioavailability of drug.

· Controlling/modifying release of drug.

· Masking bitter taste of drug.

Advantages include: ⁶

· Small equipment

· Economic and continuous process and scale up flexibility

· Solvent free manufacturing

· High mixing efficiency

· Closed process unit to prevent cross contamination

· Short processing time

· Easily controlled process parameters

· Possibility of online analytics for process control

Disadvantage includes: ⁶

· Thermal process(drug/polymer stability)

· Flow properties of polymers are essential to processing

· Limited number of available polymers

· Require high energy input

· Melt technique process cannot be applied to heat sensitive materials due to high temperature involved.

HME can be simply defined as the process of forming a new material (the extrudate) by forcing it through an orifice or die under controlled conditions, such as temperature, mixing, feed-rate and pressure. ⁷

A variety of carrier systems have been studied or used in HME dosage forms. Such carrier systems include polyvinylpyrrolidone (PVP) or its co-polymer such as polyvinylpyrrolidone-vinyl acetate, copovidone (Kollidon VA64), poly (ethylene-co-vinyl acetate), various grades of polyethylene glycols, cellulose ethers and acrylates, various molecular weight of polyethylene oxides, poly methacrylate derivatives and poloxamers. Amongst the different classes of biodegradable polymers, the thermoplastic aliphatic poly (esters) such as poly (lactide) (PLA), poly (glycolide) (PGA) and copolymer of lactide and glycolide, poly (lactide-co-glycolide) (PLGA) have been used in extrusion. Starch and starch derivatives have been applied along with low molecular weight excipients like sugars and sugar alcohols and waxes.

Plasticizers are added to HME formulations to facilitate the extrusion of the material and to increase the flexibility of the extrudate. The choice of suitable plasticizer depends on many factors, such as plasticizer-polymer compatibility and plasticizer stability. Polyethylene glycol (PEG 4000), polyoxy 35 castor oil (Cremophor EL) and sorbiton monolaurate (Montane 20 PHA), triacetin, citrate esters and low molecular weight polyethylene glycols have been investigated as plasticizers in hot-melt extruded systems.^7-15

Basic requirements for polymers used in HME:

Ø	Thermoplastic behavior	-deformability is essential
Ø	Suitable Tg	-50-180ºC
Ø	High thermal stability	-50-180ºC
Ø	Low hygroscopicity	-prevents crystallization
Ø	No toxicity	-application of large amounts possible
Ø	High or no solubilisation	-thermodynamically stable capability formulation

MATERIALS AND METHODS:

Dapsone was received as gift sample from Emcure pharmaceuticals limited, Pune, India. Copovidone (Kollidon VA64), polyoxyl 35 castor oil (Cremophor EL), PEG 4000, sorbiton monolaurate and all other reagents and chemicals used were of analytical grade.

METHODS:

1. Characterisation of Dap:

Dap was characterized by following test:

A. Description:^1-3

Dap was studied for its color and physical appearance.

B. Saturation Solubility:¹

Solubility of Dap was measured in distilled water. An excess amount of drug was added to 50 ml conical flask and was kept under shaking for 72 hrs (Rotary shaker, Biomedica). Saturated solution was filtered through 0.45 µ membrane filter, absorbance of filtered solutions was determined and amount of drug solubilised was calculated.

C. Melting Point: ^3,4

Dap melting point was determined by both the capillary method and instrumental method. Capillary method was done by taking capillary in which drug was inserted and then attached to thermometer. Both capillary along with thermometer was inserted into the parrafin bath which was heated and the melting temperature was recorded.

Instrumental method involves insertion of capillary in the paraffin bath and the melting temperature was recorded electronically (Melting point apparatus VEEGO). This method proved to be more accurate than the former method.

D. XRD:¹²

Dap was subjected to XRD (P.W. 1729, X-ray generator, Philips, Nether land). To study XRD pattern, the drug sample was placed into aluminum holder and the instrument was operated between initial and final 2θ angle of 5-50⁰respectively in an increment of 0.4⁰2θ.

E. IR:¹²

Dap, was subjected to Fourier Transform Infra Red (FTIR 8400s spectrophotometer Shimadzu) studies to check the characteristic sharp peaks of drug and its functional groups. The Pottasium bromide (KBr) disk method was used for preparation of sample. The samples were ground gently with anhydrous KBr and compressed to form pellet. The scanning range was 400-4000cm^-1.

F. DSC:¹²

Dap was subjected to DSC study using (Mettler TA 4000) DSC apparatus. First 5-10 mg of sample was weighed into aluminum crucible. This powder was analyzed by heating at scanning rate of 10⁰C / minute over a temperature range 50 to 200⁰C with nitrogen flow of 50mL/min.

2. Preparation of Calibration Curve:^{1, 13}

100µg/ml stock solution of Dap was prepared in by first dissolving 100mg of drug in 100mL of 2mL dilute HCl. Further dilutions were done to obtain solutions of 1....10 µg/mL. Respective absorbance values were measured at fixed λ max.

3. Determination of Drug :Polymer Ratio:^14,15

Solubility of Dap was checked in different solvents such as methanol, ethanol and water. Both drug and polymer were soluble in ethanol and hence selected for optimization of ratio. Drug and polymer (1:1 to 1:5) were solubilised in ethanol. The obtained solution was then poured in petri plates and films were cast by solvent evaporation method and were observed after 24 hrs at room temperature for their appearance.

4. Effect of Temperature on Decomposition of Polymers:^14,15

Polymer was subjected to different temperatures at 120, 130, 140, 150 ºC, using heating mantle (Lab Hosp. Corp., ELCON) and the molten polymer was cooled at room temperature and then milled using hammer mill(Lab Hosp).The obtained granules of different processing temperature were then compared for their appearance and discoloration .

Table 1: ND FORMULATION

Ingredient	Applications	Quantity per tablet(mg)
Dap	Drug	100
Lactose monohydrate	Diluent	33
Microcrystalline Cellulose	Diluent	33
Crospovidone	Disintegrant	8
Magnesium Stearate	Lubricant	1
Total		175

5. Preparation of Non HME (ND) Formulation[As control sample]:

Dap (as such), lactose monohydrate, microcrystalline cellulose and crospovidone as in table 1 are (sifted through #40 sieve) mixed well for 5 min. Prepared dry mix lubricated by magnesium stearate (sifted through # 60 sieve) for 3 min. Lubricated granules were evaluated for flow properties and compressed into tablets.

6. Effect of type and concentration of plasticizer on solubility and dissolution:

Dap, polymer and plasticizer as in table 2 were mixed well and taken in porcelain dish. This dry mix was subjected to melt at 175°C using heating mantle (Lab Hosp. Corp., ELCON) with mixing to get clear molten mass. Curing of molten mass was done by keeping it at room temperature for 12 hrs.

a. Size Reduction of HME Flakes:

Solid dispersion prepared by HME was then passed through 3mm screen of hammer mill, milled granules were sifted through #40 sieve. Granules retained on #40 sieve then passed through 1mm screen of hammer mill, milled granules were sifted through #40 sieve. Obtained granules were mixed well for 5min.

Table 2: HME FORMULATION

	Applications	D1	D2	D3	D4	D5	D6	D7	D8	D9
Dry mix for HME		mg/tab	mg/tab	mg/tab	mg/tab	mg/tab	mg/tab	mg/tab	mg/tab	mg/tab
Dap	Drug	100	100	100	100	100	100	100	100	100
Kollidon VA64	Thermal binder	100	100	100	100	100	100	100	100	100
PEG 4000	Plasticizer	10*	-	-	20**	30^#	-	-	-	-
Cremophor EL	Plasticizer	-	10*	-	-	-	20**	30^#	-	-
Montane 20 PHA	Plasticizer	-	-	10*	-	-	-	-	20**	30^#
HME Granules		210	210	210	220	230	220	230	220	230
Lubricants
Colloidal Silicon Dioxide	Lubricant	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3	1.3
Sodium Stearyl Fumarate	Lubricant	2.7	2.7	2.7	2.7	2.7	2.7	2.7	2.7	2.7
Total		214	214	214	224	234	224	234	224	234

*10% w/w of polymer (Kollidon VA 64)

**20% w/w of polymer (Kollidon VA 64)

^#30% w/w of polymer (Kollidon VA 64)

b. Lubrication of HME Granules:

HME granules were then lubricated by sodium stearyl fumarate and colloidal silicon dioxide (sifted through #60 sieve) for 5 min. Lubricated granules were evaluated for flow properties and compressed into tablets. Compression parameters were recorded.

c. Characterization of HME Dry Mix ,HME Granules And NHME Dry Mix:

i. DSC:

The drug, HME and NHME were subjected to DSC study using (Mettler TA 4000) DSC apparatus. First 5-10 mg of sample was weighed into aluminum crucible. These powders/granules were analyzed by heating at scanning rate of 10⁰C / minute over a temperature range 50 to 200⁰C with nitrogen flow of 50mL/min.

ii. XRD:

The drug, HME complex and NHME formulated powder were subjected to XRD (using P.W. 1729, X-Ray Generator, Philips, Nether land). To study XRD pattern, the sample was placed into aluminum holder and the instrument was operated between initial and final 2θ angle of 5-50⁰respectively in an increment of 0.4⁰2θ.

iii. IR:

The drug, HME complex and NHME formulated powder were subjected to FTIR (8400s spectrophotometer Shimadzu) studies to check the characteristic sharp peaks of drug and its functional groups. The KBr disk method was used for preparation of sample.

Prepared tablets (table 2) of formulation F1 –F9 were subjected to solubility and dissolution study.

7. In Vitro Permeability of HME and NHME Formulation:

The prepared tablets were subjected to In vitro permeability test using dialysis membrane LA401.

8. Stability Studies:¹³

Stability studies of tablets were performed as per International Conference on Harmonisation (ICH) guidelines. The tablets from the optimized batch were subjected for stability study at 40ºC/75% RH for 3 months.

RESULT AND DISCUSSION:

1. Characterisation of Dap:

A. Description:

B. It is a white, odorless crystalline powder. Hence confirms the description as per the certificate of analysis (COA).

C. Saturation Solubility:

As per literature the solutbility of Dap in water is less than 380µg/mL. Experimentally it was found to be 3.703952381µg/mL.

D. Melting point:

Melting point by capillary method and instrumental method observed was 170⁰C and 175⁰C respectively. (as per the literature 175ºC)

E. XRD:

Sharp peaks were observed from 5 to 30º of 2θ scale, which reveals the crystalline nature of drug.

F. IR:

IR spectra reveal characteristic functional groups same as reference standard.

G. DSC:

DSC studies show the peak value at 175⁰C corresponds to standard melting point (175.5ºC).

All the characteristic test of pure drug confirms the purity of Dap.

2. Preparation of Standard Curve:

Dap is soluble in dilute HCl 2mL, so this medium was used for preparation of standard curve. λ max, correlation coefficient R and calibration curve equation are as given below.

Using absorbance and concentration data Beer lamberts plot was prepared which is shown in figure 1 and table 3. Calibration curve equation has shown linear relationship and high degree of correlation in the range of 1-10 μg/mL at 288nm. This curve was utilized in Dap estimation as and when required

Figure 1: CALIBRATION CURVE FOR DAP

Table 3: CALIBRATION CURVE FOR DAP (n=3)

Concentration (µg/mL)	Average Absorbance
1	0.009±0.001
2	0.0416±0.001
3	0.0845±0.002
4	0.1331±0.001
5	0.178±0.005
6	0.2211±0.002
7	0.2612±0.003
8	0.2981±0.001
9	0.3422±0.002
10	0.3858±0.001

3. Determination of Drug :Polymer Ratio

Initially all the prepared films were transparent as in table 4. Appearance of these films even after storage at room temperature for 24hrs remained transparent except film of pure drug which on storage shows the recrystallization of drug. In all other ratios of drug: polymer (1:1 to 1:5). Dap remain in solubilised state throughout the storage period as in figure 2. So 1:1 ratio was selected for further study as Dap remains in solubilised state in this ratio.

Table 4: OPTIMISATION OF DRUG: POLYMER RATIO

	Sample	Solvent	Ratio	Solubility	Appearence	Appearance after 24 hrs
A	Drug	Ethanol	-	Clear solution	Clear, transparent	White clusters were seen
B	Polymer	Ethanol	-	Clear solution	Clear, transparent	Clear, transparent
C	Drug: Polymer(1:1)	Ethanol	1:1	Clear solution	Clear, transparent	Clear, transparent
D	Drug: Polymer(1:2)	Ethanol	1:2	Clear solution	Clear, transparent	Clear, transparent
E	Drug: Polymer(1:3)	Ethanol	1:3	Clear solution	Clear, transparent	Clear, transparent
F	Drug:Polymer(1:4)	Ethanol	1:4	Clear solution	Clear, transparent	Clear, transparent
G	Drug:Polymer(1:5)	Ethanol	1:5	Clear solution	Clear, transparent	Clear, transparent

Figure 2: Appearance of films after 24 hrs (at room temperature and in desiccators), a: pure drug, b: polymer, c: drug: polymer (1:1), d: drug: polymer (1:2), e: drug: polymer (1:3), f: drug: polymer (1:4), g: drug: polymer (1:5)

Figure 3: TEMPERATURE RANGE FOR EXTRUSION OF PURE POLYMERS

4. Effect of Temperature on Decomposition of Polymer

Extrudates of Kollidon VA64 look clear and glassy, with increasing temperature the colour turns yellowish and brownish. The actual discolouration of polymer was observed above 120⁰ C processing temperature. Above 200⁰C melting temperature extrusion becomes difficult.

5. Preparation of Non HME Formulation

Lubricated granules characterization and compression parameters are as given below in table 5.

6. Effect of type and concentration of plasticizer on solubility and dissolution:

Glass transition temperature (Tg) (figure 3) of Kollidon VA64 is 101⁰C, which may be reduced after addition of plastisizers. As a general rule, melt extrusion process should be run at temperature 20-40⁰C above the Tg. Temperature range for melt extursion of pure polymer is 155-200⁰C where as for polymer plastisizer combination it is 120-200⁰C

Table 5: EVALUATION OF LUBRICATED GRANULES AND TABLETS-ND (n=3)

Parameters for granules	Observations
Bulk Density(g/mL)	0.55±0.022
Tapped density(g/mL)	0.64±0.012
Hausner’s Ratio	1.16±0.01
Carr’s Index (%)	14.06±0.07
Angle of repose (⁰ )	25±0.034
Parameters for tablets	Observations
Machine	Lab Hosp
Punch	8mm,Tablet shape
Weight of tablet(mg)	175±0.011
Hardness(kg)	10±0.061
Disintegration Time(mins)	8±0.51
Friability (%)	0.3±0.801

In melt extrusion process drug can be either dissolved or dispersed in an amorphous or crystalline state. To obtain thermodynamically stable formulation drug must get completely dissolved below its saturation solubility in the polymer which is known as solid solution .When the main objective of melt extrusion technology is enhancement of solubility, the processing temperature should be equivalent or slightly higher than melting point of drug to get solid solution system. Melting point of Dap is approximately 175⁰C (by DSC method), so the 180⁰C temperature was selected for melt extrusion processing to get solid solution.

a. Size Reduction and Lubrication of Granules

As compared to NHME granules, dense granules were obtained by HME process. Increased bulk and tapped density values confirmed the presence of dense granules. Hausner’s ratio, carr’s index and angle of repose values reveals the good flow characteristics of granules (table 6). Disintegration time of tablets prepared by HME technology was three times higher than those prepared by NHME technology (table 6). Disintegration pattern was bursting and erosion in tablets prepared by NHME and HME technology respectively.

b. Characterization of Drug, HME Granules And NHME Dry Mix

Absence of sharp peak was observed in DSC of HME granules as compared to DSC of pure drug. This indicates the presence of amorphous drug in molten carrier, but in case of NHME the sharp peak was observed indicating the crystalline nature of drug. DSC thermograms of Dap in NHME and Dap HME granules represented in the figure 4I. The DSC thermograms of pure Dap shows sharp endotherm at 175⁰C attributed to the melting of Dap. This sharp melting endotherm indicates the crystalline nature of drug. The DSC thermogram of NHME shows melting at 159 and 181⁰C of the drug and polymer respectively. The DSC thermogram of HME PEG, HME Chremophor and HME Montane 20PHA shows absence of characteristic melting endotherm of Dap indicating the perfect miscibility of drug and polymer in the solid dispersion. As single Tg is characteristic of the thermoplastic system, the DSC thermogram of solid dispersion shows complete amorphization of drug.

Table 8: STABILITY STUDIES (n=3)

Formulation

Storage Condition

40⁰±2⁰C/75%±5%RH

Storage Period

Initial

Physical Appearance

Good

Moisture Content (%)

1.3±

0.12

1.3±

0.75

1.4±

0.34

1.4±

0.04

1.5±

0.02

2.0±

0.75

2.2±

0.05

2.8±

0.03

2.8±

0.31

2.9±

0.51

3.6±

0.024

3.7±

0.24

Drug Content (%)

99.2±

0.06

99.11±

0.01

98.97±

0.07

98.7±

0.017

99.00±

0.08

98.88±

0.28

98.12±

0.33

97.11±

0.85

98.2±

0.85

98.01±

0.50

97.99±

0.32

97.12±

0.64

Dissolution (%) at 150min

95.02±

0.22

94.11±

0.89

93.93±

0.36

93.2±

0.09

93.57±

0.21

93.10±

0.01

92.90±

0.85

91.81±

0.36

94.17±

0.28

93.88±

0.01

93.2±

0.85

92.01±

0.92

CONCLUSION:

Solubility of Dap can be increased by HME technology which is one of the method of solid dispersion. The XRD pattern shows amorphous nature of Dap in HME granules. The DSC thermogram of HME granules shows absence of characteristic melting endotherm of Dap indicating the perfect miscibility of drug and polymer in the HME granules. Dissolution rate of Dap HME tablets is not similar to that of NHME and marketed formulations, but the saturation solubility and in vitro permeability of Dap HME formulations is higher than NHME and marketed formulations. In all HME formulations HME with PEG4000 shows better enhancement in, in vitro permeability and saturation solubility. All optimized HME formulations shows good stability over the period of 3 month at 40⁰±2⁰C/75%±5%RH.

ACKNOWLEDGEMENT:

Authors are thankful to Emcure Pharma Ltd, BASF pharma ltd, Modern College of Pharmacy (For Ladies) Moshi and University of Pune for their support.

REFERENCES:

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Received on 31.10.2013 Accepted on 01.12.2013

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Pg 206-214

Parameters for granules	Observations
	D1	D2	D3
Bulk Density(g/mL)	0.52±0.15	0.512±0.11	0.501±0.033
Tapped density(g/mL)	0.67±0.04	0.64±0.001	0.63±0.019
Hausner’s Ratio	1.28±0.11	1.25±0.04	1.26±0.064
Carr’s Index (%)	22.38±0.021	20±0.061	20.48±0.27
Angle of repose(⁰)	27±0.01	26±0.023	28±0.022
Parameters for tablets	D1	D2	D3
Machine	Lab. Hosp.
Punch	9mm,Tablet shape
Weight of tablet(mg)	214mg	224mg	234 mg
Hardness(kg)	10 kg±0.06	10 kg±0.09	10 kg±0.01
Disintegration Time(min)	24mins±0.01	25mins±0.036	23mins±0.088
Friability(%)	0.3%±0.01	0.31%±0.08	0.36%±0.037