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Essay: Preformulation Studies of Drug (Chloroquine Phosphate): Physical Properties, Solubility Profile, and Spectroscopic Analysis

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The results of the various tests being carried out to determine the various aspects of the drug, the polymers and the final formulations along with an appropriate discussion as in the following sections.

6.1 PREFORMULATION STUDIES OF DRUG (CHLOROQUINE PHOSPHATE)

6.1.1 Physical properties of drug

The physical properties of drug is in compliance as reported (I.P. 2010) as the sample of Chloroquine Phosphate was found to be white colored, odorless & crystalline powder in appearance. This confirmed the identity and purity of drug.

6.1.2 Melting point

The melting point of drug is in compliance with reported value (I.P. 2010) as depicted in table 6.1. This confirmed the identity and purity of drug.

Table 6.1 Melting point of Chloroquine Phosphate

    Drug Standard Melting Point Observed Melting Point

Chloroquine Phosphate 218°C 220°C

6.1.3 Solubility profile of drug

The solubility trend (I.P. 2010) of Chloroquine Phosphate in different solvents has been shown below in table 6.2. Results of solubility studies indicate that Chloroquine Phosphate exhibited freely soluble in water, very slightly soluble in chloroform, in ethanol (95%), in methanol and in ether.

Table 6.2 Solubility profile of Chloroquine Phosphate in various solvents

Solvent Observation

Water Freely soluble

Chloroform Very Slightly soluble

Ethanol (95%) Very Slightly Soluble

Methanol Very Slightly soluble

Ether Very  Slightly  soluble

6.1.4 Partition coefficient

Partition coefficient is a measure of drug lipophilicity and an indication of its ability to cross biomembrane. For drug delivery, hydrophilic lipophilic balance (HLB) is an important factor.  Partition coefficient of Chloroquine Phosphate as determined in octanol: water (1:1) mixture showed lipophilic nature as shown in table 6.3.

Table 6.3 Partition coefficient of Chloroquine Phosphate

Parameter Standard Observed

Partition coefficient 3.73 4

6.1.5 Infra-Red (IR) spectroscopy

The structure and infra-red spectrum of Chloroquine Phosphate is shown in Figure 6.1 and 6.2 respectively. IR spectrum of the sample was compared with standard IR (Volume: 5 | Issue: 5 | May 2015 | ISSN – 2249-555X) to confirm the purity of drug. Frequency of observed bands and its interpretation is shown in Table 6.4. The IR spectrum of Chloroquine Phosphate was found to be in concordance with the reference spectrum of drug which indicates that the supplied drug was pure and free from impurities.

  .H3PO4

  Figure 6.1 Structure of Chloroquine Phosphate   

 

    Figure 6.2 IR Spectrum of Chloroquine Phosphate

Table 6.4 FTIR peaks of Chloroquine Phosphate

Standard peaks (cm-1) Observed peaks (cm-1) Interpretation

1553 1552.35 NH Absorption

1580-1520 1589.40 NH+C=N (N ring)

1614 1613.38 C=C+CH ( C ring )

2940-2945 2942.33 CH2

6.1.6 UV spectroscopy

Figure 6.3 shows standard (I.P. 2010) and observed λmax of Chloroquine Phosphate. UV spectrum of Chloroquine Phosphate is shown in Figure 6.3.

Table 6.5 Standard and observed λmax of Chloroquine Phosphate

  Drug  Standard λmax   Observed λmax

Chloroquine Phosphate   220nm   220.93nm

   

  Figure 6.3 UV spectrum of Chloroquine Phosphate

6.1.7 Standard curves of Chloroquine Phosphate

6.1.7.1 Standard curve of Chloroquine Phosphate in Simulated Gastric Fluid (SGF) pH 1.2 at λmax 220.93 nm

Table 6.6 represents standard curve of Chloroquine Phosphate in SGF 1.2 analyzed by UV spectrophotometer at 220.93 nm. Obtained result complied with the reported value, there by confirming identity and purity of procured drug and linear response was obtained in the range of 2-20 µg/ml with correlation coefficient r² = 0.992 in Figure 6.4. Results inferred that Beer’s law was obeyed in these concentration ranges at 220.93 nm.

Table 6.6 Concentration range and corresponding absorbance of Chloroquine Phosphate at 220.93 nm in Simulated Gastric Fluid (SGF) pH 1.2

Concentration (µg/ml) Absorbance at  λmax 220.93 nm

2 0.111

4 0.155

6 0.218

8 0.304

10 0.341

12 0.450

14 0.522

16 0.627

18 0.690

20 0.726

Figure 6.4 Standard curve of Chloroquine Phosphate in SGF pH 1.2

6.1.7.2 Standard curve of Chloroquine Phosphate in Simulated Intestinal Fluid (SIF) pH 6.8 at λmax 220.93 nm

Table 6.7 represents standard curve of Chloroquine Phosphate in SIF 6.8 analyzed by UV spectrophotometer at 220.93 nm. Obtained result complied with the reported value, there by confirming identity and purity of procured drug and linear response was obtained in the range of 2-12 µg/ml with correlation coefficient r² = 0.996 in Figure 6.5. Results inferred that Beer’s law was obeyed in these concentration ranges at 220.93 nm.

Table 6.7 Concentration range and corresponding absorbance of Chloroquine Phosphate at 220.93 nm Simulated Intestinal Fluid (SIF) pH 6.8

Concentration (µg/ml) Absorbance at  λmax 220.93 nm

2 0.122

4 0.218

6 0.308

8 0.403

10 0.472

12 0.586

14 0.656

16 0.792

18 0.891

20 0.929

Figure 6.5 Standard curve of Chloroquine Phosphate in SIF pH 6.8

6.1.7.3 Standard curve of Chloroquine Phosphate in Simulated Intestinal Fluid (SIF) pH 7.4 at λmax 220.93 nm

Table 6.8 represents standard curve of Chloroquine Phosphate in SIF 7.4 analyzed by UV spectrophotometer at 220.93 nm. Obtained result complied with the reported value, there by confirming identity and purity of procured drug and Linear response was obtained in the range of 5-30 µg/ml with correlation coefficient r² = 0.997 in Figure 6.6. Results inferred that Beer’s law was obeyed in these concentration ranges at 220.93 nm.

Table 6.8 Concentration range and corresponding absorbance of Chloroquine Phosphate at 220.93 nm in Simulated Intestinal Fluid (SIF) pH 7.4

Concentration (µg/ml) Absorbance at  λmax 220.93 nm

5 0.209

10 0.329

15 0.476

20 0.630

25 0.797

30 0.959

Figure 6.6 Standard curve of Chloroquine Phosphate in SIF pH 7.4

6.2 PREFORMULATION STUDIES OF POLYMERS – CHITOSAN AND EUDRAGIT S-100

6.2.1 Infrared Spectroscopy

Structure and IR spectrum of Chitosan shown in figure 6.7 and 6.8 respectively and Eudragit S100 in figure 6.9 and 6.10 respectively. Observed peaks in IR spectrum as depicted in table 6.10 and 6.11 respectively were found to be concordant with functional groups present in structure of polymer, which confirmed the purity of polymers.

   

 

   Figure 6.7 Structure of Chitosan

   

Figure 6.8 FTIR Spectrum of Chitosan

Table 6.9 FTIR peaks of Chitosan

Standard peaks (cm-1) Observed peaks (cm-1) Interpretation

1200 -1500 1426.11 CH2

1550 -1650 1593.18 NH amide

    

   

  Figure 6.9 Structure of Eudragit S100

    

  Figure 6.10 FTIR Spectrum of Eudragit S100

Table 6.10 FTIR peaks of Eudragit S100

Standard peaks (cm-1) Observed peaks (cm-1) Interpretation

1000-1300 1156.08 COOR

1385-1485 1483.77 CH2

1700-1735 1728.45 C = O

2500-3000 2952.31 OH Stretching

2900-3000 2996.71 ( C – H ) Streching

6.3 FORMULATION AND OPTIMIZATION OF MICROSPHERES

6.3.1 Formulation of CHITOSAN Microspheres

Microspheres were prepared by ionic gelation method (J.A. Ko et al., International Journal of Pharmaceutics 249 (2002) 165/174) using Spinot RQ122 Magnetic Stirrer. The method involves the drop wise addition of sodium tripolyphosphate solution in the chitosan solution with constant stirring for 2 hrs. The prepared microspheres were collected by centrifugation at 12000 rpm for 20 minutes.

6.3.2 Optimization of CHITOSAN Microspheres

Optimization of the formulation was done on the basis of particle size and size distribution and entrapment efficiency. Process parameters included polymer concentration and crosslinking agent concentration. Further uniformity of prepared Microspheres was also studied as depicted in table 6.11. CHITOSAN Microspheres with uniform size was successfully prepared at optimized concentration of 1% w/v CHITOSAN solution and TPP solution under constant stirring speed of 1000 RPM for 2 hrs.

Table 6.11 Optimization of CHITOSAN Microspheres with observation parameters

Code Chitosan

% TPP

% Drug

mg Particle size

(µm) PDI Entrapment Efficiency

(%) Uniformity

A1 0.5 0.5 10 0.044±0.004 0.097 69.80±2.03 Uncollectable

A2 0.5 0.75 10 0.091±0.001 0.258 71.37±1.52 Spherical

A3 0.5 1.0 10 0.822±0.021 0.282 72.27±2.05 Spherical

A4 0.5 1.25 10 0.325±0.028 0.184 70.32±2.06 Irregular

B1 1 0.5 10 0.980±0.019 0.546 81.17±1.26 Surface Ruptured

B2 1 0.75 10 1.477±0.016 0.489 82.87±1.80 Spherical

B3 1 1.0 10 1.878±0.031 0.384 86.12±1.04 Spherical

B4 1 1.25 10 1.730±0.013 0.652 83.77±1.66 Non Spherical

C1 1.5 0.5 10 1.680±0.019 0.546 65.67±1.53 Surface Ruptured

C2 1.5 0.75 10 2.443±0.022 0.416 69.67±1.53 Irregular

C3 1.5 1.0 10 3.539±0.031 0.589 69.67±3.06 Spherical

C4 1.5 1.25 10 2.929±0.022 0.311 63.83±1.76 Irregular

Table 6.11 reveals that B3 formulation was found to be the optimum formulation for the selected drug delivery system with mean particle size of about 1.878±0.031µm and highest entrapment efficiency of 86.12±1.04%. As concentration of chitosan increases (0.5%w/v – 1.5%w/v) caused the viscosity to increased and led to an increase in the particle size (A1, A2, A3, B1, B2, B3, C1, C2, C3) due to the availability of more cations to interact with cross linking agent. With increase in cross linking agent concentration (0.5%w/v – 1%w/v) particle size increase (A1, A2, A3, B1, B2, B3, C1, C2, C3) due to the interaction of negatively charged tpp with positively charged chitosan. Further increase in cross linking agent concentration (1.25%) results in decrease in particle size (A4, B4, C4) due to more ionic interaction between negatively charged tpp with positively charged chitosan (J.A. Ko et al 2002).The entrapment efficiency increases with the increase in cross linking agent concentration (A1, A2, A3), (B1, B2, B3), (C1, C2, C3) and decreased at higher cross linking agent concentrations (1.25%) may be due to more ionic interactions. The microspheres found to be more spherical with increase in polymer and cross linking agent concentration. Hence it is observed that particle size and entrapment efficiency of formulation is increasing with increase in polymer and cross linking agent concentration up to a certain concentration, beyond that both particle size and entrapment efficiency decreased abruptly due to increasing in ionic interactions. The drug chloroquine phosphate 10 mg along with stirring speed 1000 rpm and stirring time 2 hrs is kept constant in all the formulations.

On the selected optimised formulation (B3) further studies were carried out.

   Figure 6.11 Particle Size of prepared formulations of Chitosan Microsphere.

Figure 6.12 Percentage Entrapment Efficiency of prepared formulations of drug loaded CHITOSAN Microspheres

6.3.2.1 Particle Size

The prepared microspheres were seen under microscope for particle size determination figure 6.13 and figure 6.14 and were found to be in the range of 1.5-2.0 µm.

 

Figure 6.13 Motic image of drug loaded CHITOSAN Microspheres.

  Figure 6.14 Motic image of drug loaded CHITOSAN Microspheres.

6.3.2.1 Particle Size and Size Distribution

The prepared microspheres were observed under Zetasizer for particle size and size distribution determination figure 6.15 and were found to be in the range of 1.5-2.0 µm with PDI 0.384.

   

    Figure 6.15 Particle size analysis of drug loaded CHITOSAN Microspheres.

6.4 In-vitro EVALUATION OF DRUG LOADED MICROSPHERES

In-vitro characterization included parameters like scanning electron microscopy, infrared spectroscopy, entrapment efficiency, in-vitro drug release.

6.4.1 Scanning Electron Microscopy

Diameter and shape of the Microspheres were determined using SEM. The diameter of microspheres was found to be in the range of 1.5-2.0 µm. The loaded Microspheres showed larger diameter which could be due to the incorporation of drug in the polymeric microspheres.

 

   Figure 6.16 SEM images of drug loaded CHITOSAN Microspheres.

6.4.2 Infrared spectroscopy

Infrared spectroscopy was done for analyzing the compatibility between polymer-drug. The FTIR spectrum’s of CHITOSAN Microspheres with drug are depicted in figure 6.17. The peaks found to be concordant with functional groups present in structure of respective polymers and drug and revealed that there is no interaction between polymers and drug.

    Figure 6.17 FTIR spectrum of CHITOSAN MICROSPHERE with drug (Chloroquine Phosphate)

6.4.3 Entrapment Efficiency

Entrapment efficiency describes the efficiency of preparation method to incorporate drug into the carrier system. Theoretically entrapment efficiency increases with increase in polymer due to availability of higher surface area (Protero et al., 2002). Entrapment efficiency depicted in table 6.13.

Table 6.12 Entrapment efficiency of Microspheres (mean±SD, n=3)

Formulation % Entrapment efficiency

CHITOSAN Microspheres 86.12±1.04

6.4.4 In-vitro Drug Release

The data obtained from the in-vitro release study of CHITOSAN Microspheres is presented below in the table 6.14 and figure 6.18. The table depicts the percent cumulative drug release from Microspheres with time.

Table 6.13 Percent cumulative release from CHITOSAN Microspheres (mean±SD, n=3)

Time (minutes) % Cumulative release from CHITOSAN Microspheres

0 0

15 19.71±0.26

30 24.54±0.36

60 38.41±0.41

90 52.40±0.33

120 59.69±0.16

150 63.35±0.37

180 67.66±0.39

240 70.73±0.22

300 72.36±0.34

360 72.66±0.39

420 73.47±0.62

    Figure 6.18 In-vitro releases of CHITOSAN Microspheres

In-vitro release studies of chitosan microspheres showed the almost 60% of the drug is released in 120 minutes in SGF pH1.2 followed by total of 73 % release in total of 420 minutes in SIF 6.8 and further no release was observed. The release behavior suggesting the burst release effect in uncoated microspheres at pH 1.2 due to polymer degradation in the acidic pH which indicate that drug from uncoated microspheres is released before it reached the colon. Although the graph figure 6.18 indicates the burst release in first 30 minutes due to the fact that certain amount of drug was present on the surface of the Microspheres but further the release is sustained as drug released only after polymer degraded. Hence the release can control by giving coating with pH sensitive polymer to the formulation preventing the release in acidic pH thus release at the target site. The release could hence be summarized as primarily diffusion based for the initial period of 15-30 minutes beyond which it was based on the erosion of the polymer where maximum amount of drug got released.

6.5 COATING OF DRUG LOADED CHITOSAN MICROSPHERES

Core Microspheres was coated by Oil-in-Oil solvent evaporation method (paharia et al., 2007). The method involves dispersing core microspheres in coating solution prepared by dissolution of Eudragit S100 in ethanol and acetone (2:1). The organic phase is poured in light liquid paraffin containing 1%w/v span 80 the system is made under 1000 rpm at room temperature for 3 hours to allow evaporation of solvent. Coated microspheres were centrifuged, washed with n-hexane and freeze dried overnight.

6.5.1 Formulation and optimization of EUDRAGIT S100 COATED CHITOSAN Microspheres

Optimization of the formulation was done on the basis of particle size and size distribution figure 6.19 and in vitro release table 6.16. Process parameters included core: coat ratio table 6.14. Continuous COATED CHITOSAN Microspheres with uniform size 3.0-3.5 µm and with desired release behavior was successfully prepared using B3 Formulation at optimized coat core ratio 1:5 (E3).

Table 6.14 Optimization of EUDRAGIT COATED CHITOSAN Microspheres with observation parameters

Code Chitosan Microsphere Core

(mg) Eudragit S100 Coat

(mg) Core:Coat Ratio Particle size (µm)

E1 50 150 1:3 2.141±0.028

E2 50 200 1:4 2.588±0.060

E3 50 250 1:5 3.043±0.056

Table 6.14 and table 6.16 reveals formulation E3 was found to be the optimum formulation suggesting core coat ratio 1:5 for the selected drug delivery system with mean particle size of about 3.043±0.056µm and desired release profile .As core coat ratio increases (1:3-1:5) particle size increases (E1, E2, E3) due to increase in coat amount. Also giving the more desirable release behaviour as coat amount is increasing (1:5>1:4>1:3) due to slow or almost negligible degradation at gastric pH and degradation at selective ph. The core microspheres 50 mg is kept constant in all the formulations. On the selected optimised formulation (E3) further studies were carried out.

    Figure 6.19 Particle Size of prepared formulations of Coated Microsphere

6.5.1.1 Particle Size

The coated microspheres were seen under microscope for particle size determination figure 6.20 and figure 6.21 and were found to be in the range of 2.5-3.0 µm.

    

  Figure 6.20 Motic images of Coated Microspheres.  

 

   Figure 6.21 Motic images of Coated Microspheres.

6.6 In-vitro EVALUATION OF COATED MICROSPHERES

In-vitro characterization included parameters like scanning electron microscopy, infrared spectroscopy, entrapment efficiency, in-vitro drug release.

6.6.1 Scanning Electron Microscopy

Diameter and shape of the Microspheres were determined using SEM. The diameter of microspheres was found to be in the range of 2.5-3.0 µm.

   

   Figure 6.22 SEM images of EUDRAGIT COATED CHITOSAN Microspheres.

6.6.2 Infrared spectroscopy

Infrared spectroscopy was done for analyzing the compatibility between polymer-polymer-drug. The FTIR spectrum’s of EUDRAGIT COATED CHITOSAN Microspheres with drug are depicted in figure 6.23. The peaks found to be concordant with functional groups present in structure of respective polymers and drug and revealed that there is no interaction between polymers and drug.

    

Figure 6.23 FTIR spectrum of EUDAGIT COATED CHITOSAN MICROSPHERE with drug (Chloroquine Phosphate)

6.6.3 Entrapment Efficiency

Entrapment efficiency describes the efficiency of preparation method to incorporate drug into the carrier system. Theoretically entrapment efficiency increases with increase in polymer due to availability of higher surface area (Protero et al., 2002). Entrapment efficiency depicted in table 6.15.

Table 6.15 Entrapment efficiency of Coated Microspheres (mean±SD, n=3)

Formulation % Entrapment efficiency

Coated Microspheres 86.12±1.04

6.6.4 In-vitro Drug Release

The data obtained from the in-vitro release study of COATED Microspheres is presented below in the table 6.16 and figure 6.24. The maximum release observed is around 70 % due to the absence of rat cecal content. As after degradation of pH sensitive polymer at pH7.4 chitosan is being eroded by colonic bacteria as well as by polymer degradation. In absence of cecal content chitosan is only eroded by polymer degradation resulting in lesser release. The table depicts the percent cumulative drug release from Microspheres with time.

Table 6.16 Percent cumulative release from Eudragit Coated CHITOSAN Microspheres (mean±SD, n=3)

Time (hours) E1 E2 E3

0 0 0 0

0.5 8.78±0.03 2.34±0.03 0

1 12.67±0.02 4.56±0.06 1.23±0.05

2 18.89±0.02 8.89±0.02 4.45±0.07

4 33.33±0.03 15.56±0.05 7.56±0.02

6 50.67±0.06 23.34±0.03 14.34±0.01

8 58.89±0.12 33.45±0.02 21.23±0.09

10 67.78±0.45 46.88±0.02 33.34±0.05

12 69.98±0.05 56.67±0.05 45.56±0.04

16 – 69.99±0.03 58.89±0.06

20 – – 68.89±0.02

24 – – 71.56±0.03

   Figure 6.24 In-vitro releases of Eudragit Coated Chitosan Microspheres

In-vitro release studies of coated microspheres showed only around 20% of the drug is released in 2 hrs. (E1) in SGF pH1.2 as compared to 60% drug release in uncoated microspheres (B3) in 120 minutes indicating that release is controlled due to pH sensitive coating followed by maximum of 69 % release in 12 hours in SIF 7.4  and further no release was observed. With the increase in coat amount the release is further controlled/decreased in 2 hours as only around 10% release (E2) and around 5% release (E3) in SGF pH1.2 due to lesser polymer erosion at gastric pH. Also the time at which maximum release is observed around 70% is also increased (12 hours (E1), 16 hours (E2) and 24 hours (E3)) as core coat ratio is increasing 1:5>1:4>1:3 due to lesser pH erosion at gastric pH and retaining more drug for release at desired pH and target site due to pH sensitive properties of polymer.

The graph figure 6.19 indicates the burst release due to the fact that certain amount of drug was present on the surface of the Microspheres but further the release is sustained as drug released only after polymer degraded. Hence the release is controlled by giving coating with pH sensitive polymer to the formulation preventing the release in acidic pH thus release at the target site. The release could hence be summarized as primarily diffusion based for the initial period of 0.5 hours beyond which it was based on the erosion of the polymer where maximum amount of drug got released.

6.6.4 In-vitro release models

The kinetics of Chloroquine phosphate release from the prepared microspheres was studied by fitting the release data to Zero-order Kinetics figure 6.25 and Korsmeyer Peppas Model figure 6.26 based upon the highest regression (R) values Table 6.16. Further Korsmeyer and Peppas equation resulted into the values of n>1, which appears to indicate that the release from the prepared microspheres was by Super case-ӀӀ transport.

  Figure 6.25 Zero Order Kinetics of Eudragit Coated Chitosan Microspheres.

 

    Figure 6.26 Peppas Model for Eudragit Coated Chitosan Microspheres

Table 6.17 Regression Values for Pharmacokinetic Models

Models Zero Order First Order Higuchi Pappass n value

Regression Value 0.9721 0.6565 0.9396 0.9838 1.22

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