Results And Discussion Of Pharmokinetis

RESULTS AND DISCUSSION 3.1

Preparation and characterization of CAS

The three methods described in the experimental section produced similar product but the product from method 3 was much free of impurities, mainly CS. Thus, the drug was prepared in bulk by using this method. The characterization was carried out by analytical, physical and spectral data. The analytical results are given in Table 3.1. A sample of copper (II) acetylsalicylate did not melt but turned brown on heating to 300ºC as reported in literature [77]. The IR spectrum (Figure. 3.1) resembled that already reported in the literature [76].

Table 3.1. Analytical results of CAS. Elements % Calculated

% Found

C

51.25

51.75

H

3.35

3.34

O

30.34

30.39*

Cu

15.06

14.95

Analyzed

*

determined by difference

Transmittance (%) Figure 3.1. FTIR spectrum of CAS. Wave number (cm-1)

3.2

Pharmaceutical properties

Crystallanity The powder X-ray diffractograms of CAS and ASA are shown in Figure 3.2. It appears that crystalline character decreases on complexation of ASA with copper. Thus, the CAS is expected to possess enhanced bioavailability and improved tabletting properties [122 - 125].

Particle size analysis The results of particle size analysis are presented in Table 3.2. It appears that the particle size decreases on complexation with the metal ion. This indicates that the complex will have enhanced compactability and bioavailability. Particle size analysis indicates the free flowing nature of CAS, as more than 50% of the material was retained up to 150 μm. Moisture The moisture contents of ASA and CAS are given in Table 3.3. CAS was anhydrous. Density measurements The bulk, tap, and true densities for the powders are listed in Table 3.3. ASA has higher bulk and tap densities than those of CAS. The data indicate a reduced die-filling character of CAS. Porosity CAS had the highest porosity followed by ASA (Table 3.3). This is a positive contribution of

complexation whereby the new materials will have better compressibility.

Flow The values for these parameters are listed in Table 3.3. These are consistent with the flow rates determined by flow meter. The values of angle of repose and flow rate in case of CAS indicate an improvement in flowability with respect to ASA. Percent compressibility and the Hausner ratio methods were used for the compressibility index. The results of the test are given in Table 3.3. Taste Twenty healthy volunteers completed the taste study in a blind fashion. The taste and aftertaste were evaluated. The results indicate no bitter taste and, therefore, it could be used orally.

4000 CAS 3000 2000

Intensity

1000 0 20

40

16000 14000 14000 12000 12000

60

80

100

ASA

10000 8000 6000 4000 2000 0 0

20

40

60 2 Theta

Figure 3.2. PXRD spectra of CAS and ASA

80

100

Table 3.2. Particle size analysis of CAS by sieve analyzer. Mesh

Retention

Mesh

Retention

(μm) 20 40 60 80 100 150

(%) 0.37 1.0 1.13 1.35 1.58 50.5

(μm) 180 μ 250 μ 280 μ 325 μ 400μ

(%) 39.2 36.5 35.8 35.04 1.92

Table 3.3. Powder properties. Parameter

ASA

CAS

Moisture (% w/w)

0.05 ± 0.01

0.00

ρtrue (g mL-1)

1.97 ± 0.05

1.82 ± 0.03

ρbulk (g mL-1)

0.43 ± 0.01

0.51 ± 0.01

ρtap (g mL-1)

0.52 ± 0.01

0.65 ± 0.01

Bukliness (mL g-1)

1.99 ± 0.02

2.30 ± 0.01

ɛº (%)

69.5 ± 0.08

75.35 ± 0.06

HR

1.40 ± 0.01

1.45 ± 0.01

CI (%)

17.31 ± 0.03

21.5 ± 0.05

α(10 g, i.d. 10 mm, 20 rpm)

16˚

15˚

Flow rate (g s-1)

0.50

0.44

3.3

Evaluation of tablets

The results of hardness, friability, disintegration and dissolution are given in Table 3.4. Hardness, friability and disintegration results were found to be according to required standard specifications. Similarly, the dissolution of all batches is greater than 80% up to 30 min according to USP specifications [111]. Dissolution behaviour of these batches is also shown in Figure 3.3.

Table 3.4. Evaluation of tablets.

Product

Hardness (Kg)

Friability (%)

Disintegration time (min)

Released after 30 min (%)

ASA(WG)

9.0 ± 1.0

0.22

1.6

80.0

ASA(DC)

6.5 ± 1.5

0.35

4.0

84.0

CAS(WG)

9.4 ±1.0

0.18

1.5

83.5

CAS(DC)

6.7 ± 1.5

0.25

2.6

85.0

100 90 80

Release (%)

70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

70

80

90

100

Time (min)

Figure 3.3. Dissolution behaviour of ASA (WG) □; CAS (WG) ×; ASA (DC) ♦; CAS (WG)▲ tablets. DC = direct compression; WG = wet granulation.

110

3.4

Development and validation of assay method(s)

3.4.1

HPLC method

Three different mobile phases were used for the development of assay method on HPLC. In the first method, mobile phase A (water, acetonitrile and 0.1 M phosphate buffer pH 2.5 in 35: 25: 40 ratio) was used along with chromatographic conditions as given in experimental section. The resultant chromatogram is shown in Figure 3.4. By using this method, only two peaks representing ASA and SA were identified by spiking technique. These results indicate that CAS hydrolyzes in this mobile phase and is converted to ASA and SA. As this method did not show any peak for CAS, so it could not be used for the assay of CAS. In the second method, methanol-ethanol (50:50) was used as mobile phase for the assay of CAS according to the chromatographic conditions as given in the experimental section. The chromatogram (Figure 3.5) shows again the resolution of only ASA and SA. So, this method also could not be used for the assay of CAS. In the third method, mobile phase C i.e. methanol-acetic acid (20:1) was used. By using this method, four peaks representing CAS, ASA, SA and CS were resolved. The typical chromatogram is shown in Figure 3.6. The system optimization was carried out by using various compositions of this mobile phase as given in Table 3.5. It was found that the separation deteriorated on increasing or decreasing methanol-acetic acid ratio beyond 20:1. A test run was obtained by using the mixture of CAS, ASA, CS, and SA standards and all four components were separated. The typical chromatogram is shown in Figure 3.6. The retention time for CAS, CS, ASA, and SA

was 2.6, 2.8, 3.0 and 3.2, respectively. In this study, the resolution was 1.0 for CS and greater than 1 for CAS and ASA. The peaks in the mixture were verified by spiking with the standards. The performance parameters thus obtained are given in Table 3.6.

(mV × 100)

1

10

5 2

0 6

8

10

Time (min) Figure 3.4. Chromatogram of CAS showing separation of ASA (1) and SA (2) in the mobile phase A. (mV × 100)

10 1 2

5

0 0 1 2 3 4 5 Time (min) Figure 3.5. Chromatogram of CAS showing separation of ASA (1) and SA (2) in the mobile phase B.

(mV × 100)

3 2

1

4

2 1

1

0

00

11

22 3 3 Time(min) (min) Time

4

4

5

5

Figure 3.6. Chromatogram showing separation of CAS (1), CS (2), ASA (3) and SA (4) in the mobile phase C.

Table 3.5. Effect of mobile phase composition on performance parameters. Mobile phase Methanol-Acetic acid (20 : 1.0)

Methanol-Acetic acid (20 : 0.5)

Methanol-Acetic acid (20 : 1.5)

Parameter

CAS

CS

ASA

SA

N

2260

4659

5153

10725

Rs

1.97

0.89

1.69

̶

As

1.00

0.90

1.10

1.00

K

4.20

4.80

5.10

5.50

N

2205

4552

5150

10656

Rs

0.82

0.45

0.75

̶

As

1.80

1.00

1.50

1.30

K

8.20

9.80

10.10

10.50

N

2105

4052

4150

10206

Rs

0.65

0.45

0.75

̶

As

2.00

1.20

1.40

1.50

K

6.20

8.80

9.00

11.50

Table 3.6. Performance parameters.

Parameter

CAS

CS

ASA

SA

N

2260

4659

5153

1072

Rs*

1.97

1.01

1.69

̶

As

1.00

0.90

1.10

1.00

K

4.20

4.80

5.10

5.50

* between the adjacent peaks

3.4.2

Method validation

Accuracy and precision The accuracy ranged from 80.7 to 99.65 % in terms of recovery. The precisions were determined within the day and between the days. The analyses were performed at three different concentration levels, covering the entire linear range. The CV ranged from 0.03 to 0.08 and 0.05 to 0.11 for within the day and between the days, respectively. The results are given in Table 3.7. Linearity Concentration and peak area were found to be linearly related for CAS, CS, ASA and SA in concentration ranges under study. The linearity data are given in Table 3.8. The calibration curves in mobile phase and plasma are shown in Figures 3.7 and 3.8, respectively. Limit of detection (LOD) and limit of quantification (LOQ) The values of LOD and LOQ are given in Table 3.7. The very low levels indicate that the method is very sensitive for the determination of the substances under investigation in presence of each other. Specificity and reproducibility The method was found to be specific for the determination of a particular analyte in dosage forms, as the reproducibility of measurement (CV 0.0075 for CSA, 0.0025 for CS, 0.012 for ASA, 0.005 for SA) of five different samples spiked with standard was very high (Table 3.9)

Table 3.7. Validation parameters of analytes in the mobile phase and plasma at three different concentration levels. Matrix

Parameter

In mobile phase

Precision (CV, within day/between days)

CAS (Mean) i. 0.03/0.7 at 0.004 µg mL-1

CS (Mean) i. 0.04/0.07 at 0.09 µg mL-1

ASA (Mean) i. 0.03/0.05 at 0.03 µg mL-1

SA (Mean) i. 0.08/0.1 at 0.02 µg mL-1

ii. 0.04/0.06 at 100 µg mL-1

ii. 0.03/0.09 at 75 µg mL-1

ii. 0.04/0.07 at 100 µg mL-1

ii. 0.06/0.11 at 125 µg mL-1

iii. 0.04/0.07 iii. 0.05/0.08 iii. 0.03/0.06 iii. 0.07/0.09 at 200 µg at 150 µg at 200 µg at 250 µg -1 -1 -1 mL mL mL mL-1 Accuracy (% recovery)

i. 99.07 at 0.004 µg mL-1

i. 80.70 at 0.09 µg mL-1

i. 99.65 at 0.03 µg mL-1

i. 95.83 at 0.02 µg mL-1

ii. 99.1 at 100 µg mL-1

ii. 80.45 at 75 µg mL-1

ii. 99.55 at 100 µg mL-1

ii. 95.90 at 125 µg mL-1

iii. 99.12 at 200 µg mL-1

iii. 80.55 at 150µg mL-1

iii. 99.5 at 200 µg mL-1

iii. 95.78 at 250 µg mL-1

LOD (ng mL-1)

2.0

LLOQ (ng mL1 )

4.0 0.004-200

45.0 90.0 0.09-150

15.0

10.0

30.0

20.0

0.03-200

0.02-250

Concentration range (µg mL-1) Continued

Table 3.7 (Continued) In plasma Precision (CV, within day/between days)

Accuracy (% recovery)

i. 0.05/0.1 at 0.012 µg mL-1

i. 0.08/0.14 at 0.13 µg mL-1

i. 0.06/0.1 at 0.06 µg mL-1

i. 0.12/0.18 at 0.04 µg mL-1

ii. 0.07/0.1 at 100 µg mL-1

ii. 0.1/0.15 at 75 µg mL-1

ii. 0.05/0.1 at 100 µg mL-1

ii. 0.11/0.16 at 125 µg mL-1

iii. 0.05/0.11 at 200 µg mL-1

iii. 0.09/0.13 at 150 µg mL-1

iii. 0.04/0.09 at 200 µg mL-1

iii. 0.13/0.17 at 250 µg mL-1

i. 95.0 at 0.012 µg mL-1

i. 80.20 at 0.13 µg mL-1

i. 96.50 at 0.006 µg mL-1

i. 92.83 at 0.04 µg mL-1

ii. 95.1 at 100 µg mL-1

ii. 80.15 at 75 µg mL-1

ii. 96.45 at 100 µg mL-1

ii. 92.85 at 125 µg mL-1

iii. 95.2 at 200 µg mL-1

iii. 80.0 at 150 µg mL-1

iii. 96.56 at 200 µg mL-1

iii. 92.90 at 250 µg mL-1

LOD (ng mL-1)

6.0

65.0

30.0

20.0

LLOQ (ng mL1 )

12.0

130.0

60.0

40.0

0.012-200 Concentration range (µg mL-1)

0.13-150

0.06-200

0.04-250

Table 3.8. Linearity parameters. Parameter

CAS

CS

ASA

SA

R2

0.991

0.998

0.9914

0.994

Slope

134.2

116.4

53.6

40.3

Concentration range (µg mL-1)

0.004-200

0.004-200

0.03-200

0.02-250

Table 3.9. Specificity data, in terms of % recovery, at 100 µgmL-1 of each analyte in six plasmas. Plasma

CAS

CS

ASA

SA

1

95.1

80.15

96.55

92.8

2

95.05

80.2

96.5

92.83

3

95.07

80.16

96.45

92.72

4

95.12

80.19

96.54

92.84

5

95.08

80.15

96.6

92.81

6

95.09

80.18

96.5

92.8

CV (%)

0.0075

0.0025

73

0.012

0.005

600

500

Peak Area

400

300

200

100

0 0

50

100

150

200

250

300

-1

Concentration μg mL

Figure 3.7. Calibration curves of CAS (∆), CS (♦), ASA (□) and SA (×) in mobile phase.

74

600

500

Peak area

400

300

200

100

0 0

1

2

3

4

5

-1

Concentration μg mL

Figure 3.8. Calibration curves of CAS (∆), CS (♦), ASA (□) and SA (×) in plasma.

75

6

3.5

Stability study of pharmaceutical preparations

3.5.1

Stability of CAS in the mobile phase C and in plasma

The stability study of drug in mobile phase as well as in plasma was carried out and it was found that the chromatogram did not change significantly over the period of 6 h i.e. there was no significant change in the peak position as well as the peak areas in mobile phase, and was found to be stable for 72 h in plasma. Thus, the methanol-acetic acid system was found to be suitable for this study. 3.5.2

Stability of CAS in tablets

It appears that the CAS, when is in contact with moisture, disproportionates into CS, ASA and SA according to Scheme 5. The results of the accelerated stability study of the tablets are shown in Figures 3.9 – 3.12.

76

O

O

Cu2 + H2O OCOCH 3

4

CAS

O COOH O

O

CS

COOH

+ (CH3COO)2Cu + CH3COOH

+

Cu +

OCOCH 3

2 ASA

OH

SA

Scheme 5

77

50.4 50.3 50.2

Assay (% w/v))

50.1 50 49.9 49.8 49.7 49.6 49.5 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.9. Stability curves showing the concentration of the active ingredient in tablets vs number of days. CAS (WG) at 40 °C & 75% RH (□); CAS (WG) at 50 °C & 75% RH (×); CAS (DC) at 40 °C & 75% RH(♦); CAS (DC) at 50 °C & 75% RH (∆); DC = direct compression; WG =wet granulation.

78

4 3.5

Assay (% w/v))

3 2.5 2 1.5 1 0.5 0 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.10. Stability curves showing the concentration of the free SA in tablets vs number of days. SA in CAS (WG) tablets at 40 °C & 75% RH (□); SA in CAS (DC) tablets at 50 °C & 75% RH (×); SA in CAS (DC) tablets at 40 °C & 75% RH (♦); SA in CAS (WG) tablets at 50 °C & 75% RH (∆).

79

4.5 4 3.5

Assay (% w/v))

3 2.5 2 1.5 1 0.5 0 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.11. Stability curves showing the concentration of the free CS in tablets vs number of days. CS in CAS (WG) tablets at 50 °C & 75% RH (□); CS in CAS (DC) tablets at 50 °C & 75% RH (♦); CS in CAS (WG) tablets at 40 °C & 75% RH (×); CS in CAS (DC) tablets at 50 °C & 75% RH (∆).

80

48

47.8

Assay (%w/v))

47.6

47.4

47.2

47

46.8 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.12. Stability curves showing the concentration of the free ASA in tablets vs number of days. ASA in CAS (WG) tablets at 40 °C & 75% RH (□); ASA in CAS (DC) tablets at 40 °C & 75% RH (♦); ASA in CAS (WG) tablets at 50 °C & 75% RH (×); ASA in CAS (DC) tablets at 50 °C & 75% RH (∆).

81

The stability profiles of the tablets are discussed as follows: Wet granulation The stability was monitored by determining the concentration of the active ingredient and the decomposition products in the tablets in PVC securitainers placed at 40ºC and 50ºC with 75% RH at various intervals of time. At 40ºC, the concentration of CAS (the active ingredient) dropped slowly according to the equation y = - 0.0051x + 50.232 with r2 = 0.8709. Accordingly, the concentration of SA (the decomposition product of CAS) increased by following the equation y = 0.00102x + 1.6712 with r2 = 0.9003.

At 50ºC, the

concentration of CAS dropped slowly according to the equation y = - 0.0043x + 50.134 with r2 = 0.8521. Accordingly, the concentration of SA (the decomposition product of CAS) increased by the following trends according to the equation y = 0.0115x+2.088 with r2 = 0.7536. The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC with 75% RH was determined and it was found that the concentration of CS increased by following the equations y = 0.0222x + 0.7191 (r2=0.9604) and y = 0.0259x + 0.692 (r2 = 0.9538), respectively. There is no significant difference (F < F critical) between the two values. This indicates that the stability profile of CAS formulated by wet granulation is similar at 40ºC and 50ºC with 75% RH. The concentration of ASA (decomposition product of CAS) at 40ºC and 50ºC with 75% RH dropped slowly according to the equations y = - 0.0029x + 47.68 with r2 = 0.9254 and y = 0.0031x + 47.671 with r2 =0.8987, respectively. This trend is obvious because the ASA further decomposes into SA, which has increasing trend as shown in Figure.3.12.

82

Direct compression The stability was monitored by determining the concentration of the active ingredient and the decomposition products in the tablets placed at 40ºC, and 50ºC with 75% RH, at various intervals of time. At 40ºC, the concentration of CAS (the active ingredient) dropped slowly according to the equation y = -0.0043x + 50.259 with r2 = 0.6877. Accordingly, the concentration of SA (the decomposition product of CAS) increased by following the equations y = 0.005x + 1.8219 with r2 = 0.8304. At 50ºC, the concentration of CAS (the active ingredient) dropped slowly according to the equation y = -0.0053x + 50.28 with r2 = 0.8738. Accordingly, the concentration of SA (the decomposition product of CAS) increased by following the equation y = 0.0058x + 1.5933 with r2 = 0.8383. The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC with 75% RH was determined and it was found that the concentration of CS increased by following the equations y = 0.0153x + 0.7996 (r2 = 0.9006) and y = 0.0249x + 0.6842 (r2 = 0.9215), respectively. There is no significant difference (F < F critical) between the two values. This shows that the stability profile of CAS formulated by direct compression is similar at 40ºC and 50ºC with 75% RH. The concentration of ASA (decomposition product of CAS) at 40ºC and 50ºC with 75% RH dropped slowly according to the equations y = - 0.0028x + 47.665 with r 2 = 0.8782 and y = 0.0035x + 47.69 with r2 = 0.9065, respectively. This behaviour of ASA contents has been explained before.

83

3.5.3

Comparison of stability data of CAS and ASA tablets

Wet granulation The stability was monitored by determining the concentration of the active ingredient and the decomposition products in the tablets placed at 40ºC and 50ºC with 75% RH, at various intervals of time. At 40ºC, the concentration of CAS (the active ingredient) dropped slowly according to the equation y = 23.562e-0.0012x with r2 = 0.8222, whereas the decomposition occurred at much higher rate (F > F critical) in case of tablets of ASA and followed the trend as per equation y = 99.998x-0.0754 with r2 = 0.9214. Accordingly, the concentration of SA (the common decomposition product of CAS and ASA tablets) increased by following the equations y = 0.0085x+1.5454 (r2 = 0.9064) and y = 0.146x-0.2375 (r2 = 0.9271) for CAS and ASA tablets, respectively. These results clearly show an enhanced stability of CAS formulation as compared to that of ASA. At 50ºC, the concentration of CAS (the active ingredient) dropped slowly according to the equation y = 0.0188x+23.947 with r2 = 0.7864, whereas the decomposition occurred at much higher rate (F > F critical) in case of tablets of ASA according to the equation y = -0.183x+93.089 with r2 = 0.9404. Accordingly, the concentration of SA (the common decomposition product of CAS and ASA tablets) increased by following the trends according to equations y = 0.0085x+1.5454 (r2=0.9064) and y = 0.146x-0.2375 (r2 = 0.9271). These results also show an enhanced stability of CAS formulation as compared to that of ASA.

84

The concentration of CS (another decomposition product of CAS) at 40ºC and 50ºC with 75% RH was determined and it was found that the concentration of CS increased by following the equation y = 0.0222x +0.7191 (r2=0.9604) and y = 0.0259x +0.692 (r2 = 0.9538), respectively. There is no significant difference (F < F critical) between the two values. This indicates that the stability profile of CAS formulated by wet granulation is similar at 40ºC and 50ºC with 75% RH. Direct compression The stability was monitored by determining the concentration of the active ingredient and the decomposition products in the tablets placed at 40ºC and 50ºC (with 75% RH) and at various intervals of time. At 40ºC, the concentration of CAS and ASA (the active ingredient) dropped slowly according to the equations y = 25.041e-0.0005x with r2 = 0.73, and y = 98.867x0.076

with r2 = 0.7265 (F < F critical), respectively. Accordingly, the concentration of SA (the

common decomposition product of CAS and ASA tablets) increased by following the equations y= 0.0195x+1.3391 (r2 = 0.745) and y = 0.3714Ln(x) + 0.2708 (r2 = 0.8579) for CAS and ASA tablets, respectively. These results clearly indicate that there is no significant difference in the stability of CAS formulation as compared to that of ASA. At 50ºC, the concentration of CAS and ASA (the active ingredient) dropped slowly according to the equations y = -0.0128x+25.092 with r2 = 0.6816 and y = -0.0505x+96.584 with r2 = 0.9399 (F < F critical), respectively. Accordingly, the concentration of SA (the common decomposition product of CAS and ASA tablets) increased by following the trends according to equations y = 0.0124x+2.0862(r2 = 0.7874) and y = 0.0493x-0.426 (r2 = 0.9775). These results also indicate no significant difference in the stability of CAS formulation as compared to that of ASA.

85

The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC (with 75% RH) was determined and the concentration of CS increased by following the equation, y= 0.0153x+0.7996 (r2 = 0.9006) and y = 0.0249x +0.6842 (r2 = 0.9215), respectively. There is no significant difference (F < F critical) found between the two values. This indicates that the stability profile of CAS formulated by direct compression is similar at 40ºC and 50ºC with 75% RH. The comparisons of the stability study data of CAS and ASA tablets are shown in Figures 3.13 – 3.16.

86

100 95 90 85

Assay(%w/v))

80 75 70 65 60 55 50 45 40 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.13. Stability curves showing the concentration of the active ingredient in tablets vs number of days. ASA (DC) at 40 °C & 75% RH (*); ASA (DC) at 50 °C & 75% RH (●); ASA (WG) at 40 °C & 75% RH (-); ASA (WG) at 50 °C &75% RH (×); CAS (DC) at 40 °C & 75% RH(♦); CAS (DC) at 50 °C & 75% RH(▲); CAS (WG) at 40 °C & 75% RH (■); CAS (WG) at 50 °C & 75% H(+); DC = direct compression; WG = wet ganulation.

87

18 16 14

Assay (%w/v))

12 10 8 6 4 2 0 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.14. Stability curves showing the concentration of the free SA in tablets vs number of days. SA in ASA (DC) tablets at 40 °C & 75 % RH (■); SA in ASA (DC) tablets at 50 °C & 75% RH (●); SA in ASA (WG) tablets at 40 °C & 75% RH(-); SA in ASA (WG) tablets at 50 °C & 75% RH (×); SA in CAS (DC) tablets at 40 °C & 75% RH (♦); SA in CAS (DC) tablets at 50 °C & 75% RH (*); SA in CAS (WG) tablets at 40 °C & 75% RH (▲); SA in CAS (WG) tablets at 50 °C & 75% RH (+).

88

100

90

Assay(%w/v))

80

70

60

50

40 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.15. Stability curves showing the concentration of the active ingredient in wet granulated tablets vs number of days. ASA (WG) at 40 °C & 75% RH (■); ASA (WG) at 50 °C & 75 % RH (×); CAS (WG) at 40 °C & 75% RH (♦); CAS (WG) at 50 °C & 75% RH (▲).

89

18 16 14

Assay (%w/v))

12 10 8 6 4 2 0 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.16. Stability curves showing the concentration of the free SA in wet granulated tablets vs number of days. SA in ASA (WG) tablets at 40 °C & 75% RH (■) ;SA in ASA (WG) tablets at 50 °C & 75% RH (×); SA in CAS (WG) tablets at 40 °C & 75% RH (♦); SA in CAS (WG) tablets at 50 °C & 75% RH (▲).

90

3.5.4

Stability comparison of CAS and ASA capsules

The stability was monitored by determining the concentration of the active ingredient and the decomposition product in the capsules placed at 40ºC and 50ºC with 75% RH, at various intervals of time. The results of the accelerated stability study of capsules are shown in Figures 3.17 – 3.19. At 40ºC, the concentration of CAS and ASA (the active ingredient) dropped slowly according to the equation, y = 25.021e-0.0005× with r2 = 0.78 and y = 98.856x-0.076 with r2 = 0.765 (F < F critical), respectively. Accordingly, the concentration of SA (the common decomposition product of CAS and ASA capsules) increased by following the equations y = 0.0018x+0.1287 (r2 = 0.8328) and y = 0.0013x -0.0014 (r2 = 0.9065) for CAS and ASA capsules, respectively. These results clearly indicate no significant difference in the stability of CAS capsules as compared to that of ASA. At 50ºC, the concentration of CAS and ASA (the active ingredient) dropped slowly according to the equation y = -0.0122x+24.092 with r2 = 0.7816 and y = -0.0405x+98.584 with r2 = 0.94 (F < F critical), respectively. Accordingly, the concentration of SA (the common decomposition product of CAS and ASA capsules) increased by following the trends according to equations y = 0.0016x+ 0.121(r 2 = 0.8228) and y = 0.0016x-0.0085 (r2 = 0.9689). These results also indicate similar stability of CAS and ASA formulation. The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC with 75% RH was determined and it increased by following the equations y = 0.0155x+0.7992 (r2 = 0.9106) and y = 0.0245x +0.6642 (r2 = 0.9015), respectively. There is no significant

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difference (F < F critical) found between the two values. This shows that the stability profile of CAS capsules is similar at 40ºC and 50ºC with 75% RH.

92

100

90

Assay(%w/v))

80

70

60

50

40 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.17. Stability curves showing the concentration of the active ingredient in capsules vs number of days. CSA capsules at 40 °C & 75% RH (■); ASA capsules at 40°C & 75% RH (●); ASA capsules at 50 °C & 75% RH (×); CAS capsules at 50 °C & 75% RH (▲).

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0.8 0.7

Assay (% w/v))

0.6 0.5 0.4 0.3 0.2 0.1 0 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.18. Stability curves showing the concentration of free SA vs number of days: in ASA capsules at 50 °C & 75 % RH (□); in ASA capsules at 40 °C & 75% RH (♦); in CAS capsules at 50 °C & 75% RH (×); in CAS capsules at 40 °C & 75% RH (∆).

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4 3.5

Assay (% w/v))

3 2.5 2 1.5 1 0.5 0 0

20

40

60

80

100

120

140

160

180

Days

Figure 3.19. Stability curves showing the concentration of free CS vs number of days: in CAS capsules at 50 °C & 75% RH (□); in CAS capsules at 40 °C & 75% RH (∆).

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3.5.5

Stability comparison of CAS and ASA aqueous suspension

Stability study of CAS and ASA aqueous suspension was carried out under the same conditions as used for tablets and capsules; however, it was observed that the suspension of both the products were not stable. The results are shown in Figure 3.20. 3.5.6

Study of commercial CAS preparation

Samples of commercially available CAS preparation, Nuhas capsules (Sigma Herbals, Lahore), 60 mg CAS, were analyzed for the CAS, ASA, CS and SA contents. The results are given in Table 3.10. The data indicate excellent stability of the product over the period under study.

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100 90 80

Assay(%w/v))

70 60 50 40 30 20 10 0 0

2

4

6

8

10

12

14

Hours

Figure 3.20. Stability curves showing the concentration of the active ingredient in ASA and CAS syrup vs time (hours). ASA at 40 °C & 75% RH (■); ASA at 50 °C & 75% RH (×); CAS at 40 °C & 75% RH (♦); CAS at50°C&75%RH(▲).

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Table 3.10. Three years data of commercially available 60 mg CAS capsule (Nuhas, Sigma Herbals). The contents of various analytes are given as mg per capsule. Age (Months) Analyte

CAS

0

6

12

24

36

59.0

58.92

58.51

59.85

59.12

0.35

0.3

0.28

0.20

0.14

CS

0.10

0.13

0.12

0.11

0.10

SA

0.05

0.06

0.10

0.15

0.2

ASA

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3.6

Pharmacokinetic studies

The pharmacokinetic data following the single oral administration of 60 mg CAS (tablet or capsule) is given in Tables 3.11 and 3.12. The pharmacokinetic data of both the tablets and capsules was almost similar and the reported results are an average of both dosage forms. The chromatograms of blank plasma and sample plasma are shown in Figure 3.21. The plasma concentration-time curves are shown in Figure 3.22. It was noted that about 1.5% (i.e. AUC0-∞h × 100/Dose) of unconverted CAS of the oral dose reaches systemic circulation. This behavior was found to be similar to that of ASA, indicating similar bioavailability after oral administration [126]. The extrapolated AUC∞ was < 10% in case of CAS and ASA (as metabolite). The areas under the curves (AUC0-∞h) of the metabolites including ASA, SA and CS (0.45, 0.31 and 2.35 hmgL-1, respectively) show that significant amounts of these species remain available in plasma for longer periods of time; the concentration of CS being the highest, whereas, after administration of 900 mg ASA, the level of ASA in plasma rises rapidly to reach a maximum, with only small amounts remaining after 2 h [126]. The elimination of CAS, ASA, CS and SA follows the first order kinetics with r 2 0.960, 0.930, 0.999 and 1.000, respectively. The mean pharmacokinetic data of CAS, ASA, CS and SA is given in the Tables 3.11 and 3.12. The Cmax was found to be 0.38 mgL-1 at a tmax 0.72 h, which is about 0.6% of the administered dose. In case of aspirin (ASA), normally, it is about 4% of the dose between 14-15 min of administration of 900 mg [126]. The t1/2 was 8.67 h, which is ideal for once a day dosing. The Vd and Cl values for CAS were 829 Lkg -1 and 66.30 Lh-1, respectively. The large Vd may be due to uptake by a specific tissue or membrane, as highly lipophilic compounds are known to distribute into lipids in cell membranes and fat stores; these effectively form slow release

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depots of the drug and prolong the plasma levels [127]. The relatively high clearance may lead to low exposure and low plasma average concentrations during chronic dosing. Theses findings offer an understanding of the enhanced anti-inflammatory activity of CAS as compared to that of ASA alone [126]. The plasma copper level was determined by atomic absorption spectrophotometer at 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 12 h after administration of a single dose of 60 mg CAS. The results are given in Table 3.13 and the trend is shown in Figure. 3.23. It can be seen that the plasma copper level raised about two times the normal plasma copper level, which remained constant over a period of 12 h.

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Table 3.11. Pharmacokinetic data after a single oral dose of 60 mg CAS and 900 mg aspirin (ASA). Parameter CAS ASA [126] tmax, h

0.72

0.24

0.38

36.62

8.67

0.22

AUC0- ∞h, hmgL

0.91

15.32

Vd, Lkg-1

829

150-200[127]

66.30

13.33[127]

Cmax, mgL

-1

t1/2, h -1

-1

Cl (Lh )

Vd: volume of distribution ; Cl: total body clearance for extra- vascular administration.

Table 3.12. Pharmacokinetic data of CAS metabolites after a single oral dose of 60 mg. Parameter tmax, h Cmax, µg mL-1 AUC0- ∞h, h. µg mL-1

ASA 1.41 0.178 0.45

SA 2.13 0.022 0.31

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CS 3.02 0.13 2.35

Plasma concentration (mgL-1)

Figure 3.21. Chromatograms of blank and test plasma showing separation of CAS(1), CS(2), ASA(3) and SA(4).

1 CS SA ASA

0.1

CAS 0.01 Figure. 3.22. Plasma concentration-time curve of CAS, ASA, SA and CS. 0.001

0.0001 0

2

4

1026 Time (h)

8

10

12

Table 3.13. Plasma copper concentration data.

Time (h)

(Concentration (µg mL-1

Time (h)

(Concentration (µg mL-1

0.25

0.887522 ±0.00118

4.00

0.714411 ±0.00116

0.50

0.740773 ±0.00117

8.00

0.819859 ± 0.00118

1.00

0.670475 ±0.00118

12.00

0.688040 ± 0.00115

2.00

0.793497 ±0.00119

̶

̶

103

2

-1

Plasma copper ( µg mL )

1.5

1

0.5

0 0

2

4

6

8 Time (h)

Figure 3.23. Copper plasma level-time curve.

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10

12

14

CONCLUSIONS This work was aimed at the study of some of the important pharmaceutical and pharmacological properties of copper (II)-acetylsalicylate, a potential anti-inflammatory drug, with regard to its development as a drug. The work was divided into three parts. In the first part, pharmaceutical properties of copper (II)-acetylsalicylate including crystallanity, particle size distribution, porosity, flow, compressibility, moisture uptake and taste were studied and compared with those of acetylsalicylic acid. The results indicated that copper (II)-acetylsalicylate possesses reduced crystalline character, particle size and density, better flow, no taste, and enhanced porosity. The complex was found to be hydrophobic. In the second part, copper (II)-acetylsalicylate was converted into various dosage forms including tablets (through wet granulation and direct compression methods), capsules and aqueous suspension. These dosage forms were subjected to accelerated stability studies and compared with the profile of acetylsalicylic acid. The third part consists of development and validation of new HPLC method for simultaneous determination of copper (II)-acetylsalicylate and its decomposition products or metabolites in dosage forms and plasma samples. The method was found to be suitable for stability and pharmacokinetic studies. This method was successfully applied to determine the various pharmacokinetic parameters in humans. The results showed that copper (II)-acetylsalicylate possessed higher values of the time to peak drug concentration, the half life of the drug, volume of distribution, and clearance. The data obtained through these studies clearly demonstrate that copper (II)-acetylsalicylate

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possesses better properties for formulation into the dosage forms studied. The results also further our understanding regarding the enhanced anti-inflammatory effect of copper (II)acetylsalicylate as compared to acetylsalicylic acid.

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