The effect of pharmaceutical excipients on isoniazid release from chitosan beads

Figure 2.3: Results of the different TPP concentrations on the percentage drug loading in

cm.

From the results obtained it is clear that a TPP concentration of 5% w/v has the best encapsulation properties of all four concentrations that were tested. Beads of the 6% w/v TPP batch were not quit spherical and very brittle. Because of the brittleness and weak crosslinking of beads in the 6% solution in showed lower drug loading. The supernatant of the 6% w/v batch was a milky white colour when compared to the supernatant of the other batches. The reason for this phenomenon is that the beads of the 5% batch had better crosslinking of the chitosan which lead to better encapsulation of INH. The milky supernatant of the 6% batch indicates that the INH leaked out of the beads into the TPP solution. Beads from this batch also appeared lighter and hollow compared to other. Figure 2.3 indicates that the 4% batch showed lower encapsulation efficiency than the 3% and the 5%. A logical explanation for this is that the stirring time for the 4% batch may have not been sufficient enough to allow proper crosslinking, which lead to a decrease in INH encapsulation.

2.5.1.2 Swelling of beads with different TPP concentrations

The degree of swelling of beads prepared during this experiment was conducted by the method as described in section 2.4.2 above. The swelling of the beads were studied at two

29 50 45 40 35 30 X2520 15 0 10 5 0 3% 4% 5% 6% [TPP]

different pH levels, pH 5.6 and pH 7.4, to determine in which part of the intestine the swelling would be the best. Results from this study is shown in figure 2.4 and figure 2.5.

2.5 'i' III !!:!. 2 ... .E '; 1.5_III '0 .. I.'! ... ~ 0.5 3 o !I 3% 04% 11.'15% 06% 10 60 360 Time (minutesl

Figure 2.4: Degree of swelling at pH 5.6 for different TPP concentrations.

Figure 2.5: Degree of swelling at pH 7.4 for different TPP concentrations.

The results in figure 2.4 indicate the degree of swelling for each of the different TPP concentrations at pH 5.6. The 3% and 5% batch showed the most promising swelling over the 360 minute period. The 3% batch showed good swelling over the first 10 minutes with more swelling over the next 50 minutes. A maximum was reached after 60

---30 ----2.5

!

2 _

I

11

I

Il

i':1111

3%

m4%

5% 06%

i

g

0.5 0 10 60 360 Time (minutes)

section 1.4.2 and the pH of the TPP solutions was adjusted to 2, 4, 5, 6, 7, 8, 9, and 10 respectively. The natural pH of TPP (8.7) was also used to determine the drug loading to see if any pH alteration would be necessary.

2.5.2.1 Drug loading

The percentage drug loading was determined by the method described in section 2.4.1.1 to determine at which pH of the TPP solution the maximum quantity of isoniazid would

be captured into the chitosan-gellingmedium, the results are presented in figure2.6.

45 40 35 C) c 30 :c .2 25 C) 20 2 C 15 ~ 10 5 o 2 4 5 6 7 pH 8 8.7 9 10

Figure 2.6: Percentage drug loading at different pH levels ofTPP solution.

From the data shown in figure 2.6 there is a steady increase in drug loading with an increase in the pH of the TPP solution reaching a maximum at pH 8. A further increase in the pH resulted in a decrease in the % drug loading. The best explanation for this phenomenonis that INH solubilitywas best at pH 6- 8. The amountof drugthat was incorporated into the beads at these levels was best, however a pH of 8 showed the best drug loading capability. At pH levels 2, 3, 4, 5, 9 and 10 the % drug loading was less because of a smaller amount of INH that was soluble. At pH 7 there might have been one or more deformed beads which led to an inaccurate analysis. The natural pH ofTPP is 8.7

32

---and showed good encapsulation efficiency for INH. It was decided that the remainder of the study would be conducted at a pH level of 8.

2.5.3 Different isoniazid concentrations

In order to optimize the quantity of isoniazid that could be encapsulated in chitosan beads it was necessary to conduct an experiment to determine which concentration of isoniazid the optimum quantity could be incorporatedinto the beads.

2.5.3.1 Drug loading

The drug loading method as described in section 2.4.1.1 was used to determine the percentage drug that could be encapsulated at different INH concentrations. It was decided to use a percentage value of 1% (w/v)

-

7% (w/v) INH to determine the effect of an increase in drug quantity on the encapsulationefficiency of the drug. Figure 2.6 shows the results.

INH Concentration (I!glml)

Figure 2.7 Percentage drug loading of different INH concentrations.

There was no particular pattern in this experiment. An INH concentration of 1% (w/v) showed a drug loading capacity of 40%. This quantity will be sufficient to proceed with

33 50 45 C) 40

.:

35

i

₀ 30 .J_C) 25 2 20 C 15 0 10 5 0 1% 2% 3% 4% 5% 6% 7%

the experiment, but a larger % drug loading is preferred. When beads prepared of the 4% batch were pulverised and tested for drug loading capacity, it showed a 45% capability to encapsulate INH. It was decided to continue the study using a 4% INH concentration. It is not clear why there is such a difference between the % drug loading of consecutive concentrations and this needs further investigation.

2.5.3.2 Swelling

Swelling of the beads was done according to the method described in section 2.4.2. Results of the swelling capability are shown in figure 2.8 and 2.9.

Figure 2.8: Degree of swelling of chitosan beads containing different concentrations

Figure 2.9: Degree of swelling of chitosan beads containing different concentrations of INH at a pH level of 7.4. 34 2.5

l

2 .1%02% 1.5 .3% 1 1:14% b 1 m5%

i

1Z16% 0.5 Ii;]7% 0 10 60 360 Time (minutes) of INH at a pH level of 5.6. 2.5

I

2 .1%o 2%

g

1.5

.

3% 1 CI 4% '8 1 g;j 5%

I

C 6% 0.5 7% 0 10 60 360 Time (minutes)

3.5.1.1 Morphology of beads that contains vitamin C

The morphology studies were carried out as described in section 3.3 using a scanning electron microscope (SEM). During this study full view, cross-cut and microscopicviews of the beads were carried out.

Figure 3.1: Full view ofa 4% (w/v) CIB

Figure 3.3: Microscopicview ofINH incorporated into the bead.

Figure 3.5: Cross view of a 4% (w/v) INH and 0.25% (w/v) VC bead.

Figure 3.2: Cross view of a 4% (w/v)CIB

INH crystals.

Isoniazid crystal

Figure 3.6: Microscopicview of a 4% (w/v) INH and 0.25% (w/v) VC bead.

Figures 3.1 and 3.5 shows that the beads prepared for this study remained almost spherical throughout the study. Beads that only contain chitosan and isoniazid (INH) presented a surface that had little porosity as shown in figures 3.2 and 3.3. This could be attributed to the fact that there was proper crosslinking of the chitosan and INH. These figures also show how well INH was incorporated in the beads. When the excipient VC was added, it was depicted that there will be a few changes in the morphology and characteristicsof the beads. Beads that contained VC had a rough outer surface. This was the result of INH particles encapsulated in the outer surface of the beads. When VC was added the porosity of the beads increased as can be seen in figure 3.5, which could be contributed to the fact that the VC helped to release substances from chitosan beads which made the beads more porous. Figure 3.6 showed that there was a large amount of INH crystals encapsulated in the pores of the beads.

3.5.1.2 Drug loading of beads that contains vitamin C

The drug loading method as described in section 2.4.1.1 was used to carry out this experiment. The following figure shows the drug loading capability of chitosan/INH/VC beads compared to that of CIB.

Single pharmaceutical excipient (SPE)

Figure 3.7: Percentage drug loading ofCIB and 0.25% (w/v) vitamin C SPE beads.

---42 50 45 40 35 30 m 25 .5 20 -oe 15 10 5 0 ()<Q .J.0

From figure 3.7 it is clear that cm had a better encapsulation ability than beads which also contained VC. This fact is also observed in the other single- and multiple

pharmaceutical excipient beads throughout the study. The lower drug loading capability can be the cause of drug that was lost in the TPP solution during crosslinking due to extra excipients that needed to be incorporated into the beads, or due to competition between the drug and the excipient for binding position during crosslinking.

3.5.1.3 Swelling of beads that contain vitamins C

The swelling behaviour studies of chitosan beads were conducted in triplicate over a period of 360 min (6 hours). The method as described in section 2.4.2 was used for this swelling study. 2.5 2

I

_C)

,

'0

I

1.5

g

1 60 Time (minutes)

Figure 3.8: Degree of swelling for cm and 0.25% (w/v) vitamin C SPE beads at pH 5.6.

10 360

2.5

l

.~

1 '5

I

1.5

g

2 1 10 60 Time (minutes) 360

Figure 3.9: Degree of swelling for CIB and 0.25% (w/v) vitamin C SPE beads at pH 7.4.

The swelling observed with VC was more or less the same as was observed with CIB beads after 10 minutes. As time increased VC beads showed better swelling capabilities because of the acidic environment of VC. After 60 minutes the swelling of the beads still increased to a maximum over the 360 minute period. Swelling of VC beads were more prominent in pH 5.6 than at pH 7.4. There was a visual increase in the swelling of beads that contained VC as time increased to that of CIB. This result was as expected from the morphology study. Because the VC beads were more porous than the chitosan beads, it was expected to have a better degree of swelling.

3.5.2 The effect of Ac-Di-Sol@on the characteristics of CIB

The effects that 0.5% (w/v) ADS had on the morphology, drug loading and swelling of CIB was examined and will be discussed in this section.

44

--3.5.2.1

Morphology of beads that contains ADS

The method described in section 3.4.1.1 was used for this morphology study. The following figures show the SEM results of CHiINHIADS beads.

Figure 3.10: Cross cut view of 4% (w/v) INH

and 0.5% (w/v) ADS beads.

Figure 3.11: Microscopicview of 4%

(w/v) INH and 0.5% (w/v) ADS beads.

Beads that were prepared according to the ionotropic gelation method as described in section 2.3 and which contain ADS, presented beads that were not quit spherical as can be seenin figure3.10.Thesebeadshad agoodchitosancross-linkedsurfacewitha good degree of porosity and the INH crystals was caught in these pores as figure 3.11 shows. It was expected that these beads would have a better degree of swelling than CIB beads. The result of less spherical beads may be because of a higher quantity of the excipient and drug on the outer surface of the beads.

3.5.2.2 Drug loading of beads that contains ADS

The drug loading capability of CHiINHIADS beads was determined by the method described in section 2.4.1.1. The result of this experiment is shown in figure 3.12.

Figure 3.12: % Drug loading of cm and 0.5% (w/v) Ac-Di~Sol@ SPE beads.

The phenomenon described in section 3.4.1.2 would also be seen in figure 3.12. Due to the addition of a pharmaceutical excipient to the normal cm formula, the drug loading capability of chitosan beads decreased. The direct effect of this phenomenon could clearly be seen in figure 3.12 as the beads that contain ADS had a lower capability to encapsulate INH. Another explanation for the lower drug loading results may be that some of the INH leaked out into the TPP solution during the.formulationphase.

3.5.2.3 Swelling of beads that contains ADS

The swelling experiment was done at two different pH levels, at pH 5.6 and 7.4. This was done to simulate different parts of the intestine and to determine the effect of the beads in a alkaline and acetic area. Swelling studies were done according to the method as described in section 2.4.2. 46 50 45 40 35

PO]

I I

.

I_CIS

I

m 25 f!J A OS .5 20 15 0 10 5 0 a<Q

2.5 I r 1 '5 ~ 1.5 g 2 I~CISEIADS I 1 10 60 360 Time (m inutes)

Figure 3.13: Degree of swelling for CIB and 0.5% (w/v) Ac-Di-Sol@SPE beads at pH 5.6. 2.5

I

.~

1 '5

~

1.5

g

2 I_CISmADS I 1 10 60 Time(minutes)

Figure 3.14: Degree of swelling for CIB and 0.5% (w/v) Ac-Di-Sol@ SPE beads

at pH 7.4.

360

The swelling pattern of ADS beads at pH 5.6 and pH 7.4 showed quit a few resemblances. At pH 5.6 there was an initial swelling in the first 10 minutes and the swelling was then almost kept constant for the duration of the experiment. At pH 7.4 there was a quick onset of swelling over the first 10 minutes and over the next 50 minutes the beads showed even more swelling capabilities. After 60 minutes the swelling stayed

at this level and the beads had reached its maximum swelling potential. The reason why the SPE swelling is better than the cm is because of a higher degree of porosity as described in section 3.3.2.1.

3.5.3 The effect of Explotab@on the characteristics of CIB

The effects that 0.25% (w/v) EXPL had on the morphology, drug loading and swelling of cm was examined and will be discussed in this section.

3.5.3.1 Morphology of beads that contains EXPL

When EXPL beads were prepared for the morphology study as described in section 3.4.1.1, it was noticed that these beads were very brittle. The results of the SEM photos are given below.

Figure 3.15: Cross-cut view of a 0.25%

(w/v) EXPL and 4% (w/v) INH bead.

Figure 3.16: Microscopicview of a 0.25%

(w/v) EXPL and 4% (w/v) INH bead

EXPL beads were less spherical and more porous than ADS beads. It was noticed that these beads were brittle and this may be the result of insufficient chitosan during and or stirring time during preparation of the beads. The lNH particles that were captured in the pores can clearly be seen in figure 3.16. There is a few large pores that can be seen in figure 3.16 which may also indicate why these beads were brittle.

3.5.3.2

Drug loading of beads that contains EXPL

When EXPL beads were prepared according the drug loading method described in section 2.4.1.1, the supernatant of the beads were cloudier compared to the other prepared beads. 50 45 40 g' 35 :g 30_o ~ 25

~

20 ~ 15 10 5 o

#v

o

Single pharmaceutical excipient (SPE)

Figure 3.17: Percentage drug loading of cm and 0.25% (w/v) Explotab@SPE beads.

The result of the drug loading test for EXPL is the worst of all the SPE beads. The addition of the excipient which interferes with the uptake of INH during the preparation phase can be a possible reason for this occurrence. Because the beads were porous, it indicates that some of the INH leaked out through the pores during the crosslinking phase. Because the morphology study showed that the beads were brittle, it might also contribute to this lack of INH uptake or release. There might not have been sufficient chitosan during the preparation phase which produced beads that were brittle and no proper crosslinking could take place. Because of this reason insufficient amount of INH was encapsulated into the beads. Because of the cloudier supernatant during preparation, it can be assumed that some of the INH leaked out into the TPP solution during preparation.

3.5.3.3

Swelling of beads that contains EXPL

The swelling study for EXPL beads was done as for all the single pharmaceutical excipients as described in section 2.4.2. The result of the study is given in figure 3.18 and 3.19. 2.5 ~ C)

t

'IS

~

1.5

g

2 60 Time (minutes) 360

Figure 3.18 Degree of swelling for cm and 0.25% (w/v) Explotab@SPE beads at pH 5.6. 2.5

~

~

1.5

1

~_o 1

~

g

0.5 1 10 2 o 60 Time (minutes) 360

Figure 3.19: Degree of swelling for cm and 0.25% (w/v) Explotab@SPE beads At pH 7.4.

10

0.25% (w/v) VC and 0.5% (w/v) ADS bead.

Figure 3.20: Cross cut view of a 4% (w/v) IND, Figure 3.21: Microscopicview of a 4% (w/v)

IND, 0.25% (w/v) VC and 0.5% (w/v) ADS bead.

Multi pharmaceutical excipient (MPE) beads that contain VC and ADS produced beads that were spherical with a higher degree of porosity than the SPE beads. Figure 3.21 shows that there is a large quantity of drug on the inner surface and in the chitosan network. The figure also shows a good dispersion of the drug throughout the bead. The beads had a higher capability to encapsulate INH than the SPE beads because it is more porous. The higher degree of porosity was due to the combination of the uneven structures ofVC and ADS.

3.6.1.2 Drug loading of beads that contains vitamin C and

ADS

The result of the drug loading test that was done on VC/ADS beads is shown in figure 3.22. The figure also shows the drug loading for SPE beads ADS and VC to enable a comparison ofthe SPE and the MPE beads.

DVC+ADS C! CIS

ClVC

DADS

Single pharmaceutical excipient (SPE)

Figure 3.22: Percentage drug loading for CIB, SPE beads 0.25% (w/v) VC and 0.5% (w/v) ADS and MPE beads 0.25% (w/v) VC/0.5% (w/v) ADS.

A comparison of the drug loading capability of CIB, the SPE beads and the MPE beads is shown in figure 3.22, a marked difference could be observed. The MPE beads with a drug loading of (37.16%) exhibited a higher drug loading capability than the SPE beads of ADS with a drug loading of (33.14%). The drug loading capability of SPE beads VC (36.96%) was almost the same as the MPE beads. The most logical explanation for this was that the MPE beads were more porous than the SPE beads and the MPE beads therefore had a greater capability to incorporate/encapsulate a higher quantity of INH crystals into the pores. The CIB had a greater capability to encapsulated INH according to figure 3.22. An explanation for this observable fact was that during preparation of the SPE and MPE beads some of the INH leaked out into the TPP solution. The reason for the inclusion of the excipients is to improve the swelling of the beads and not necessary the drug loading, it is thus expected that the SPE and MPE beads would show better swelling than CIB.

53 50 45 40 2'35 '6 30 25 C) 20 '# 15 10 5 0 a<Q 0 _.p'

3.6.1.3 Swelling of beads that contains vitamin C and ADS

The swelling behaviour study of VC/ADS beads was done in triplicate as described in section 2.4.2. The results of this swelling study compared to the swelling of the SPE beads in the two media are given in figure 3.23 and 3.24.

..._o

~

1.5

g

2.5 2 18 CIS eVC DADS lEIADS+VC 1 10 60 Time (minutes) 360

Figure 3.23: Degree of swelling for CIB, SPE beads 0.25% (w/v) VC and 0.5% (w/v) ADS and MPE beads 0.25% (w/v) VC/0.5% (w/v) ADS at pH at 5.6.

2.5 1 III CIS eVC CJADS c ADS+VC 10 60 Time (minutes) 360

Figure 3.24: Degree of swelling for CIB, SPE beads 0.25% (w/v) VC and 0.5% (w/v) ADS and MPE beads 0.25% (w/v) VC/0.5% (w/v) ADS at pH of7.4.

- - -- -

Figure 3.25: Cross view of a 0.25% (w/v) VC / Figure 3.26: Microscopic view of a 0.25% (w/v)

0.25% (w/v) EXPL CIB. VC / 0.25% (w/v) EXPL CIB.

Beads that contain the MPE combinationof VC/EXPL were not exactly spherical but had a smooth outer surface as can be seen in figure 3.25. In figure 3.26 the large quantity of INH that was encapsulated in the beads can clearly be seen. This micrograph also shows that there were proper crosslinking of the chitosan which meant that there was proper stirring time during bead formation. The beads had a high degree of porosity which accounted for the large quantity of INH encapsulated in the beads. It is not clear why this bead is not quit spherical, when compared to other beads from the same batch. The most logical explanation for this is that there might still have been some air bubbles in the matrix, even though enough time was allowed for bubbles to dissolve from the matrix beforebeadformation.

3.6.2.2 Drug loading of beads that contains vitamin C and

EXPL

The results of the drug loading test from the beads containing VC/EXPL is shown in figure 3.27.

1mCIS

cEXR..

DVC D VC+EXR..

Single pharmaceutical excipient (SPE)

Figure 3.27: Percentage drug loading for cm, SPE beads 0.25% (w/v) VC and 0.25% (w/v) EXPL and MPE beads 0.25% (w/v) VC/0.25% (w/v) EXPL.

The combination of VC and EXPL produced beads with a drug loading of (37.86%) which is better than that of EXPL (31.81%) and VC (36.96%). The drug loading was almost the same for the MPE beads and that of the VC beads. From this figure it is also clear that cm still has the better capability to encapsulate INH. The only explanation for this is that the INH leaked into the TPP solution during preparation as was also the case in section 3.5.1.2. The combinationofVC and EXPL did not improvethe drug loading as much as was expected.

3.6.2.3 Swelling of beads that contains vitamin C and EXPL

The swelling behaviour study of VCIEXPL beads was done in triplicate as described in section 2.4.2. Results of the swelling study are given in figure 3.23 and 3.24 for the two media (PH 5.6 and pH 7.4). 57 50 45 40 g» 35 30 C,25

B

20 ;!. 15 10 5 0 a<Q <v'v 0

... o

~

1.5

§

2.5 2 II CIS eVC oEXPL EI EXPL +VC 1 10 60 Time (minutes) 3.60

Figure 3.28: Degree of swelling for CIB, SPE beads 0.25% (w/v) VC and 0.25% (w/v)

EXPL and MPE beads 0.25% (wIv) VCI0.25% (wIv) EXPLat pH 5.6.

2.5 1 .CIS eVC oEXPL iii EXPL+VC 10 60 Time (minutes) 3.60

Figure 3.29: Degree of swelling for CIB, SPE beads 0.25% (w/v) VC and 0.25% (w/v) EXPL and MPE beads 0.25% (w/v) VCIO.25% (w/v) EXPL at pH 7.4.

The results of the swelling study for VC/EXPL beads as given in figure 3.28 and figure 3.29, shows that this is not a suitable MPE combination. It is clear from figure 3.28 that in a pH level of 5.6 VC/EXPL beads has the worst degree of swelling, even CIB showed better swelling capabilities. Figure 3.29 shows that at a pH level of 7.4 VC/EXPL beads have a slight better degree of swelling than CIB. There is no logical explanation for this manifestationand it needs further investigation.

3.6.3 The

effect

of

Ac-Di-Sol@

characteristics

of chitosan beads

and

_Explotab@

on

the

The effects that 0.5% (w/v) ADS and 0.25% (w/v) EXPL had on the morphology, drug loading and swelling of CIB was examined and will be discussed in this section.

3.6.3.1 Morphology of beads that contains ADS and EXPL

The micrographs obtained from the SEM study for beads that contain ADS and EXPL is given in figures 3.30 and 3.31. The method described in section 3.4.1.1 was used for this study.

Figure 3.30: Cross cut view of a 0.5% (w/v) ADS / 0.25% (w/v) EXPL CIB.

Figure 3.31: Microscopicview of a 0.5% (w/v)

ADS / 0.25% (w/v) EXPL CIB.

Beads that were prepared with the MPE combination of ADS/EXPL produced almost spherical beads as can be seen in figure 3.30. The beads had a rough outer surface which is the result of INH encapsulated on the outer surface of the bead as well as the uneven structure of the excipients powder. Figure 3.31 shows a strong chitosan network which is the result of proper crosslinking of the chitosan. It also shows that the bead has a high porosity and a large number of INH crystals are encapsulated in the pores. This is the largest quantity of INH encapsulated according to all the micrographs. It is expected that these beads will show good drug loading and swelling.

3.6.3.2 Drug loading for beads that contains ADS and EXPL

The result of the drug loading test as described in section 2.4.1.1 which was done on ADS/EXPL beads, is shown in figure 3.32.

50 45 40

2'

35 ~ 30

-

25 ~ 20 ~ 15 10 5 o .CIS 121ADS [JEXPL [J ADS+ EXPL #v

~

?' I

Figure 3.32: Percentage drug loading for cm, SPE beads 0.25% (w/v) EXPL and 0.5%

(w/v) ADS and MPE beads 0.25% (w/v) EXPL/O.5%(w/v) ADS. Single pharmaceutical excipient (SPE)

From the drug loading test that was done above, the cm combination showed the best drug loading capability (43.92%) of all. The MPE beads combination of ADS/EXPL showed the second best drug loading capabilities (33.95%) above that of the SPE beads of ADS (33.14%) and EXPL (31.81%). It was expected that the ADS/EXPL combination would have the best drug loading capability, due to its greater porosity. This phenomenon repeated itself in every drug loading study that was conducted in this chapter. The best and most logical explanation for this is that some of the INH must have leaked out into the TPP solution during preparation. The combination of two excipients with uneven structures may contribute to the lower drug loading because of competition between INH, ADS and EXPL for binding space in the chitosan matrix.

60

-.

3.6.3.3 Swelling of beads that contains ADS and EXPL

The swelling behaviour study of ADS/EXPL beads was done in triplicate and as described in section 2.4.2. Results of the swelling study are given in figure 3.23 and 3.24.

Figure 3.33: Degree of swelling for cm, SPE beads 0.25% (w/v) EXPL and 0.25%

(w/v) EXPL and MPE beads 0.25% (w/v) EXPL I 0.25% (w/v) EXPL at pH 5.6.

2.5 1 mGIS EIADS oEXA... ~ EXA...+ADS 10 60 Time (minutes) 360

Figure 3.34: Degree of swelling for cm, SPE beads 0.25% (w/v) EXPL and 0.25%

(w/v) EXPL and MPE beads 0.25% (w/v) EXPL I 0.25% (w/v) EXPL at pH 7.4.

61 2.5 I 2 . '0 1.5 B 1 10 60 360 Time (minutes)

~

~<t-' x~ #" «t<t-' ~ ..fJ .f! .pi

Pharmaceuticalexipients Figure 3.35: Drug loading of all SPE and MPE beads

mCIS .vc I!iiIADS [J EXPL ESIVC+ADS Ia VC+EXPL Q ADS+ EXPL

Conclusion of the swelling studies that was done can best be explained in figure 3.36 and

60min Time (minutes)

360min

Figure 3.36: Degree of swelling for SPE and MPE beads at pH 5.6.

II3CIB .VC mADS o EXPL

. ADS+VC

m EXPL+VC If:!EXPL+ADS

At pH 5.6 of the SPE beads, ADS showed good swelling over 60 minutes with a constant increase and kept this swelling for the entire experiment. VC is the only excipient in the entire experiment which showed promising swelling between the 60 minute and 360 minute period. The best swelling however was observed with MPE combination of EXPL/ADS. Excessive swelling was observed for the first 10 minutes with a constant

63 50 45

f

40 ;. 35 30 25 B 2015 10 0 5 0 () ..f-' figure 3.37 below. 3 l' 2.5 an --. 2 1 1.5 I/) '0 ₁

I

§

0.5 0 10min

increase over the entire 360 minutes. The reason for this excessive swelling is the high porosity of the beads due to more uneven structures in the chitosan matrix.

Figure 3.37: Degree of swelling for SPE and MPE beads at pH 7.4.

At pH 7.4 ADS again showed the moat promising swelling of all the SPE beads, it also showed better swelling that ADS/VC and EXPLlVC beads. The MPE combination of EXPLIADS showed the best swelling as it did at pH 5.6. Again the beads showed excessive swelling over the first 10 minutes with a constant increase for the remainder of the experiment.

Beads that exist over fast swelling capabilities are of great importance for target specific delivery of any drug. Dissolution studies on the prepared beads will be discussed in chapter 4. 64 3 12.5 19CIS .VC C) 2 .5 IDADS 1 ' , 1.5 , o EXPL ". 'e

.

ADS+VC 1 13EXPL+VC

g

0.5 ;5< [I] EXPL+ADS 0

10min 60min 360min

4.3.1 Dissolution of chitosan - isoniazid beads (CIB)

The first 60 minutes of the results from the release study for cm in dissolution medium pH 5.6 and pH 7.4 is depicted in figure 4.2. The 360 minute dissolution results are given in comparison with the SPE in figures 4.5 and 4.6. Data for the dissolution study is given in annexure A. The experiment was conducted over a period of 6 hours to monitor the release of INH over a substantial amount of time. From the results it is clear that there was a burst effect of the beads within the first 5 minutes of the experiment. From this quick onset of drug release it could be concluded that there were drug particles on the surface of the beads. There was a slight better release of INH in the acidic than in the alkaline media. Figure 4.2 indicatesthat a large amount of the drug was released over the first 10minutesof dissolutionin the pH 5.6 medium.Eventhough56.86:!:0.71% (12.76 ~g/ml) of the drug was already released after 10 minutes, a maximum of 58.38:!:0.20% (13.10 ~g/ml) was released over the 6 hour experiment. Drug release in the pH 7.4 mediumalso showeda burst effect and reached52.26:!:0.97% (11.84 ~g/ml)release after only 5 minutes of dissolution. Even though this large amount of drug was released after only 5 minutes, there is still a slight increase in the release of INH and reached a

value of 57.47 :!:0.79% (13.02 ~g/ml) after 6 hours. These results indicated only a slight

sustained release of INH from cm which was preceded by a burst effect of the beads which lead to the release of most of the drug that was incorporated in the beads within the first 10minutes.

Figure 4.2: INH release from 4% (w/v) cm in pH 5.6 and pH 7.4 after 60 minutes.

68 70 "0' C 60

i

50

i

40]1 I-+- CISpH5.6

I

30 _CIS pH 7.4

f

2010 <C 0 0 20 40 60 80 Time (minutes)

60

-~ ~ 50

i

_> '0 40 :g :s_CI 30 2

"

20 (I) CI

e

10 (I)

~

--k- ADSpH5.6 VC pH 5.6 -+- EXPLpH 5.6 o o 20 40 60 Time (minutes) 80

Figure 4.3: Dissolution results for SPE 0.5% (w/v) ADS, 0.25% (w/v) EXPL and 0.25% (w/v) VC beads after 60 minutes in pH 5.6.

70

-~

60

~

50 '0 :g 40 :s CI 30 2

"

(I) 20 CI IV ... 10 ~ ~ ADS pH 7.4 ~VC pH 7.4 EXPL pH 7.4 o o 20 40 60 80 Time (minutes)

Figure 4.4: Dissolution results for SPE 0.5% (w/v) ADS, 0.25% (w/v) EXPL and 0.25% (w/v) VC beads after 60 minutes in pH 7.4.

4.3.2.1 Comparison of the dissolution data for SPE beads

A comparison of results for the SPE beads dissolution is given in figures 4.5 and figure

o ADS pH 5.6 .VC pH 5.6

151EXPL pH 5.6

IIICIS pH 5.6

Figure 4.5: Comparison of dissolutionresults from cm and SPE beads in pH 5.6.

70

.-C

60

J

50

i

40 :s en 30 2 "C Q) 20 en m 10

~

o

Il]ADS pH 7.4 .VC pH 7.4 51 EXPL pH 7.4 E] CIS pH 7.4

Figure 4.6: Comparison of dissolution results from cm and SPE beads in pH 7.4.

10 60

Time (minutes)

360

From the comparison of data in figure 4.5 it is clear that the excipients had no major effect on cm in a pH of 5.6. In pH 5.6 the release of INH compared as follow: CIB >

EXPL > ADS> /VC. The cm showed the best release of INH over a period of 6 hours.

This is the result of higher drug loading, as was shown in figure 3.35, of cm to that of

-71 4.6. 70 60

i

2; 50 ._ 40 "C en 30 2 "C

&

20 I! 10 « 0 10 60 360 Time (minutes)

ADS +EXPL pH 5.6 _ADS+VC pH 5.6 EXP L +VC pH 5.6 20 40 60 80 Time (minutes)

Figure 4.7: Average drug dissolved from MPE beads at pH 5.6.

ADS +EXPL pH 7.4 _ADS+VC pH 7.4 EXP L + VC pH 7.4 20 40 60 80 Tim e (m inutes)

Figure 4.8: Average drug dissolved from MPE beads at pH 7.4.

4.3.3.1 Comparison of the dissolution data for MPE beads

In figures 4.9 and 4.10 there is a comparison of all the MPE combinations that was examined in this experiment.

73 70 g!: 60 ..-15040

r

30

f

2010 0 0 60 50 ..-14030

:

20 '10 0 0

Figure 4.9: Comparison of the average drug released by MPE beads in a pH 5.6 media. a:lADS+EXPL pH 7.4 8ADS+VC pH 7.4 [J EXP L +VC pH 7.4

Figure 4.10: Comparison of the average drug released by MPE beads in a pH 7.4 media.

From the comparison above it is clear that the VC/EXPL formulation produced the best dissolution of all the MPE. This may be because there was a higher drug loading as was shown in figure 3.35 than all the other MPE beads. The combination of the acidic microenvironmentcreated by VC and the disintegrationproperties ofEXPL led to greater swelling of the beads which resulted in better dissolution of the beads.

---74 - ----70 "0' es. 60

150LL

.

I

ElADS+EXPLpH 5.6 ae

40l

I

8ADS+VC

f

30 pH 5.6 II5IEXPL+VC

f

20J pH 5.6 10 0 10 60 360 Time (minutes) 60 50

-I

40 30

:

20

I

10 0 10 60 360

4.4 Conclusion

Figure 4.11 shows the comparison of the results obtained from the dissolution studies. From the dissolution data it is clear that there is no major difference between the dissolution of any combination in an acidic or basic medium. This can be the result of the physical properties of isoniazid as given in section 2.2.1 which indicates that a 5% INH solution is stable in a pH range of 6.0 to 8.0. Because it was decided that the isoniazid concentration used in the formulations should be 4% as described in section 2.5.3.1, it indicates why there is no major difference between the dissolution in the pH 5.6 and 7.4 media. This indicates that all of these formulations can be used for the release of INH over a wide range of basic and acidic media. From figure 4.11 the dissolution of SPE and MPE formulationscan be arranged in the followingorder: VCIADS< VC < ADSIEXPL <

ADS < VCIEXPL < CIB < EXPL. The addition of pharmaceutical excipients to the cm

formulation showed no promising effect on the dissolution of INH, except for the formulationsthat contained EXPL. The formulationsthat contained EXPL and VC/EXPL were comparable with the cm in both pH media.

!! pH 5.6 m pH 7.4

Figure 4.11: Comparison of INH released at pH 5.6 and pH 7.4 after 6 hours.

75 70 60

I

50 40

g

30

J

20 10 0

p

_P/

/

0<0

/

Pharmaceutical excipients

Annexure B: Certificate for Analysis of Isoniazid

.

.

_.' .'<- -.~.,f,",-!r .Y .. . '0°' ~ 1. ~. :)a. t.. ,~ '-"001~ I " 1, . "'".r;" .. ." ;0,-1 ., ;. .._I ~ 84

ANNEXURE C 2: Dissolution data of CIB containing SPE

Table C 3: Amount of INH released from CIB containing SPE at pH 5.6 after 6 hours.

Figure C 3: INH release from CIB containing SPE at pH 5.6.

87

EXPL ADS VC

Time 0/0 Standard % Standard 0/0 Standard

(min) Release Deviation Release Deviation Release Deviation

0 0 0 0 0 0 0 2 34.58004 2.184179 39.0617 0.082642 35.18739 1.058332 5 43.7429 2.06776 44.4888 0.747397 40.7877 0.385409 10 50.14614 1.377167 50.22 1.108639 45.13373 0.272966 20 53.57337 1.746529 52.16223 0.316133 46.68145 0.755403 30 54.1167 1.103504 52.69775 0.503605 47.08778 0.290454 60 54.21309 0.44762 52.8315 0.357258 47.53405 0.478302 120 54.29134 0.502249 53.96267 0.111267 47.73004 0.444415 180 54.53655 0.768982 54.26012 0.2352 47.83585 0.252757 240 54.56154 0.964856 54.45599 0.20572 47.82131 0.583073 300 54.81833 0.528484 54.62944 0.176164 48.12147 0.523458 360 54.91032 0.509823 54.64502 0.362765 48.11224 0.430336

I-+-EXPL

_ADS

---.-VC -X-CIS I

70 l "0' c.;;: X X X X

i

uv l.XXX-X X

i

:c CI 30 2 'tI 20

f

10 C( 0 X 0 50 100 150 200 250 300 350 400 Time (minutes)

Table C 4: Amount ofINH released from cm containing SPE at pH 7.4 after 6 hours.

-a-EXPL ~ADS ~VC -o-CIB

Figure C 4: INH release from cm containingSPEatpH 7.4.

88

T I

EXPL ADS VC

Time 0/0 Standard 0/0 Standard 0/0 Standard

(min) Release Deviation Release Deviation Release Deviation

0 0 0 0 0 0 0 2 35.08936 1.592756 35.8763 2.980282 33.20135 2.131227 5 44.80507 1.256157 42.0721 2.381091 40.72268 0.993287 10 50.9475 1.197004 47.18422 1.713605 44.848 0.646225 20 53.96645 1.604619 50.14984 1.853688 47.24487 0.568025 30 55.14477 1.415867 50.91423 1.719296 47.84295 0.45556 60 56.18962 1.255883 51.26135 1.979117 48.23778 0.943161 120 56.78061 1.212526 51.39724 1.890305 48.3835 0.736744 180 57.02599 1.235848 51.88615 1.792969 48.8999 0.599042 240 57.67199 1.058401 52.1951 1.685348 48.81983 0.223711 300 57.97557 0.837186 53.13237 1.739215 49.2145 0.344259 360 58.49221 1.169383 52.92859 1.897914 49.47072 0.389155 70 60 e...

I

50 40

:

30

f

20 10 0 0 50 100 150 200 250 300 350 400 Time (minutes)

ANNEXURE C 3: Dissolution data of CIB containing MPE

Table C 5: Amount of INH released from CIB containing MPE at pH 5.6 after 6 hours.

-+-ADS+EXPL __EXPL+VC ~ADS+VC

Figure C 5: INH release from CIB containing MPE at pH 5.6.

89

Expl / ADS EXPL / VC ADS/VC

Time % Standard % Standard % Standard

(min) Refease Deviation Release Deviation Release Deviation

0 0 0 0 0 0 0 2 39.60548 5.336131 47.35049 2.952329 29.56811 0.646723 5 45.03301 6.298227 54.35223 5.062952 35.12249 1.037341 10 46.81333 4.686681 56.89548 6.408683 37.05729 0.810668 20 47.50983 3.836669 57.45327 6.514153 37.52368 0.76811 30 47.5447 3.378711 57.96802 6.099753 37.64037 0.815825 60 47.56369 3.277706 57.15977 6.237468 38.22021 0.731086 120 48.86366 3.952431 56.91722 6.299349 38.28488 0.668102 180 48.60524 3.541658 56.88169 6.346729 38.5228 0.634555 I 240 49.44479 3.829471 57.14685 6.512931 38.72008 0.4612 300 49.35551 3.356566 57.54139 6.657899 38.90195 0.76517 360 49.99899 3.536862 57.71002 6.454571 39.45407 1.333112 70 601

I

50 40 I

__

---m-:

30

f

201 10 OU 0 100 200 300 400 Time (m Inutes)

Table C 6: Amount of INH released from CIB containing MPE at pH 7.4 after 6 hours.

-+- ADS+EXPL __ EXPL+VC -~ ADS+VC

70

~

60 ~

!

50

i

40

f

' ~

:

30 . & 20 , 10-; C( I

0*

o

100 200 300 400 L Time (minutes)

Figure C 6: INH release from CIB containing MPE at pH 7.4.

90

Expl / ADS EXPL / VC ADS / VC

Time 0/0 Standard 0/0 Standard 0/0 Standard

(min) Release Deviation Release Deviation Release Deviation

0 0 0 0 0 0 0 2 35.9857 1.491217 43.43896 1.806822 36.27954 5.873564 5 42.55176 1.582267 48.79324 0.811841 39.00862 3.861797 10 45.8838 1.503714 51.47237 3.60124 40.33965 3.150594 20 47.222 1.483302 53.58909 2.828473 41.63327 3.759017 30 47.37794 1.491727 53.38234 3.273011 41.48988 2.427339 60 47.40679 1.210782 53.65518 1.637297 41.92838 2.984346 120 47.75882 1.415101 54.40632 2.309903 41.88323 3.275833 180 49.16172 1.361292 54.93015 1.9711 41.93406 2.649188 240 49.30774 2.00668 55.7724 1.89941 42.86908 1.430634 300 49.62537 1.997459 55.53453 1.987135 42.82644 1.619722 360 50.09105 1.913979 55.30848 1.282114 42.86243 1.735095