PIGESTIVE MULTIENZYME PREFORBULATIGN STABILITY SCREENING USING DIFFERENTIAL

SCANNING CALORIIllETRY

Hamed Hamed El-Shattawy

Department of Pharmaceutics, Faculty of Pharmacy Al-Azhar University, Nasr-City, Cairo, Egypt.

ABSTRACT

Pifferential scanning calorimetry was used for assessing the compatibility in the solid state of pepsin- diastase, pepsin-pancreatin, pancreatin-diastase and pepsin-diastase-pancreatin mixtures using uncoated and Eudragit coated enzymes. The enthalpy change (cal/g) of the resulting thermogram transitions indicated pepsin- diastase and pancreatin-diastase mixtures to be compati- ble, and pepsin-pancreatin and pepsin-diastase-pancreatin mixtures to be incompatible when uncoated enzymes were used while compatible when coated enzymes with Eudragit were used.

I NTRODUCT I ON El-Shattawy et al. previously used differential

scanning calorimetry (DSC) as a screening technique for assessing the compatibility of aspartame' ' 2, anhydrous ampicillin3, cephalexin4 and erythromycin5 with some of the direct compression excipients. The compatibilities of anhydrous ampicillin, ampicillin trihydrate and cephalexin with anhydrous dextrose and with aspartame were also investigated by the same authors . In 6-8

1435

Copyright 6 1983 by Marcel Dekker, Inc.

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1436 EL-SHATTAWY

this investigation, the author used r S C in preformulation stability studies involving pepsin, diastase and pancreatin.

studied extensively with regard to protein structures and biochemical functions9-'' . Pepsin solutions are reduced in proteolytic activity by agitation and storage,particul- arly at or above normal room temperatures12. The proteo- lytic activity of pepsin is also destroyed by pancreatic

enzymes in neutral solution . Enzyme stability in the solid state is complicated

and is readily affected by factors such as temperature,

described the application of the Weibull distribution function to solid-state enzyme inactivation in a study involving microbial lipases, microbial a-amylase, pancreatin and diastase.

Graf and Sakr" studied the effect of direct compre- ssion excipients on the characteristics of Stomach Extract tablets produced under standardized manufacturing condit- ions. problems involved in the manufacture of satisfactory pan- creatic enzymes oral solid dosage forms due to the very complex nature of the enzymes and their great liability to quickly loose activity during manufacture, on storage and in the gastro-intestinal tract before reaching their specific site of action.

effects of formulation factors on the stability and bio-

availability of pancreatic enzymes tablets manufactured

by the direct compression technique,

mixed digestive enzymes, no information is available in the pharmaceutical literature about the problem of enzyme-

enzyme interaction, in the solid state. In this study the author investigated the compatibility in the solid state of pepsin-diastase, pepsin-pancreatin, pancreatin- diastase and pepsin-diastase-pancreatin mixtures using

Enzyme inactivation in aqueous systems has been

12

humidity, and co-existing substances13 . Sugiura et al. 13

The same authors" reported that there are many

Graf et al. 15-17 studied the

Although many commercial preparations containing

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DIFFERENTIAL SCANNING CALORIMETRY 1437

uncoated and coated enzymes. This is achieved by compar- ing the PSC thermograms of the individual enzymes in the mixture and finally on the mixture itself. Although it cannot be’conclusively stated that an interaction incom- patibility will occur during storage at room temperature , PSC can distinguish between enzyme mixtures unlikely to cause a problem and those that may cause trouble and thereby a more rational approach to early formulation designs can be established.

18

EXPERILEIiTAL Mate r i a1 s

The following materials were used: pepsin,diastase (supplied by Arab Drug Company), pancreatin (supplied by Memphis Chemical Company), Eudragit E and Eudragit S (Rohm and Haas Co.,Inc.). All other chemicals were USP or analytical grade.

Jvlanufacture of Enzyme Granules

pepsin, 95% ethanol for diastase and 90% isopropanol for pancreatin. The mass was forced through an 800 p sieve and the granules were dried in a vacuum desiccator. The obtained irregular granules were rounded by placing in an ERWEKA coating pan (type LJG) f o r 15 minutes at constant speed. The granules were then passed through an 800 p

sieve and retained over a 630 p sieve using an ERWEKA screen analyzer (type VT). These retained granules were

collected f o r direct coating.

Coating of the Granules Eudragit E in chloroform, Eudragit E in 2:3 acetone-

isopropanol mixture and Eudragit S in 1:l acetone-isopro- panol mixture were used for the coating of pepsin,diastase and pancreatin, respectively.

The rounded granules(200g) were sprayed in an ERWEKA coating pan with a solution of 10 g Eudragit in 500 ml of the respective organic solvent, by means of an air-pressure

The enzyme powder was massed with chloroform for

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1438 EL-SHATTAWY

spray gun. app l i ed i n 10 m l p o r t i o n s over a pe r iod of 2-3 hours .

spray ing was stopped a t occas iona l i n t e r v a l s , and t h e con ten t s of t h e pan were d r i e d wi th co ld a i r . coated g ranu le s were d r i e d overn ight i n a vacuum d e s i c c a t o r .

P i f f e r e n t i a l Scanninf?: Calor imetry Samples (3 -9 mg) were weighed and encapsula tea i n

f la t -bot tomed aluminum pans wi th crimped-on l i d s . The samples were hea ted i n a n atmosphere o f n i t r o g e n and t h e r - mograms were obta ined on a Perkin-Elmer TSC-1B D i f f e r e n t i a l Scanning Calor imeter . Thermograms were obta ined by h e a t i n g

a t a cons t an t h e a t i n g r a t e of 10°C p e r minute, a cons t an t range s e t t i n g o f 8 mcal p e r second and recorded a t a cons t an t c h a r t speed of one inch p e r minute. The ind iv id - u a l enzymes and t h e mixtures the reo f were heated over t h e temperature range 30 t o 3 O O O C .

The t o t a l amount of t h e polymer s o l u t i o n was The

F i n a l l y t h e

The a r e a under t h e d i f f e r e n t i a l scanning c a l o r i m e t r i c

hea t ing curve was measured us ing a K 6; E p lan ime te r and t h e hea t of t r a n s i t i o n w a s t hen c a l c u l a t e d as desc r ibed

1 prev ious ly . A t least two r e p l i c a t e s made f o r each DSC thermogram.

RESULTS AND LISCUSSION

The DSC thermogram o f peps in (Trace 1 of F igure 1) showed two endothermic peaks, t h e first one wi th a t r a n - s i t i o n temperature range from 134-147OC and wi th a maximum peak of t r a n s i t i o n a t 14OoC, t h e second one w i t h a t r a n - s i t i o n temperature range from 188-222OC and wi th a maximum peak o f t r a n s i t i o n a t 208OC. The DSC thermogram of coated peps in (Trace 2 of F igure 1) showed t h e same two enuo- thermic peaks wi th a s l i g h t s h i f t t o h ighe r tempera tures as regard t h e t r a n s i t i o n temperature range (134-1500 and 190-228OC) and t h e maximum peak of t r a n s i t i o n (143°C and 21OOC) of t h e two t r a n s i t i o n s , r e s p e c t i v e l y .

Uncoated and coated d i a s t a s e e x h i b i t no t r a n s i t i o n when scanned over t h e temperature range of 30 t o 300°'C.

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DIFFERENTIAL SCANNING CALORIMETRY 1439

100 170 240

Temperature (OC)

Figure 1: Thermo rams o f the i nves t i ga ted enzymes and mixtures thereof . Key: 9 1) uncoated pepsin, 3 mg; ( 2 ) coated pepsln, 3 mg; (3) uncoated 1:l pepsin-diastase mixture, 6 mg; (4 ) coated 1:l pepsin-diastase mixture, 6 mg; ( 5 ) uncoated pancreat in, 3 mg; (6 ) coated pancreat in, 3 mg; ( 7 ) uncoated 1:l pepsin-pancreat in mixture, 3 mg; (8) coated 1:l pepsin-pancreat in mixture, 3 mg; (9) uncoated 1:l pancreat in-diastase mixture, 6 mg; (10) coated 1:l pancreat in-diastase mixture, 6 mg; (11) uncoated 1:l: l pepsin- diastase-pancreat in mixture, 9 mg; (12) coated 1: l : l pepsin-diastase- pancreat in mixture, 9 mg.

Therefore , rSC thermograms o f peps in -d ia s t a se mixtures w i l l r e f l e c t t h e c h a r a c t e r i s t i c f e a t u r e s of t h e thermo- grams o f peps in i f no i n t e r a c t i o n occurred. T h i s i s indeed t h e case as seen i n Traces 3 and 4 o f F igure 1. Some change i n peak shape and height- to-width r a t i o w a s expected because of p o s s i b l e d i f f e r e n c e s i n t h e mixture

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1440 EL- SHATTAWY

sample geometry19. peps in -d ia s t a se , uncoated and coated enzyme mixtures was found t o be c l o s e t o t h e p red ic t ed va lues c a l c u l a t e d from t h e exac t percentage c o n t r i b u t i o n of peps in t o t h e t o t a l en tha lpy change of t h e mixtures (Table 1) i n d i c a t - ing no i n t e r a c t i o n between peps in and d i a s t a s e i n the s o l i d s t a t e under t h e experimental c o n d i t i o n s .

The en tha lpy change ( c a l / g ) of

The rSC thermogram of p a n c r e a t i n (Trace 5 of F igure 1 ) showed two endothermic peaks, t h e f i rs t one wi th a t r a n - s i t i o n temperature range from 12E-145"C and w i t h a maximum peak of t r a n s i t i o n a t 1380C, t h e second one wi th a t r a n - s i t i o n temperature range from 191-213OC and wi th a maximum peak of t r a n s i t i o n a t 2 O 7 O C . The CSC thermogram of coated p a n c r e a t i n (Trace 6 of F igure 1 ) showed t h e same two endothermic peaks wi th a s l i g h t s h i f t t o h ighe r tempera- t u r e s as regard t h e t r a n s i t i o n temperature range (126-147O and 191-219OC) and t h e maximum peak of t r a n s i t i o n (140°C and 208OC) of t h e two t r a n s i t i o n s , r e s p e c t i v e l y .

The DSC thermogram of peps in-pancrea t in uncoated enzyme mixture (Trace 7 o f F igure 1 ) showed t h r e e enclo- thermic peaks. The first one wi th a t r a n s i t i o n temperature range from 129-149OC and wi th a maximum peak of t r a n s i t i o n a t 14OOC corresponding t o t h e f i r s t peaks o f peps in and panc rea t in . The second peak with a t r a n s i t i o n tempera- t u r e range from l50-166OC and wi th a maximum peak of t r a n - s i t i o n a t 160OC is a new t r a n s i t i o n . The t h i r d peak wi th a t r a n s i t i o n temperature range from 180-213OC and wi th a maximum peak of t r a n s i t i o n a t 2OOOC corresponding t o t h e second peaks of peps in and panc rea t in . The en tha lpy change (cal/g) of t h e f irst peak was found t o be 62.23% t h e p red ic t ed v a l u e , c a l c u l a t e d from t h e exac t percentage c o n t r i b u t i o n of peps in and p a n c r e a t i n t o t h e t o t a l en tha lpy change of t h e mixture , while t h e en tha lpy change of t h e t r a n s i t i o n corresponding t o t h e second peaks of peps in and p a n c r e a t i n was found t o be 49.E7% t h e p red ic t ed va lue (Table 1) i n d i c a t i n g i n t e r a c t i o n between peps in and pan- c r e a t i n i n t h e s o l i d s t a t e under t h e exper imenta l condi-

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i3

.F

8 F 1 4.90

5.25

93.33

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5.70

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108.57

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0

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8.09

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62.23

10.18

20.41

49.87

I4 13.01

13.00

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20.09

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98.43

3

7.41

7.75

95.61

9.46

10.30

91.84

I 7.73

7.75

99.74

10.26

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99.61

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1 I

8.76

I 8.66

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13.61

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* Th

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and 20.22

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and

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and 20.60

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1442 EL- S HATTAWY

12 t i o n s . Th i s f i n d i n g i s i n agreement wi th l i t e r a t u r e t h a t t h e p r o t e o l y t i c a c t i v i t y of peps in i s des t royed by p a n c r e a t i c enzymes i n n e u t r a l s o l u t i o n .

mixture (Trace 8 of Figure 1 ) showed a l s o t h r e e endothermic peaks as i n t h e case of t h e uncoated enzyme mix tu re ,bu t t h e t r a n s i t i o n temperature range and t h e maximum peak of t r a n s i t i o n s h i f t e d t o h ighe r temperature and t h e two main peaks corresponding t o peps in and p a n c r e a t i n a r e more d i s t i n c t . The en tha lpy change ( c a l / g ) o f t h e s e two peaks was found t o be c l o s e t o t h e p red ic t ed va lues (Table 1 ) i n d i c a t i n g no i n t e r a c t i o n between peps in coated wi th Eudragi t E , and p a n c r e a t i n coated wi th Eudragi t S i n t h e s o l i d s t a t e under t h e experimental cond i t ions .

p a n c r e a t i n - d i a s t a s e , uncoated and coated enzyme mix tu res , r e s p e c t i v e l y . The en tha lpy change ( c a l / g ) of panc rea t in - d i a s t a s e , uncoated and coated enzyme mixtures w a s found t o be c l o s e t o t h e p red ic t ed va lues c a l c u l a t e d from t h e exac t percentage c o n t r i b u t i o n o f p a n c r e a t i n t o t h e t o t a l en tha lpy change of t h e mixture (Table 1) i n d i c a t i n g no i n t e r a c t i o n between p a n c r e a t i n and d i a s t a s e i n t h e s o l i d s t a t e under t h e experimental cond i t ions .

Traces 11 and 12 of F igure 1 are t h e thermograms of pepsin-diastase-pancreatin, uncoated and coated enzyme mixtures , r e s p e c t i v e l y . The en tha lpy change ( c a l / g ) of t h e two main peaks of t h e uncoated enzyme mixture was found t o be 85.68 and 63.19%, r e s p e c t i v e l y , t h e p r e d i c t e d va lues while t hose of t h e coated enzyme mixture was found t o be 101.15 and loo%, r e s p e c t i v e l y (Table 1 ) r e f l e c t i n g t h e s t a b i l i t y o f t h e i n v e s t i g a t e d coated enzymes mixture .

The PSC thermogram of peps in-pancrea t in coated enzyme

Traces 9 and 1 0 of F igure 1 a r e t h e thermograms o f

ACKNOWLEDGEMENTS

The au tho r acknowledges t h e Arab Crug Company and Memphis Chemical Company, Ca i ro , Egypt. Thanks t o t h e IPPH Department, School o f Pharmacy, Purdue Un ive r s i ty , West La faye t t e , I N 47907, U.S.A. Thanks and g r a t e f u l

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DIFFERENTIAL SCANNING CALORIMETRY 1443

appreciation are extended to Cr. Dane 0. Kildsig, Dr. Garnet E. Peck and Judi A. Yost f o r continuous help.

REFERENCES

1. H.H. El-Shattawy, G.E. Peck and D.O. Kildsig, Drug

2 . H.H. El-Shattawy, D . O . Kildsig and G.E. Peck, ibid,

3. H.H. El-Shattawy, ibid, in press. 4. H.H. El-Shattawy, D . O . Kildsig and G.E. Peck, ibid,

5. H.H. El-Shattawv, C . O . Kildsig and G.E. Peck, ibid,

6. H.H. El-Shattawy, D.0. Kildsig and G.E. Peck, ibid,

7. H.H. El-Shattawy, D . O . Kildsig and G.E. Peck, ibid,

8. H.H. El-Shattawy, D.O. Kildsig and G.E. Peck, ibid,

9. S. Nakayama and Y. Kano, J. Biochem. (Tokyo) &, 25

Dev. and Industrial Pharm., u, 605 (1981) .

in press.

in press.

in press.

in press.

in press.

in press.

(1957). 10. C. Tanford, J. Am. Chem. SOC., 86, 2.050 (1964) . 11. C . Tanford, K.C. Anue and A. Ikai, J. Mol. Biol.,

12. A . Wade and J.E.F. Reynolds, eds., "Extra Pharmaco- 2.2, 185 (1973) .

peia-Martindale ", 27th ed., The Pharmaceutical Press, London, 1977, p. 582.

J. Pharm. Sci., 68, 1381 (1979) . 13 . M. Sugiura, M. Kurobe, S. Tamura and S. Ikeda,

14. E. Graf and A. Sakr, Pharm. Ind., 4.0, 165 (1978) , 15. E. Graf and A. Sakr, ibid, 4 l , 66 (1979) . 16. E. Graf, A. Sakr and A. Nada, ibid, 42, 1031 (1980). 17 . E. Graf, A. Sakr and A. Nada, ibid, Q, 282 (1981) . 18. K.C. Lee and J.A. Hersey, J. Pharm. Pharmac., 29,

515 (1977). 19. E.S. Watson, M.J. O'Neill, J. Justin and N. Brenner,

Anal. Chem., s, 1233 (1964) .

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