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The adsorption of selected antibiotics by Kaolin Aswakun, Penpan 1975

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THE ADSORPTION OF SELECTED ANTIBIOTICS BY KAOLIN by PENPAN ASWAKUN B. PHARM., MAHIDOL UNIVERSITY BANGKOK, THAILAND, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Di v i s i o n of Pharmaceutical Chemistry of the Faculty of Pharmaceutical Sciences We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER, 1975 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f or reference and study. I f u r t h e r agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s re p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date t4 @&foL>r /f7r ABSTRACT In t h i s i n v e s t i g a t i o n , the i n v i t r o a d s o r p t i o n of t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin s u l f a t e , l i n c o m y c i n h y d r o c h l o r i d e , chloramphenicol, and a m p i c i l l i n t r i h y d r a t e by k a o l i n were s t u d i e d . A d s o r p t i o n s t u d i e s were c a r r i e d out a t 37.0°C. i n water and i n pH 1.2, 3.0, and 5.0 s o l u t i o n s . The a d s o r p t i o n isotherms f o r t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin s u l f a t e , and l i n c o m y c i n h y d r o c h l o r i d e i n aqueous s o l u t i o n were of the Langmuir type, w i t h the f o l l o w i n g e x c e p t i o n s . (a) A E r e u n d l i c h a d s o r p t i o n isotherm was obtained f o r t e t r a c y c l i n e h y d r o c h l o r i d e i n pH 1.2 s o l u t i o n s . (b) An S type a d s o r p t i o n isotherm, (according t o the c l a s s i f i c a t i o n o f isotherms by G i l e s and h i s co-workers (1960) was obtained f o r l i n c o m y c i n h y d r o c h l o r i d e i n pH 1.2 s o l u t i o n s . In g e n e r a l , a d s o r p t i o n v a r i e d w i t h the pH of the s o l u t i o n s . Chloramphenicol and a m p i c i l l i n t r i h y d r a t e are not adsorbed by k a o l i n . D e s o r p t i o n s t u d i e d f o r the adsorbed a n t i b i o t i c s were c a r r i e d out i n . water. The r e s u l t s i n d i c a t e d t h a t the a d s o r p t i o n of t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n i s a r e v e r s i b l e p r o c e s s , the a d s o r p t i o n o f neomycin s u l f a t e by k a o l i n i s an i r r e v e r s i b l e p r o c e s s , and the a d s o r p t i o n of l i n c o m y c i n h y d r o c h l o r i d e i s a p a r t i a l l y r e v e r s i b l e p r o c e s s . On the b a s i s of data o b t a i n e d , i t was found t h a t 47.21% of 250 mg. t e t r a c y c l i n e h y d r o c h l o r i d e dose and, 57.98% neomycin base (220.5 mg.) would be adsorbed by s i x gm. of k a o l i n . On the b a s i s of a 500 mg. dose of 1incornycin h y d r o c h l o r i d e / 11.44% would be.expected t o be adsorbed b y . s i x gm. o f k a o l i n . T h i s v a l u e i n c r e a s e s t o 33.94% i f 17.76 gm. o f k a o l i n are' a d m i n i s t e r e d to the p a t i e n t . I f the above two v a l u e s are compared to the i n v i v o data r e p o r t e d by Wagner (1966), i t becomes e v i d e n t t h a t the decrease i n plasma l e v e l s (by about 90%) i s much g r e a t e r than t h a t which would be p r e d i c t e d on the b a s i s of i n v i t r o a d s o r p t i o n s t u d i e s . However, Wagner (1966) s t u d i e d a commercial p r e p a r a t i o n (Kaopectate) and it-may be t h a t other i n g r e d i e n t s c o n t r i b u t e to the dramatic decrease i n b l o o d l e v e l s . T h e r e f o r e , i n v i t r o data cannot b e . d i s r e g a r d e d but, a t the same time > should not be e x t r a p o l a t e d t o i n v i v o drug e f f e c t s . The f i n a l p r oof must come from c a r e f u l l y c o n t r o l s t u d i e s i n man. T h i s a b s t r a c t r e p r e s e n t s the t r u e contents of the t h e s i s submitted. S u p e r v i s o r - iv -TABLE OF CONTENTS Page I . INTRODUCTIONS 1 I I . LITERATURE . SURVEY ^ ' 4 1. K a o l i n . . . . . . ^ . . . . . . . i . . . i . . . . . . . . 4 2. T h e o r y . . . . . . . . . . i ^ . . . ^ . . . . . . . i . . . . . . . • 10 3. A d s o r p t i o n o f D r u g s b y K a o l i n ............... 15 (a) I n . V i t r o . A d s o r p t i o n . S t u d i e s ............ . 1 5 (b) I n V i v o A d s o r p t i o n S t u d i e s ............. 21 4. The T e s t D r u g s . . . . . . ^ . . . . . . . . . . . . . . . i . . . i . 23 (a) T e t r a c y c l i n e H y d r o c h l o r i d e (TC-HC1) .... 23 (b) N e o m y c i n S u l f a t e (NC-S0 4) 27 (c) L i n c o m y c i n H y d r o c h l o r i d e (LC-HC1) ...... 31 (d) C h l o r a m p h e n i c o l (CM) 33 (e) A m p i c i l l i n T r i h y d r a t e (AM) 36 I I I . EXPERIMENTAL 39 1. A p p a r a t u s 39 2. C h e m i c a l s and R e a g e n t s ...................... 41 3. P r e p a r a t i o n o f The S o l u t i o n s Use i n The E x p e r i m e n t 42 4. A n a l y s i s a n d S t a b i l i t y C h a r a c t e r i s t i c s o f The T e s t D r u g s . . . . . . . i....... i . . . ^ . . . . . . . 43 5. A d s o r p t i o n C h a r a c t e r i s t i c s o f The T e s t D r u g s 59 6. D e s o r p t i o n C h a r a c t e r i s t i c s o f The T e s t D r u g s 61 - V -Page IV. RESULTS AND DISCUSSION 64 -1. P r e l i m i n a r y E v a l u a t i o n o f K a o l i n ............ 64 2. "determination : of E q u i l i b r i u m Times 64 (a) E q u i l i b r i u m Time f o r TC-HC1 64 (b) E q u i l i b r i u m Time f o r NC-S04- 65 (c) E q u i l i b r i u m T i m e : f o r LC-HC1 ............ • 65 3. The A d s o r p t i o n and D e s o r p t i o n C h a r a c t e r i s t i c s o f The T e s t Drugs 65 (a) T e t r a c y c l i n e H y d r o c h l o r i d e ............. 65 (li)j A d s o r p t i o n Isotherm, i n Water a t 37.0°C. .. v ; .- ... . 65 (2) A d s o r p t i o n Isotherms i n pH 1.2, 3.0> and 5.0 S o l u t i o n s ................. 71 (3) D e s o r p t i o n C h a r a c t e r i s t i c s o f TC-HC1 79 (4) A d s o r p t i o n I n t e r a c t i o n s between T h e r a p e u t i c Doses of TC-HC1 and K a o l i n 81' (b) Neomycin S u l f a t e ....................... 82 (1) A d s o r p t i o n Isotherm i n Water at 37.0°C. 82 (2) Adsorption.Isotherms i n pH 1.2, 3.0, and 5.0 S o l u t i o n s ................. 86 C3) Desorption. . C h a r a c t e r i s t i c s of NC-S0 4. 90 (4) A d s o r p t i o n I n t e r a c t i o n s between T h e r a p e u t i c Doses o f NC^S0 4- and K a o l i n ^ 90 (c) Lincomycin H y d r o c h l o r i d e 93 (1) . A d s o r p t i o n Isotherm i n Water a t 37.0°C. .. ; ; . 93. (2) A d s o r p t i o n Isotherms i n pH 1.2, 3.0, and 5.0 S o l u t i o n s ................. 93 (3) D e s o r p t i o n C h a r a c t e r i s t i c s o f LC-HC1 102 (4) A d s o r p t i o n I n t e r a c t i o n s between, T h e r a p e u t i c Doses of LC-HC1 and K a o l i n i . .• ....... i. . 104 (d) Chloramphenicol . 105 (e) A m p i c i l l i n T r i h y d r a t e .................. 105 V. SUMMARY AND' CONCLUSIONS 108 VI. REFERENCES ... i ... i . . . . . . . . . . . i . . . i i . . . i . . APPENDIX i ... i ... ^  .. . i ... i 119 - v i i LIST OF TABLES Table P age 1 The E f f e c t o f Time on The Amount o f T e t r a -c y c l i n e H y d r o c h l o r i d e Adsorbed per Gm. o f K a o l i n 66 2 The E f f e c t o f Time on The Amount of Neomycin S u l f a t e Adsorbed per Gm. of K a o l i n ... w ..... . 66 3 The E f f e c t o f Time on The Amount of Lincomycin H y d r o c h l o r i d e Adsorbed per Gm. o f K a o l i n • •• • • 67 4 A d s o r p t i o n Data f o r T e t r a c y c l i n e H y d r o c h l o r i d e by K a o l i n . . . . ; ;.. i ....... i i .. i i .. . i .. 69 5 A d s o r p t i o n of T e t r a c y c l i n e H y d r o c h l o r i d e by K a o l i n a t 37°C. i ... i ... i ....... i. . 73 6 De s o r p t i o n of T e t r a c y c l i n e H y d r o c h l o r i d e from K a o l i n ^ ^ . 80 7 A d s o r p t i o n Data f o r Neomycin S u l f a t e by K a o l i n ...a. : ... : ... : ....... i i . . 83 8 A d s o r p t i o n of Neomycin S u l f a t e by K a o l i n a t 37°C ..... i w ;.. 89 9 D e s o r p t i o n of Neomycin S u l f a t e from K a o l i n .. 91 10 A d s o r p t i o n Data f o r Lincomycin H y d r o c h l o r i d e by K a o l i n ... i ... i ^  ...... i i ... i ...... . 94 11 . A d s o r p t i o n of Lincomycin H y d r o c h l o r i d e by K a o l i n a t 37°C 101 12 De s o r p t i o n o f Lincomycin H y d r o c h l o r i d e from K a o l i n . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . . ; . 103 - v i i i -LIST OF FIGURES F i g u r e Page 1 Diagrammatic s k e t c h of the s t r u c t u r e of the k a o l i n i t e l a y e r , a f t e r Grunner (1932) 5 2 System of Isotherm C l a s s i f i c a t i o n ........... 12 3 T e t r a c y c l i n e Degradation 25 . 4 Chemical s t r u c t u r e of neomycin§C ............ 28 5 A water bath w i t h a tumbler used t o determine a d s o r p t i o n of drug by k a o l i n ................ 40 6 S p e c t r a l C h a r a c t e r i s t i c s of T e t r a c y c l i n e H y d r o c h l o r i d e 45 7 S p e c t r a l C h a r a c t e r i s t i c s o f Neomycin S u l f a t e •-• - 48 8 S p e c t r a l C h a r a c t e r i s t i c s of Lincomycin H y d r o c h l o r i d e ............................... 52. 9 S p e c t r a l C h a r a c t e r i s t i c s o f Chloramphenicol 55 10 S p e c t r a l C h a r a c t e r i s t i c s of A m p i ^ c i l l i n T r i h y d r a t e i.. i ... i i i 57 11 . A d s o r p t i o n i s o t h e r m a t 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by kaolin,suspended i n water .. 70 12 Langmuir is o t h e r m a t 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n water .. 72 13 A d s o r p t i o n isotherm a t 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n pH 1.2, 3.0, and 5.0 s o l u t i o n s .......... ^  .......... . 75 14 Langmuir is o t h e r m at 37°C. f o r t e t r a c y c l i n e , h y d r o c h l o r i d e by k a o l i n suspended i n . pH 3.0 and pH 5.0 s o l u t i o n s 76 15 F r e u n d l i c h ; i s o t h e r m a t 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n pH 1.2 s o l u t i o n i . . . . . . . . . . . i ; 77 16 A d s o r p t i o n i s o t h e r m a t 37°C. f o r neomycin s u l f a t e by k a o l i n suspended i n water ... 84 - ix -F i g u r e : Page 17 Langmuir is o t h e r m a t 37°C. f o r neomycin s u l f a t e by k a o l i n suspended i n water . ; . 85 18 A d s o r p t i o n isotherm a t 37°C. f o r neomycin s u l f a t e by k a o l i n suspended i n pH 1.2, 3.0,. and 5.0 s o l u t i o n s ........................... 87 19 Langmuir isotherm a t 37°C. f o r neomycin s u l f a t e by kaolin.suspended i n pH 1.2, 3.0, and 5.0 s o l u t i o n s 88 20. A d s o r p t i o n . i s o t h e r m a t 37°C. f o r l i n c o m y c i n h y d r o c h l o r i d e by k a o l i n suspended i n water .. 95 21 Langmuir is o t h e r m a t 37°C. f o r l i n c o m y c i n . h y d r o c h l o r i d e by k a o l i n suspended i n water .. 96 22 A d s o r p t i o n isotherm at 37°C. f o r l i n c o m y c i n h y d r o c h l o r i d e by. k a o l i n suspended i n pH 1.2, 3.0, and 5.0 s o l u t i o n s 97 23 Langmuir isotherm, a t 37°C. f o r l i n c o m y c i n h y d r o c h l o r i d e by k a o l i n suspended i n pH 3.0 s o l u t i o n . . . . i i . . i . . . i . . . i . . . i . . . i . . . i . . . . . . . 98 24 Langmuir is o t h e r m a t 37°C.•for l i n c o m y c i n h y d r o c h l o r i d e by k a o l i n suspended i n pH 5.0 s o l u t i o n .................................... 99 ACKNOWLEDGEMENTS I wish t o express my s i n c e r e g r a t i t u d e and a p p r e c i a t i o n t o Dr. M. Pernarowski f o r h i s encouragement and guidance d u r i n g my s t u d i e s . Dr. T.H. Brown f o r h i s suggestions and c r i t i c i s m s . Dean B.E. ,R-igdel committee member. Yuvadee Khaodhiar f o r her a s s i s t a n c e i n t y p i n g t h i s manuscript. I. INTRODUCTION K a o l i n , a n a t i v e hydrated aluminum s i l i c a t e w i t h an approximate e m p i r i c a l formula of A^O^ . 2 S i 0 2 . 2H 20, i s d e s c r i b e d i n the N a t i o n a l Formulary as a s o f t , white or y e l l o w i s h - w h i t e powder which i s f r e e d from g r i t t y p a r t i c l e s by e l u t r i a t i o n . Because o f i t s a d s o r p t i v e p r o p e r t i e s , i t has been used, u s u a l l y i n combination w i t h p e c t i n , f o r the treatment o f d i a r r h e a and d y s e n t r y . I t i s claimed t h a t k a o l i n adsorbs some, but not a l l , b a c t e r i a , t o x i n s , and v i r u s e s and p r o v i d e s a p r o t e c t i v e c o a t i n g f o r the i n t e s t i n a l mucosa. Although, i n most i n s t a n c e s , i t i s used alone, antispasmodics, a n t i c h o l i n e r g i c s or a n t i b i o t i c s may be a d m i n i s t e r e d to the p a t i e n t c o n c u r r e n t l y or i n c o r p o r a t e d i n t o the p harmaceutical dosage form by the manufacturer. Since i t s i n v i v o e f f e c t i s by way of a d s o r p t i o n , i t i s p o s s i b l e t h a t i t can adsorb drugs of the type d e s c r i b e d above and, thus, decrease t h e i r i n v i v o e f f e c t . In v i t r o s t u d i e s have shown t h a t k a o l i n can adsorb such u n r e l a t e d substances as, f o r example, s t r y c h n i n e s u l f a t e q u i n i n e s u l f a t e , and b e n z o i c a c i d . Such s t u d i e s have shown the importance of a d s o r p t i o n and d e s o r p t i o n of drugs by k a o l i n but the r e s e a r c h e r s i n v o l v e d i n these i n v e s t i g a t i o n s have only s p e c u l a t e d on p o s s i b l e i n v i v o e f f e c t s . However, Wagner (1966) d i d show t h a t l i n c o m y c i n h y d r o c h l o r i d e b l o o d - 2 -l e v e l s decreased i f Kaopectate, the K a o l i n Mixture w i t h P e c t i n . N.F. XIII manufactured by the Upjohn Company, i s ad m i n i s t e r e d w i t h the a n t i b i o t i c . On the b a s i s o f Wagner's r e s u l t s , t h i s i s now r e p o r t e d i n the l i t e r a t u r e as a drug i n t e r a c t i o n . Lincomycin h y d r o c h l o r i d e i s not the drug o f c h o i c e i n the treatment o f dyse n t r y . F u r t h e r , Kaopectate i s ad m i n i s t e r e d because the drug i t s e l f may cause d i a r r h e a . However, Neomycin s u l f a t e has been i n c o r p o r a t e d i n t o a commercial p r e p a r a t i o n ? ( f o r example, Kaomycin, Upjohn Company) and, ac c o r d i n g to Goodman and Gilman (19 70), chloramphenicol and a m p i c i l l i n are the drugs of choice i f severe symptoms are encountered and an a n t i b i o t i c i s i n d i c a t e d (as i n the treatment o f s a l m o n e l l a i n f e c t i o n s ) . However, there i s no i n v i t r o or i n v i v o evidence t o j u s t i f y the co n c u r r e n t a d m i n i s t r a t i o n or i n c o r p o r a t i o n o f such drugs i n t o k a o l i n c o n t a i n i n g p r e p a r a t i o n s . The purpose of t h i s study i s , t h e r e f o r e , t o i n v e s t i g a t e the i n v i t r o a d s o r p t i o n o f t e t r a c y c l i n e h y d r o c h l o r i d e , l i n c o m y c i n h y d r o c h l o r i d e , c h l o r m a p h e n i c o l , a m p i c i l l i n t r i h y d r a t e , and neomycin s u l f a t e by k a o l i n . Since the pH of the g a s t r o i n t e s t i n a l t r a c t can vary from approximately one to seven, a d s o r p t i o n s t u d i e s were c a r r i e d out a t f o u r pH values i n t h i s range. F u r t h e r , a drug may be adsorbed but, i f i t i s desorbed r a p i d l y , i t s f u l l i n v i v o e f f e c t should not be a l t e r e d . T h e r e f o r e , a f t e r a d s o r p t i o n s t u d i e s - 3 -were completed, d e s o r p t i o n s t u d i e s were i n i t i a t e d , where necessary, i n order t o determine the amount of drug r e l e a s e d from the k a o l i n w i t h r e s p e c t to time. Although the r e s u l t s h e r e i n cannot be d i r e c t l y e x t r a p o l a t e d t o the i n v i v o c o n d i t i o n , they do i n d i c a t e the p o s s i b i l i t y o f an i n t e r a c t i o n between k a o l i n and t e t r a c y c l i n e h y d r o c h l o r i d e , l i n c o m y c i n h y d r o c h l o r i d e and neomycin sulfate.. The f i n a l p r o o f o f such an i n t e r a c t i o n must come from c a r e f u l l y c o n t r o l l e d s t u d i e s i n man. - 4 -I I . LITERATURE SURVEY 1. K a o l i n K a o l i n i s a n a t i v e hydrated aluminum s i l i c a t e , powdered and f r e e d from g r i t t y p a r t i c l e s by e l u t r i a t i o n . I t occurs as a s o f t , white or y e l l o w i s h - w h i t e powder, o r as lumps. I t has a c l a y - l i k e or earthy t a s t e and, when moistened w i t h water, assumes a darker c o l o r and develops a marked c l a y -l i k e odor. I t i s i n s o l u b l e i n water, i n c o l d d i l u t e a c i d s , and i n s o l u t i o n s o f the a l k a l i h ydroxides. The name " k a o l i n " i s d e r i v e d from the Chinese word " k a u l i n g " , meaning "high r i d g e " , the name o f a h i l l near Jauchau Fu, China, where, c e n t u r i e s ago, the m a t e r i a l was f i r s t o b t a i n e d . Ross and Kerr (19 31) c l a s s i f i e d the k a o l i n group o f m i n e r a l s i n t o three d i s t i n c t s p e c i e s — k a o l i n i t e , d i c k i t e and n a c r i t e . However, the name of the o f f i c i a l c l a y , k a o l i n , i s g e n e r i c r a t h e r than s p e c i f i c and i s used t o d e s c r i b e a mixture o f a t l e a s t f o u r separate m i n e r a l s — k a o l i n i t e , n a c r i t e , d i c k i t e and anauxite. The f i r s t proposed s t r u c t u r e of k a o l i n i t e (the c h i e f s c o n s t i t u e n t o f . k a o l i n ) was t h a t p u b l i s h e d by P a u l i n g (19 30) . A r e v i s e d s t r u c t u r e was proposed by Gruner (19 32) and, l a t e r , by B r i n d l e y and h i s c o l l e a g u e s (19 46; 1951). K a o l i n i t e c o n s i s t s o f a s i n g l e s i l i c a t e t r a h e d r a l sheet and a s i n g l e alumina o c t a h e d r a l sheet combined i n t o one u n i t i n such a way t h a t the t i p s of the s i l i c a . t e t r a h e d r o n and one of the - 5 -l a y e r s of the o c t a h e d r a l sheet form a common l a y e r (Figure 1 ) . A l l the t i p s of the s i l i c a t etrahedrons p o i n t i n the same d i r e c t i o n and toward the" c e n t e r o f the u n i t made up of s i l i c a and o c t a h e d r a l sheets. The sheets o f t e t r a h e d r a l and octahe-d r a l u n i t s are s i m i l a r i n t h e i r a and b dimensions and, con-se q u e n t l y , composite o c t a h e d r a l - t e t r a h e d r a l l a y e r s are r e a d i l y formed. F i g u r e 1. Diagrammatic sketch o f the s t r u c t u r e o f the k a o l i n i t e l a y e r , a f t e r Gruner (1932). - 6 -In the l a y e r common t o the o c t a h e d r a l and t e t r a h e d r a l groups, tw o - t h i r d s of the atoms are shared by s i l i c o n and aluminum. However, only two-thirds of the p o s s i b l e p o s i t i o n s f o r aluminum i n the o c t a h e d r a l sheet are f i l l e d and, a t the same time, there are three p o s s i b l e planes of r e g u l a r p o p u l a t i o n o f the t e t r a h e d r a l l a y e r w i t h t h i s p a r t i c u l a r atom. The aluminum atoms are co n s i d e r e d t o be so p l a c e d t h a t two aluminums are separated by an OH above and below, thus producing a hexagonal d i s t r i b u t i o n i s a s i n g l e plane i n the cente r o f the o c t a h e d r a l sheet. The OH groups are p l a c e d so t h a t each OH i s d i r e c t l y below the p e r f o r a t i o n of the hexagonal net of oxygens i n the t e t r a h e d r a l sheet. The charge d i s t r i b u t i o n i n the l a y e r i s as f o l l o w s : 6 0"~ 12-4 S i + + 16+ 4 0 + 2 (OH) 10- (Layer common to t e t r a h e d r a l and o c t a h e d r a l sheets) 4 A l 3 + 12+ 6 (0H)~ 6-The charges w i t h i n the s t r u c t u r a l u n i t are balanced. The e m p i r i c a l formula i s ( A l ^ S i ^ O ^ (OH) g) and the t h e o r e t i c a l composition (expressed as oxides) i s 46.45% Si02; 39.50% A ^ O and 13.96% H 20. The m i n e r a l s of the k a o l i n i t e group c o n s i s t of sheet u n i t s o f the type j u s t d e s c r i b e d continuous i n the a and b - 7 -d i r e c t i o n s and stacked one above the other i n the c d i r e c t i o n . Because of the s u p e r p o s i t i o n of 0 and OH planes i n a d jacent u n i t s , the u n i t s are h e l d t ogether f a i r l y t i g h t l y by hydrogen bonding. The plane between the u n i t l a y e r i s a cleavage p l a n e . However, i n k a o l i n i t e , the cleavage i s not as pronounced as i t i s i n o t h e r c l a y m i n e r a l s where 0 planes are adjacent at u n i t boundaries and, hence, cannot undergo hydrogen bonding. The a d s o r p t i v e c a p a c i t y of k a o l i n has been a t t r i b u t e d to e l e c t r o s t a t i c charges which may a r i s e by two d i f f e r e n t mechanisms. Grim (1953) has suggested t h a t such charges may a r i s e from broken bonds at the edge of the k a o l i n p a r t i c l e s . On the o t h e r hand, S c h o f i e l d and Samson (1953) have suggested t h a t the major s i t e o f e l e c t r o s t a t i c charges i s on the s u r f a c e o f the k a o l i n p a r t i c l e and i s caused by the isomorphous replacement o f s i l i c o n and aluminium atoms i n the t e t r a h e d r a l and o c t a h e d r a l l a y e r s , r e s p e c t i v e l y , by aluminium and magnesium (and to a l e s s e r e x t e n t , c a l c i u m ) . Where the r e p l a c i n g atoms are mono or d i v a l e n t , the r e s u l t i n g charges w i l l i n v a r i a b l y be n e gative and, because the area of the faces i s g r e a t e r than t h a t of the edges ( Mering, M a t t h i e u - S i c a r d and Perrin-Bonnet (1953)), the o v e r a l l charge on the p a r t i c l e i s l i k e l y to be n e g a t i v e . Whatever the s i t e o f the charge, i t i s e x t e r n a l l y compensated f o r by a t t r a c t i o n of c a t i o n s to the k a o l i n s u r f a c e . - 8 -• I t is., claimed t h a t k a o l i n adsorbs some, but not a l l , b a c t e r i a , t o x i n s , and v i r u s e s and p r o v i d e s a p r o t e c t i v e c o a t i n g f o r the i n t e s t i n a l mucosa. For example: Walker (1921) r a t i o n a l i z e d that, the u s e f u l n e s s o f k a o l i n i n the treatment o f i n t e s t i n a l i n f e c t i o n was due, i n most i n s t a n c e s , t o . a d s o r p t i o n o f b a c t e r i a . Mutch (1937) found t h a t k a o l i n adsorbed mussel, mushroom, and potato p o i s o n s . Jordan (1925), McRobert (19 34) and Schwartz (19 46) have r e p o r t e d on the s u c c e s s f u l use of k a o l i n i n the treatment o f p a t i e n t s s u f f e r i n g from b a c t e r i a l food p o i s o n i n g . K a o l i n has been u t i l i z e d f o r i t s a d s o r p t i v e p r o p e r t i e s i n i n t e s t i n a l adsorbent p r e p a r a t i o n s f o r many c e n t u r i e s . I t i s used f o r the treatment o f d i a r r h e a and dy s e n t r y , and i s u s u a l l y a dministered as K a o l i n Mixture w i t h P e c t i n , N.F. X I I I , a mixture of k a o l i n (20%) and p e c t i n (1%) i n a sweetened, p e p p e r m i n t - f l a v o r e d t r a g a c a n t h suspension. The usu a l o r a l dose i s 30 ml., repeated as necessary. Although k a o l i n i s g e n e r a l l y c o n s i d e r e d t o be innocuous, granuloma o f the stomach has been', r e p o r t e d f o l l o w i n g prolonged use /(^e^'.Pharmacblpgi'cal. 'Basis-,.off* Therapeutics, 4th E d i t i o n ) . Commercial p r e p a r a t i o n s o f t e n c o n t a i n , i n a d d i t i o n to the substances mentioned above, antispasmodics, a n t i -c h o l i n e r g i c s , and a n t i b i o t i c s . The use o f a combination c o n t a i n i n g an a n t i b a c t e r i a l drug or drugs or an a n t i b i o t i c i s i n d i c a t e d only f o r p a t i e n t s i n f e c t e d w i t h a c u l t u r a l l y demonstrable s e n s i t i v e organism or d u r i n g epidermics - 9 -p r e v i o u s l y shown to be caused by a s e n s i t i v e organism ( A M A Drug E v a l u a t i o n s (1971)). Examples o f such combinations are: Creomycin (Merck Sharp & Dohme) i s a suspension which c o n t a i n s 500 mg. of s u c c i n y l s u l f a t h i a z o l e , 500 mg. of c o l l o i d a l k a o l i n , 45 mg. of p e c t i n , and 50 mg. of neomycin s u l f a t e i n each 5 ml. of the suspension. The us u a l dose i s 22.5 to 30 ml., s i x times d a i l y . Donnagel (Robins) i s a suspension which co n t a i n s k a o l i n 6 gm., p e c t i n 142.8 mg.> hyoscyamine s u l f a t e 0.1037 mg., a t r o p i n e s u l f a t e 0.0194 mg., hyoscine hydrobromide 0.0065 mg. i n each 30 ml. of the suspension. The us u a l dose i s 30 ml. i n i t i a l l y , then 15 to 30 ml. a f t e r each bowel movement. Donnagel w i t h Neomycin (Robins) i s a suspension which c o n t a i n s the same i n g r e d i e n t s as Donnagel, p l u s 300 mg. of neomycin s u l f a t e ( e q u i v a l e n t t o 210 mg. of neomycin base) i n each 30 ml. of the suspension. The u s u a l dose i s 15 to 30 ml., every four hours. Donnagel PG (Robins) i s a suspension which c o n t a i n s the same i n g r e d i e n t s as Donnagel, p l u s 24 mg. o f powdered opium i n each 30 ml. of the suspension. The dose i s 30 ml. every three hours or as needed. Kaomycin (Upjohn) i s an a n t i d i a r r h e a l mixture c o n t a i n i n g neomycin s u l f a t e 300 mg. ( e q u i v a l e n t t o 210 mg. of neomycin b a s e ) , k a o l i n 5.8 gm., and 130 mg. of p e c t i n i n each 30 ml. of the mixture. The u s u a l o r a l dose i s 30 to 60 ml. four times d a i l y . 2. Theory "Adsorption" i s d e f i n e d as the c o n c e n t r a t i o n or accumulation o f substances a t s u r f a c e s or i n t e r f a c e s . The adsorbing phase i s c a l l e d the "adsorbent"; the adsorbed phase, the "adsorbate". I f the atoms or m o l e c u l e s - o f one phase p e n e t r a t e , more or l e s s u n i f o r m l y , i n t o the i n t e r -atomic or molecular spaces of a second phase, the phenomena are termed " a b s o r p t i o n " or s o l u t i o n . McBain (1932) suggested the use o f the term " s o r p t i o n " t o d e f i n e both a d s o r p t i o n and a b s o r p t i o n , and, i n the German l i t e r a t u r e , " s o r p t i o n " i s used i n t h i s c ontext but only i f the nature of the phenomena i s not known. Two types of a d s o r p t i o n processes have been d e s c r i b e d i n the l i t e r a t u r e . " P h y s i c a l a d s o r p t i o n " , a s s o c i a t e d w i t h van der Waals f o r c e s , i s r e v e r s i b l e — the removal of the adsorbate from the adsorbent b e i n g known as " d e s o r p t i o n " . Chemical a d s o r p t i o n or "Chemisorption", i n which the adsorbate i s a t t a c h e d to the adsorbent by primary chemical bonds, i s i r r e v e r s i b l e . The r e l a t i o n s h i p between the amount of substance adsorbed on a s o l i d and the e q u i l i b r i u m c o n c e n t r a -t i o n a t c o n s t a n t temperature y i e l d s an " a d s o r p t i o n isotherm". The f i r s t c l a s s i f i c a t i o n o f a d s o r p t i o n isotherms was probably t h a t proposed, i n 1922, by Ostwald and de I z a g u i r r e ( J i r g e n s p n and Straumanis (1954)). Isotherms f o r the a d s o r p t i o n o f o r g a n i c s o l u t e s are d i v i d e d i n t o four main - 11 -c l a s s e s and are based on the nature o f s l o p e o f the i n i t i a l p o r t i o n o f the.curve ( G i l e s , MacEwan, Nakhwa and Smith (I960)). The f o u r main c l a s s e s (see F i g u r e 2) a r e : (a) S curves, i n d i c a t i v e of v e r t i c a l o r i e n t a t i o n of adsorbed molecules at the s u r f a c e . (b) L curves, the normal or "Langmuir" isotherms, u s u a l l y i n d i c a t i v e o f molecules adsorbed f l a t - o n the s u r f a c e ; o r , a t times, of v e r t i c a l l y o r i e n t e d adsorbed ions w i t h p a r t i c u l a r l y s t r o n g i n t e r m o l e c u l a r a t t r a c t i o n . (c) H curves ("high a f f i n i t y " ) (commencing at a p o s i t i o n value on the " c o n c e n t r a t i o n i n s o l i d " a x i s ) , o f t e n given by s o l u t e s adsorbed as i o n i c m i c e l l e s , and by h i g h - a f f i n i t y i o n s exchanging w i t h low a f f i n i t y i o n s . (d) C curves ("constant p a r t i t i o n " ) , l i n e a r c u r v e s , g i v e n by s o l u t e s which p e n e t r a t e i n t o the s o l i d more r e a d i l y than does the s o l v e n t . L2 curves (see F i g u r e 2) are u s u a l l y o b t a i n e d when data from s t u d i e s based on a d s o r p t i o n - f r o m d i l u t e s o l u t i o n i s p l o t t e d i n t h e . a p p r o p r i a t e manner (see Eq. 4 ) . Numerous attempts have been made to d e s c r i b e mathemati-c a l l y the e x p e r i m e n t a l l y o b t a i n e d a d s o r p t i o n isotherms. In a d s o r p t i o n a t a s o l i d - g a s i n t e r f a c e , the three equations which are most f r e q u e n t l y used are those a t t r i b u t e d to F r e u n d l i c h , to Langmuir, and to Brunauer, Emmett. and T e l l e r (BET). In g e n e r a l , the F r e u n d l i c h and Langmuir equations are used to d e s c r i b e a d s o r p t i o n a t a s o l i d - l i q u i d i n t e r f a c e . - 12. -F i g u r e 2. System of Isotherm C l a s s i f i c a t i o n . - 13 -(a) F r e u n d l i c h (or c l a s s i c a l ) A d s o r p t i o n Isotherm The F r e u n d l i c h isotherm s t a t e s t h a t , a t a given temperature, the amount of substance adsorbed per weight of the adsorbent i s i n v e r s e l y p r o p o r t i o n a l to some power o f the c o n c e n t r a t i o n of unadsorbed substance. £ = k c 1 / n (Eg. 1) m where x i s the amount o f substance adsorbed by mass m of adsorbent, c i s the s o l u t i o n c o n c e n t r a t i o n , and k and n are c o n s t a n t s . (The constant n i s g e n e r a l l y g r e a t e r than u n i t y . ) T h i s e m p i r i c a l equation i s u s u a l l y c a l l e d the F r e u n d l i c h a d s o r p t i o n equation but, a c c o r d i n g to McBain (19 32) , F r e u n d l i c h was not the f i r s t r e s e a r c h e r to use the above r e l a t i o n s h i p . E q u a t i o n 1 i s u s u a l l y w r i t t e n i n the l o g a r i t h m i c form: l o g = l o g k + i l o g c (Eq. 2) A s t r a i g h t l i n e i s obtained by p l o t t i n g l o g — a g a i n s t l o g c. The c o n s t a n t , l o g k, i s equal to t h e ' l i n t e r c e p t value on the o r d i n a t e s c a l e and ~ to the slope value of the s t r a i g h t l i n e , (b) The ijangmuir A d s o r p t i o n Isotherm P r i o r to 1916, a d s o r p t i o n t h e o r i e s p o s t u l a t e d e i t h e r a condensed l i q u i d f i l m or a compressed gaseous layer, which decreased i n d e n s i t y as the d i s t a n c e from the s u r f a c e i n c r e a s e d . Langmuir (1916) claimed t h a t , because of the r a p i d i t y w i t h which i n t e r m o l e c u l a r f o r c e s decrease w i t h d i s t a n c e , adsorbed l a y e r s are not l i k e l y t o be more than one molecular l a y e r i n t h i c k n e s s . T h i s view i s g e n e r a l l y accepted f o r che m i s o r p t i o n and f o r p h y s i c a l a d s o r p t i o n a t low p r e s s u r e s (or at low c o n c e n t r a t i o n o f s o l u t e ) and moderately h i g h temperature. The Langmuir a d s o r p t i o n i s o t h e r m i s based on the assumption t h a t : (1) o n l y monomolecular a d s o r p t i o n takes p l a c e , (2) a d s o r p t i o n i s l o c a l i z e d , and (3) the heat of a d s o r p t i o n i s independent o f s u r f a c e coverage. x abc m 1 + ac (Eq. 3) Equa t i o n 3 i s known as the Langmuir isotherm. — i s the weight of the adsorbate i n mg. adsorbed per gm. of adsorbent; c i s the e q u i l i b r i u m c o n c e n t r a t i o n of adsorbate i n mg.; and a and b are c o n s t a n t s . Constant a i s r e l a t e d t o the f o r c e s i n v o l v e d i n b i n d i n g the adsorbate molecules to the s u r f a c e of the adsorbent and constant b i s the maximum amount of adsorbate which can be adsorbed per gm. of4adsorbent. The l a t t e r v alue i s , t h e r e f o r e , a measure of the a d s o r p t i v e c a p a c i t y o f adsorbents. A c c o r d i n g t o Brunauer (1943), these constants are not e m p i r i c a l o r a r b i t r a r y . - 15 -The l i n e a r form of the Langmuir equation i s : x/m c _1_ ab + b c (Eq. 4) and an i n t e r c e p t a g a i n s t c. The main c r i t i c i s m of the Langmuir equation i s t h a t the assumption t h a t the heat o f a d s o r p t i o n i s independent o f s u r f a c e area is. npt always v a l i d . S o l i d s u r f a c e s tend to be non-uniform i n n a t u r e . A d s o r p t i o n a t the more a c t i v e s i t e s w i l l occur f i r s t and the process w i l l become somewhat l e s s exothermic w i t h i n c r e a s i n g s u r f a c e area. N e v e r t h e l e s s , many experimental a d s o r p t i o n isotherms can be e x p l a i n e d w i t h a f a i r degree o f accuracy by u t i l i z i n g the Langmuir equation (Shaw (1966)). The a d s o r p t i v e e f f e c t o f c l a y s on c e r t a i n o r g a n i c and i n o r g a n i c substances was f i r s t r e p o r t e d i n the l i t e r a t u r e p a s t two decades. Jensen (1920) found t h a t c e r t a i n a l k a l o i d s were adsorbed by f u l l e r ' s e a r t h . Evcim and B a r r (1955) i n v e s t i g a t e d 3 . A d s o r p t i o n o f Drugs by K a o l i n (a) In V i t r o A d s o r p t i o n S t u d i e s d u r i n g the f i r s t few decades o f t h i s century. However, many of the more important s t u d i e s were c a r r i e d out d u r i n g the - 16 -the a d s o r p t i o n of s t r y c h n i n e , a t r o p i n e , and q u i n i n e from aqueous s o l u t i o n by s i x d i f f e r e n t c l a y s : These c l a y s were permagel, a t t a p u l g i t e , h a l l o y s i t e , d i c k i t e , k a o l i n i t e , and K a o l i n NF. They r e p o r t e d t h a t permagel, a t t a p u l g i t e , and h a l l o y s i t e were b e t t e r a d s o r b e n t s . f o r these a l k a l o i d s than was K a o l i n NF.. C e t y l p y r i d i n i u m and benzalkonium c h l o r i d e are adsorbed by t a l c and k a o l i n (Batuyios and Brecht (1957)). The l o s s of a n t i b a c t e r i a l a c t i v i t y of c e r t a i n quaternary ammonium compounds i n the presences of c l a y s has been observed. Nakashima, M i l l e r , and H a r r i s (1955) r e p o r t e d t h a t benzalkonium c h l o r i d e i s completely i n a c t i v a t e d by m o n t m o r i l l o n i t e . In a s i m i l a r study, Pinck (1962) s t u d i e d the decrease i n a n t i b a c t e r i a l a c t i v i t y of a s e r i e s of a n t i b i o t i c s by the same adsorbent. He arranged the a n t i b i o t i c s i n t o three groups. Group I i n c l u d e d the s t r o n g l y b a s i c a n t i b i o t i c s (e. g., streptomycin s u l f a t e ) ; Group I I , . t h e amphoteric • Y a n t i b i o t i c s (e.g., c h l o r t e t r a c y c l i n e h y d r o c h l o r i d e ) , and Group I I I , the a c i d i c and n e u t r a l a n t i b i o t i c s (e.g., p e n i c i l l i n and c h l o r a m p h e n i c o l ) . On the b a s i s of t h i s c l a s s i f i c a t i o n , one gm. of m o n t m o r i l l o n i t e absorbed 185 mg. (Group I ) , 307 mg. (Group II) , and 9 mg. (Group III) .of the g a n t i t o i p t i c s . - ; He concluded t h a t the complexes of Group I c o n t a i n e d monolayers and those of Group I I c o n t a i n e d d i l a y e r s of the a n t i b i o t i c . He f u r t h e r noted t h a t the a d s o r p t i o n of a n t i b i o t i c s by m o n t m o r i l l o n i t e i n v o l v e d a base exchange - 17 -r e a c t i o n . El-Nakeeb and Yousef (196 8) s t u d i e d the e f f e c t o f supernatant l i q u i d s o b t a i n e d from c l a y - a n t i b i o t i c mixtures on staphylococcus aureus. T h e i r r e s u l t s i n d i c a t e d t h a t the a n t i b a c t e r i a l a c t i v i t y of neomycin and t e t r a c y c l i n e were reduced by 59% and 55%, r e s p e c t i v e l y . B a r r and A r n i s t a (1957) s t u d i e d the a d s o r p t i o n of s t r y c h n i n e and a t r o p i n e by a c t i v a t e d a t t a p u l g i t e , h a l l o y s i t e , and k a o l i n . They r e p o r t e d t h a t the a d s o r p t i v e p r o p e r t i e s of the c l a y s were not reduced even a f t e r they had been washed wi t h 0.05 N H y d r o c h l o r i c A c i d or Simulated G a s t r i c F l u i d T.S., U.S.P. XV. Sorby and P l e i n (1961) i n v e s t i g a t e d the a d s o r p t i o n of p h e n o t h i a z i n e d e r i v a t i v e s by k a o l i n , t a l c , and N o r i t A, an a c t i v a t e d c h a r c o a l . They found t h a t these d e r i v a t i v e s were adsorbed to a s i g n i f i c a n t e x t e n t by a l l of the three c l a y s i n v e s t i g a t e d . Sorby, P l e i n , and Benmaman (1966) found t h a t the a d s o r p t i o n of promazine h y d r o c h l o r i d e by t a l c and k a o l i n v a r i e s w i t h the pH o f the medium but t h a t the a d s o r p t i o n of t h i s drug by a c t i v a t e d c h a r c o a l was l e s s a f f e c t e d by pH. A d s o r p t i o n of promazine h y d r o c h l o r i d e by a l l three adsorbents i s s e n s i t i v e to the e l e c t r o l y t e c o n c e n t r a t i o n o f the medium. The a d s o r p t i o n c h a r a c t e r i s t i c s of a t r o p i n e from aqueous s o l u t i o n have been d e s c r i b e d by Ridout (1968a, 1968b). These p u b l i c a t i o n s i n d i c a t e t h a t the a d s o r p t i o n isotherms are d i s c o n t i n u o u s and t h a t the heterogeneous nature o f the - 18 -k a o l i n s u r f a c e accounted f o r t h i s d i s c o n t i n u i t y ^ Armstrong and C l a r k e (19 71) i n v e s t i g a t e d the a d s o r p t i o n of c r y s t a l v i o l e t , a b a s i s substance, by k a o l i n . S t u d i e s were c a r r i e d out over a pH range o f 2.5 to 9.5. They found t h a t there was an i n c r e a s e i n the amount of c r y s t a l v i o l e t adsorbed by the k a o l i n when the pH o f the medium was i n c r e a s e d and suggested t h a t t h i s may be due to -(1) an i n c r e a s e i n the d e n s i t y of the n e g a t i v e charges on the edge o f the k a o l i n p a r t i c l e due to a change i n the charge on the aluminum atoms from p o s i t i v e to n e g a t i v e , and (2) the i n c r e a s e d c o n c e n t r a t i o n of sodium hydroxide i n the system needed to achieve h i g h e r pH v a l u e s . T h i s appears t o promote replacement on the e x t e r i o r of the c l a y l a t t i c e o f the charge-compensating c a t i o n s (magnesium, cal c i u m , and p o s s i b l y aluminum) by sodium i o n s . T h i s r e p r e s e n t s , i n t e s s e n c e a c a t i o n exchange mechanism;. The r e s u l t i n g e l e c t r i c a l imbalance i n the c l a y l a t t i c e i n c r e a s e s the n e g a t i v e charge on the p a r t i c l e as a whole and consequently f a v o r s the a d s o r p t i o n of a p o s i t i v e l y charged i o n such as c r y s t a l v i o l e t . - 19. -These authors concluded t h a t the major a d s o r p t i v e . mechanism i s by e l e c t r o s t a t i c charges a r i s i n g from c a t i o n replacement i n the c l a y l a t t i c e and t h a t charges a r i s i n g from the amphoteric nature of the aluminum atom i n k a o l i n c o n t r i b u t e l i t t l e t o the o v e r a l l a d s o r p t i v e p r o c e s s . The i n f l u e n c e o f pH on the a d s o r p t i o n o f b e n z o i c a c i d by k a o l i n has been s t u d i e d by C l a r k e and Armstrong (1972). They found t h a t the e x t e n t of a d s o r p t i o n i n c r e a s e d markedly wit h i n c r e a s e i n system pH and t h a t maximum a d s o r p t i o n occured a t about pH 5.0. I f the uptake of b e n z o i c a c i d i s expressed i n terms o f the degree of d i s s o c i a t i o n of the a c i d (pK = 4.2), i t would appear t h a t the n e g a t i v e l y charged anion i s adsorbed onto the c l a y p a r t i c l e . Primary i n c r e a s e i n a d s o r p t i o n w i t h i n c r e a s i n g pH may be due t o the changing c o n c e n t r a t i o n o f benzoate i o n s . Although d i s s o c i a t i o n i n c r e a s e s a f t e r pH.5.0, a d s o r p t i o n decreases. Such decreases may be due t o changes o c c u r i n g on the k a o l i n s u r f a c e . The o r i g i n of the e l e c t r o s t a t i c charges on k a o l i n , . t o which the a d s o r p t i v e c a p a c i t y of the c l a y has been a t t r i b u t e d , has been d i s c u s s e d by Armstrong and C l a r k e (19 71). The charges on the face of the k a o l i n p a r t i c l e are b e l i e v e d t o be due t o isomorphous replacement o f l a t t i c e c a t i o n s and, hence, the s u r f a c e w i l l i n v a r i a b l y . b e n e g a t i v e . However, - 20 -Ridout (1968a, 1968b) viewed the exposed aluminum ions as being comparable to an alumina p a r t i c l e . Because of the amphoteric nature of the aluminum ion, the kaolin surface would be p o s i t i v e l y charged i n acid solution. Ridout also observed that the charge on the broken edge of the tetrahedral sheet was dependent on the s i l i c o n atom, was p o s i t i v e ; and, therefore, unaffected by pH. He concluded that, at low pH, the • edger su-rf aGecwouidlBebposivtiveityiGhar geded. Flocculation studies indicated that the edge ZPC (the zero point of charge) of kaolin occurs between pH 4.2 and 4.9 (Clarke and Armstrong (1972)). Oh the basis of t h e i r r e s u l t s obtained, i t was suggested that the p o s i t i v e edge s i t e s of the kaolin lamellae are responsible for the adsorption of bezoic acid. With an increase i n system pH beyond the. ZPC, the adsorption of •bezjb^ of the acid molecules. At these higher pH values, the p o s i t i v e edge s i t e s on the kaolin are no longer present. No i n v i t r o adsorption•studies of a n t i b i o t i c s by kaolin have been reported i n the l i t e r a t u r e . However, a n t i b i o t i c s are adsorbed onto other insoluble substances. In 19 49 , Schoehbach aaridhhisccq^workersU.-:(.Mar.tiri 1'(19 5 5) ) reported the effectiveness of aluminum hydroxide gel i n r e l i e v i n g aureomycin-induced nausea and Di Gangi and Rogers (1949) found that the a n t i b i o t i c was strongly adsorbed by the antacid.- Terramycin hydrochloride as well as aureomycin hydrochloride are adsorbed by aluminum hydroxide g e l i n - 21 -amounts ranging from 25% to 90% (Paul and H a r r i n g t o n (1952)). (k) In 'Vivo A d s o r p t i o n S t u d i e s ; Few r e s e a r c h e r s have s t u d i e d the e f f e c t of k a o l i n on the a b s o r p t i o n or c l i n i c a l e f f e c t of drugs. However, Wagner (1966) d i d study the e f f e c t of Kaopectate (the Upjohn Company brand of a k a o l i n w i t h p e c t i n mixture) on the a b s o r p t i o n o f l i n c o m y c i n h y d r o c h l o r i d e . E i g h t a d u l t subjects® were used i n the study. A f o u r way c r o s s o v e r study was c a r r i e d out u ^ i n g r L i n c ^ (the Upjohn Company brand of l i n c o m y c i n h y d r o c h l o r i d e ) , 0.5 gm. taken o r a l l y w i t h , three f l u i d ounces of water (Treatment A ) , three f l u i d ounces of Kaopectate, taken two hours b e f o r e the capsule (Treatment B), three f l u i d ounces of Kaopectate, taken two hours a f t e r the capsule (Treatment C ) , and three f l u i d ounces of Kaopectate, taken a t the same time as the capsule (Treatment D). Twelve hour b l o o d l e v e l s and 48 hour u r i n a r y e x c r e t i o n p a t t e r n s were determined. The r a t i o s o f the average areas under the serum c o n c e n t r a t i o n curves were found to be 1.0, 0.83, 0.53, and 0.03 and the r a t i o s of the average amount o f drug e x c r e t e d i n 48 hours i n the u r i n e was equal t o 1.0, 0.71> 0.69, and 0.20, f o r Treatments A, B, C, and D, r e s p e c t i v e l y . There were no s i g n i f i c a n t d i f f e r e n c e s between Treatments A and B. However., when Kaopectate i s a d m i n i s t e r e d at the same time as the drug product, the l i n c o m y c i n serum c o n c e n t r a t i o n was - 22 -o n l y about one-tenth o f t h a t observed when the a n t i b i o t i c was administered alone. The serum l e v e l was reduced by about 50% when Kaopectate was ad m i n i s t e r e d two hours a f t e r the c a p s u l e . Serum c o n c e n t r a t i o n s d i d not change when Kaopectate was admi n i s t e r e d two hours p r i o r t o the a d m i n i s t r a t i o n o f the drug product. The i n f l u e n c e of a t t a p u l g i t e and c h a r c o a l on promazine h y d r o c h l o r i d e a d s o r p t i o n has been s t u d i e d (Sorby (1965)). Equal doses of the drug were admi n i s t e r e d t o human s u b j e c t s i n s o l u t i o n and i n two l i q u i d p r e p a r a t i o n s c o n t a i n i n g e i t h e r a t t a p u l g i t e or c h a r c o a l . Approximately 80% of the drug was adsorbed by the a t t a p u l g i t e p r e p a r a t i o n . The value f o r the c h a r c o a l p r e p a r a t i o n was about 50%. The i n i t i a l r a t e o f drug a b s o r p t i o n decreased but there was no e f f e c t on t o t a l a v a i l a b i l i t y when the drug was admi n i s t e r e d i n mixtures c o n t a i n i n g a t t a p u l g i t e . C h a r c o a l decreased both the r a t e and extent o f promazine a b s o r p t i o n . In v i t r o r e l e a s e s t u d i e s i n d i c a t e d t h a t the promazine-charcoal adsorbate d i d not desorb and, t h e r e f o r e , o n l y the drug i n s o l u t i o n i n the dosage form was a v a i l a b l e f o r a b s o r p t i o n . However, the r a t e of r e l e a s e o f drug from the p r o m a z i n e - a t t a p u l g i t e adsorbate was r a p i d . Thus, the e x t e n t o f a d s o r p t i o n i s not d i r e c t l y r e l a t e d to e x t e n t o f a b s o r p t i o n . I t would a p p e a r , . t h e r e f o r e , t h a t both a d s o r p t i o n and d e s o r p t i o n must be c o n s i d e r e d and together r e l a t e d t o i n v i v o e f f e c t . In a s i m i l a r study, Sorby and L i u (1966) a d m i n i s t e r e d - 23 -promazine hydrochloride and attapulgite with pectin separately, one half-hour hour apart, i n an attempt to simulate a more usual approach to the consumption of drug products. Both the rate and extent of promazine hydrochloride absorption were decreased. In v i t r o adsorption studies established that the mixture had a strong a f f i n i t y for promazine hydrochloride., In s i m i l a r investigations, Waisben and Hueeke1 (1950) and Seed and Wilson (1950) demonstrated that aureomycin blood lev e l s were greatly reduced i f the a n t i b i o t i c and aluminum hydroxide gel were administered concurrently. S i m i l a r l y , s;§heiner and Altemeir (19 62) reported that the ingestion of aluminum hydroxide gel with declomycin s i g n i f i c a n t l y lowered the. serum leve l s of t h e . a n t i b i o t i c . 4. The Test Drugs (a) Tetracycline.Hydrochloride Tetracycline hydrochloride i s a yellow, odorless, moderately hygroscopic, c r y s t a l l i n e powder. I t i s f r e e l y soluble i n water, soluble i n solutions of a l k a l i hydroxides and carbonates, s l i g h t l y soluble}:,in alcohol, and p r a c t i c a l l insoluble i n chloroform and i n ether. Its chemical name i s 4-(Dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12, 12a-pentahydroxyl-6-methyT-r, ll^dioxo-2-naphthacenecarboxamide monohydrochloride and, based on a molecular formula, of C22 H24 N2°8 H C"'"' "*"tS m o l e c u l a r weight i s equal t o 480.91. The a n t i b i o t i c decomposes a t from 170° to 175° C. The pH of a 2% aqueous s o l u t i o n i s between 2.1 and 2.3 (Merck Index, 8th E d i t i o n ) . T e t r a c y c l i n e h y d r o c h l o r i d e , when d i s s o l v e d i n water or i n 0.1 N h y d r o c h l o r i c a c i d s o l u t i o n , absorbs a maximum of r a d i a n t energy a t 2 72 and 357 nanometers. T e t r a c y c l i n e h y d r o c h l o r i d e i s s t a b l e i n a i r but becomes dark brown i n c o l o r when exposed t o moisture and s t r o n g s u n l i g h t . I t s potency i s decreased i n s o l u t i o n s w i t h pH values below two and i t i s r a p i d l y destroyed by a l k a l i hydroxide s o l u t i o n s . The s t a b i l i t y of the powdered a n t i b i o t i c has been i n v e s t i g a t e d by B a r r i n g e r e t a l (1974). I f the a n t i b i o t i c i s s t o r e d a t room temperature, i t i s s t a b l e f o r three to four y e a r s . However, i n s o l u t i o n , the drug degrades i n mannered i n d i c a t e d i n F i g u r e 3. The mode of degradation most f r e q u e n t l y encountered i s e p i m e r i z a t i o n a t carbon 4. This occurs under a wide v a r i e t y of c o n d i t i o n s and r e s u l t s i n the formation o f " e p i t e t r a c y c l i n e " ( I I ) . In a c i d i c media, i n which a change occurs a t the h y d r o x y l group on carbon 6, t e t r a c y c l i n e i s converted t o the corresponding anhydro compound (III) . O x i d a t i o n i s not a major problem i n s o l u t i o n s w i t h pH values o f l e s s than f i v e . However, a t hi g h e r pH v a l u e s , o x i d a t i o n becomes the predominant pathway f o r l o s s of a c t i v i t y . For example, a t pH 2.8 and 9.0 drug p o t e n c i e s are equal to 90% and 8%, r e s p e c t i v e l y , i f the I I I . Anhydro F i g u r e 3. T e t r a c y c l i n e Degradation - 26 -solutions are stored at room temperature for one week. Tetracycline hydrochloride i s a broad spectrum a n t i b i o t i c and antiprotozoal agent which suppresses the growth of most Gram-positive bacteria, many Gram-negative bacteria, spirochetes, amoebae, and certain large viruses. Its c l i n i c a l spectrum encompasses most the infections amenable to therapy with p e n i c i l l i n and streptomycin, with the important exception of tuberculosis. Among the b a c t e r i a l infections i n which tetracycline hydrochloride may be employed, although not necessarily as the drug of choice, are most coccal i n f e c t i o n s , including those caused by sensitive hemolytic streptococci, staphylococci, pneumococci, meningococci, and gonococci, many urinary t r a c t i n f e c t i o n s , b a c i l l a r y infections such as b r u c e l l o s i s , pertussis, and p e r i t o n i t i s , and some of the infections caused by Salmonella and S h i g e l l a . The a n t i b i o t i c i s used to " s t e r i l i z e " the bowel p r i o r to bowel surgery. R i c k e t t s i a l diseases such as Rocky Mountain spotted fever, murine and endemic typhus, and Q-fever respond well to th i s a n t i b i o t i c . Tetracycline hydrochloride i s incompletely absorbed from the g a s t r o i n t e s t i n a l t r a c t . Absorption occurs i n the stomach and upper small i n t e s t i n e , to a lesser extent i n the lower portions of the i n t e s t i n a l t r a c t , and i s negl i g i b l e from the colon. I t i s impaired, to a variable degree, by milk and milk products, by aluminum hydroxide g e l , and by calcium and magnesium s a l t s (The: .Pharmacological Basis of Therapeutics,. 4 th Edition) . Absorption can also be reduced by sodium bicarbonate which contains no polyvalent cations and cannot form chelates with t e t r a c y c l i n e (Barr, Adiq?i?h,and Garrettson (1971) ) . Such observations are consistent with the mechanism of absorption which requires a low g a s t r i c pH for complete d i s s o l u t i o n of t e t r a c y c l i n e and suggest that any substance or condition which s i g n i f i c a n t l y increases g a s t r i c pH may decreases-dissolution and, hence, absorption. The o r a l dose of t e t r a c y c l i n e hydrochloride varies with the nature and the severity of the disease. The adult dose i s from one to two gm. of the drug per day. (b) Neomycin Sulfate Neomycin sulfate i s the sulfate s a l t of an a n t i b a c t e r i a l substance produced by the growth of Streptomyces fardiae Waksman (Fam. Streptomycetaceae). The a n t i b i o t i c normally contains 80 to 85 per cent of neomycin B, 15 to 20 per cent of neomycin C and usually less than 2 per cent of neamine, neomycin A, and traces of other unknown but b i o l o g i c a l l y active substances. The t o t a l structure and the common names of the component parts of neomycin C are shown i n Figure 4. The structure of neomycin B i s reported to be i d e n t i c a l except for the configuration of the indicated aminoethyl group. - 28 -CH2NH2 F i g u r e 4. Chemical s t r u c t u r e of neomycin C Neomycin s u l f a t e U.S.P. c o n t a i n s an amount of a n t i b i o t i c e q u i v a l e n t t o not l e s s than 60.0 per cent of neomycin base, c a l c u l a t e d w i t h r e s p e c t to the d r i e d substance. I t i s a white or s l i g h t l y y e l l o w powder. I t i s o d o r l e s s and h y g r o s c o p i c . I t i s very s o l u b l e i n water (1 gm./r ml. f^O); very s l i g h t l y s o l u b l e i n a l c o h o l ; and i n s o l u b l e i n acetone, chloroform, and e t h e r . I t s s o l u t i o n s are d e x t r o r o t a t o r y . The pH of an aqueous . s o l u t i o n , ( 3 3 mg./ml.), i s between 5.0 to 7.5. Neomycin i s f a i r l y s t a b l e over the pH range 2.0 - 9.0 (Swart, Hutchison and Waksman (1949)). T h e i r r e s u l t s i n d i c a t e d t h a t aqueous s o l u t i o n s w i t h pH values of 2.0 and 7.05, when s t o r e d at room temperature, remain s t a b l e f o r up to two days. However, o s o l u t i o n s s t o r e d at 5 C w i l l show no l o s s i n potency f o r - 29 -up t o one w e e k . A c h e m i c a l a s s a y p r o c e d u r e f o r t o t a l n e o m y c i n B a n d C , p r o p o s e d b y D u t c h e r , e t a l ( 1 9 5 3) , i s b a s e d o n t h e o b s e r v a t i o n t h a t b o t h s u b s t a n c e s , when h e a t e d w i t h s u l f u r i c a c i d , y i e l d f u r f u r a l . T h e q u a n t i t a t i v e e s t i m a t i o n o f f u r f u r a l c o n t e n t i s b a s e d o h a b s o r b a n c e m e a s u r e m e n t s a a t 2 2 - 8 0 n n a n o m e . t e B s . C T h i s m e t h o d o f a n a l y s i s was u s e d i n t h i s i n v e s t i g a t i o n a n d i s d e s c r i b e d i n d e t a i l i n t h e n e x t s e c t i o n . N e o m y c i n s u l f a t e i s a b r o a d s p e c t r u m a n t i b i o t i c . S u s c e p -t i b l e m i c r o o r g a n i s m s a r e u s u a l l y i n h i b i t e d b y c o n c e n t r a t i o n s o f 5 t o 10 m e g . d r u g / m l . s o l u t i o n , / I t i s u s e d t o p i c a l l y a n d f o r i t s l o c a l a n t i b a c t e r i a l a c t i o n i n t h e l u m e n o f t h e b o w e l . P a r e n t e r a l t h e r a p y i s c o n t r a i n d i c a t e d , u n l e s s t h e o f f e n d i n g o r g a n i s m i s n o t s u s c e p t i b l e t o o t h e r l e s s t o x i c c h e m o t h e r a -p e u t i c a g e n t s . N e o m y c i n i s e f f e c t i v e a g a i n s t a v a r i e t y o f G r a m - p o s i t i v e a n d G r a m - n e g a t i v e b a c t e r i a a n d a g a i n s t a c i d - f a s t bia.cil ' . i i-iandiyaGti^nomycetes. T h e a n t i b i o t i c i s p o o r l y a b s o r b e d f r o m t h e g a s t r o i n t e s t i n a l t r a c t . A n o r a l d o s e o f 3 gm. p r o d u c e s p e a k p l a s m a , l e v e l s o f f r o m 1 t o 4 m e g . d r u g / m l . . A t o t a l d a i l y i n t a k e o f t e n gm. f o r t h r e e d a y s y i e l d s a b l o o d c o n c e n t r a t i o n b e l o w t h a t a s s o c i a t e d w i t h s y s t e m i c t o x i c i t y . A b o u t 9 7% o f t h e o r a l l y a d m i n i s t e r e d n e o m y c i n i s e l i m i n a t e d u n c h a n g e d i n t h e f e c e s . T h e d r u g i s w e l l a b s o r b e d a f t e r i n t r a m u s c u l a r i n j e c t i o n a n d w i d e l y d i s t r i -b u t e i n b o d y f l u i d s a n d t i s s u e s ; a d o s e o f 1 gm. p r o d u c e s a p e a k s e r u m l e v e l o f 20 m e g . p e r m l . . A l t h o u g h n e o m y c i n c a n - 30 -be g i v e n o r a l l y t o very young c h i l d r e n , i n doses as h i g h as 100 mg./kg. per day, i t s use i n such p a t i e n t s f o r longer than three weeks should be avoided because o f p a r t i a l a b s o r p t i o n from the i n t e s t i n a l t r a c t . O r a l therapy w i t h neomycin s u l f a t e e i t h e r f o r " p r e p a r a t i o n " of the bowel f o r surgery or of the management o f . h e p a t i c coma r e q u i r e s the i n g e s t i o n o f 4 to 8 gm. d a i l y , i n d i v i d e d doses. Neomycin s u l f a t e can. a l s o be used f o r the t r e a t -ment of i n t e s t i n a l i n f e c t i o n s , p r i m a r i l y i n c h i l d r e n , due to pathogenic s t r a i n s of E. c o l i . The dose f o r t h i s purpose i s 100 mg./kg. per day, given o r a l l y f o r about 10 days but never longer than f o r three weeks. The a n t i b i o t i c i s r a p i d l y e x c r e t e d i n the u r i n e . The p a r e n t e r a l i n j e c t i o n of 0.5 gm. of neomycin every f o u r hours r e s u l t s i n a c o n c e n t r a t i o n o f 100 to 200 meg. of the drug per ml. o f u r i n e . From 30 to 50% of a p a r e n t e r a l dose i s e x c r e t e d i n • t h e u r i n e . Plasma c o n c e n t r a t i o n s approximating those o c c u r i n g a f t e r p a r e n t e r a l i n j e c t i o n of the drug may occur when the drug i s given o r a l l y (4 to 8 gm. d a i l y ) to c i r r h o t i c persons w i t h r e n a l d y s f u n c t i o n (TherPharmacol'pgicail-xBasis o f T h e r a p e u t i c s , 4th E d i t i o n ) . Neomycin s u l f a t e has been i n c o r p o r a t e d i i h t o a a h t i d i a r r h e a l p r e p a r a t i o n c o n t a i n i n g k a o l i n and p e c t i n . For example, Kaomycin (Upjohn Company) co n t a i n s 52,5 mg. neomycin s u l f a t e ( e q u i v a l e n t to 36.75 mg. of neomycin b a s e ) , 985.0 mg. k a o l i n , 22.0 mg. p e c t i n , and other p h a r m a c e u t i c a l adjuvants i n each o f 5 ml. o f the suspension. The u s u a l dose i s two to f o u r t a b l e s p o o n f u l s f o u r times d a i l y . - 31 -(c) L i n c o m y c i n H y d r o c h l o r i d e M o n o h y d r a t e L i n c o m y c i n HC1 i s t h e m o n o h y d r a t e d h y d r o c h l o r i d e s a l t o f l i n c o m y c i n , an a n t i b i o t i c p r o d u c e d b y t h e g r o w t h o f a member o f t h e l i n c o l n e n s i s g r o u p o f s t r e p t o m y c e s l i n c o l n e n s i s (Fam. S t r e p t o m y c e t a c e a e ) . I t h a s a p o t e n c y e q u i v a l e n t t o n o t l e s s t h a n 790 meg. o f l i n c o m y c i n p e r mg., c a l c u l a t e d o n t h e a n h y d r o u s b a s i s . I t i s a w h i t e o r p r a c t i c a l l y w h i t e , c r y s t a l l i n e p o w d e r . I t i s o d o r l e s s o r h a s a f a i n t o d o r a nd a b i t t e r t a s t e . I t s s o l u t i o n s a r e a c i d i c a n d a r e d e x t r o r o t a t o r y . I t . i s f r e e l y s o l u b l e i n w a t e r ; s o l u b l e i n d i m e t h y 1 f o r m a m i d e ; a n d v e r y s l i g h t l y s o l u b l e i n a c e t o n e . I t s c h e m i c a l name i s M e t h y l 6, 8 - d i d e o x y - 6 - ( l - m e t h y l - 4 - p r o p y l - 2 - p y r r o l i d i n e c a r b o x a m i d o ) - 1 -t h i o - D - e r y t h r o - D - g a l a c t o - o c t o p y r a n o s i d e M o n o h y d r o c h l o r i d e a n d , b a s e d on a m o l e c u l a r f o r m u l a o f C ^ g H ^ ^ O g S . HC1, i t s m o l e c u l a r w e i g h t i s e q u a l t o 4 4 3 . 0 1 . The m e l t i n g p o i n t r a n g e i s f r o m 145°-147°C. The p k a o f t h e f r e e b a s e i s 7.6. A q u e o u s s o l u t i o n s o f t h i s a n t i b i o t i c show no c h a r a c t e r i s t i c a b s o r p t i o n i n t h e u l t r a v i o l e t o r v i s i b l e r e g i o n s o f t h e e l e c t r o m a g n e t i c s p e c t r u m . L i n c o m y c i n . h y d r o c h l o r i d e i s more s t a b l e t h a n l i n c o m y c i n b a s e . D r y l i n c o m y c i n , s t o r e d a t 70°C f o r s i x m o n t h s , showed no d e t e c t a b l e d e g r a d a t i o n . I n 0.1 N h y d r o c h l o r i c a c i d s o l u t i o n a n d a t 7 0 ° C , l i n c o m y c i n was f o u n d t o h a v e a h a l f - l i f e o f 39 h o u r s (Hoeksema, e t a l ( 1 9 6 4 ) ) . - 32 -The a n t i b a c t e r i a l a c t i v i t y of l i n c o m y c i n c l o s e l y resembles t h a t of erythromycin. I t has a r a t h e r broad spectrum o f a c t i v i t y a g a i n s t Gram-positive organisms, e s p e c i a l l y s t a p h y l o c o c c i , pheumococci, pyogenic s t r e p t o c o c c i , and c o r y n e b a c t e r i a . However, i t has l i t t l e e f f e c t on e n t e r o c o c c i , which are Gram-positive. Lincomycin i s used t o t r e a t i n f e c t i o n s o f the r e s p i r a t o r y and u r i n a r y t r a c t s or of s o f t t i s s u e s when the i n f e c t i n g organism i s a p p r o p r i a t e l y s u s c e p t i b l e . Lincomycin may be of value i n the management of c e r t a i n types of i n t e s t i n a l y d i s o r d e r (e.g. the b l i n d - l o o p syndrome) because of i t s a c t i v i t y a g a i n s t B a c t e r o i d e s and other anaerobes. O r a l a d m i n i s t r a t i o n of the drug i n man produces a s t r i k i n g decrease or e l i m i n a t i o n of these b a c t e r i a from the i n t e s t i n a l t r a c t but the number of e n t e r o c o c c i may be i n c r e a s e d 100,000-fold (The:,Pharmac6logieal~BasisTof -Therapeutics ,4th E d i t i o n ) . Lincomycin i s r a p i d l y but o n l y p a r t i a l l y absorbed from the g a s t r o i n t e s t i n a l t r a c t . Although the a n t i b i o t i c i s i n c o m p l e t e l y absorbed from the gut, o r a l a d m i n i s t r a t i o n p r o v i d e s adequate b l o o d l e v e l s . From 20 to 35% of an o r a l dose i s absorbed; peak plasma c o n c e n t r a t i o n s range from about 2 to 5 meg./ml. a f t e r an o r a l dose of 500 mg.. The b i o l o g i c a l h a l f - l i f e of l i n c o m y c i n a f t e r o r a l , i n t r a m u s c u l a r , or intravenous a d m i n i s t r a t i o n i s about 5 t o 6 hours (The Pharmacological B a s i s o f T h e r a p e u t i c s , 4th E d i t i o n ) . - 33 -O r a l a d m i n i s t r a t i o n o f l i n c o m y c i n can cause d i a r r h e a , , nausea and vom i t i n g , and abdominal cramps. I f d i a r r h e a does occur, a k a o l i n p r e p a r a t i o n , i s o f t e n administered t o the p a t i e n t . I f drug therapy i s continued, decreased b l o o d l e v e l s w i l l be observed (Wagner (1966)). On the b a s i s o f t h i s , the Food and Drug A d m i n i s t r a t i o n Panel on Over-The-Counter Drugs has recommended t h a t a l l k a o l i n c o n t a i n i n g products be so l a b e l l e d t h a t the p a t i e n t i s aware of the i n t e r a c t i o n j (Federa l R e g i s t e r (1975)). The recommended o r a l dose of l i n c o m y c i n f o r a d u l t s i s 500 mg. every s i x or e i g h t hours, depending on the s e v e r i t y o f the i n f e c t i o n ; f o r c h i l d r e n , i t i s 30 to 60 mg./kg. d i v i d e d i n t o three o r f o u r e q u a l doses each day. (d) Chloramphenicol Chloramphenicol i s marketed as f i n e , white t o g r a y i s h white o r y e l l o w i s h - w h i t e , n e e d l e - l i k e c r y s t a l s o r - e l o n g a t e d p l a t e s . I t i s s l i g h t l y s o l u b l e i n water, f r e e l y s o l u b l e i n a l c o h o l , acetone, b u t a n o l , propylene g l y c o l , and e t h y l a c e t a t e , s l i g h t l y s o l u b l e i n et h e r and ch l o r o f o r m , and i n s o l u b l e i n benzene and petroleum et h e r . I t s aqueous s o l u t i o n s are n e u t r a l t o lit m u s paper. I t has a m e l t i n g p o i n t o o range o f 150.5 t o 151.5 C. (Merck Index, 8th E d i t i o n ) . I t s chemical name i s D (-) -thireo-2 > 2- d i c h l o r o - N (B-hydroxy-a-(hydroxymethyl)-p-nitrophenethyl) acetamide and, based on - 34 -a molecular formula o f C-^H^C^^O,-, i t s m o l e c u l a r weight' i s e q u a l to 323.13. I t s aqueous s o l u t i o n s absorb a maximum of r a d i a n t energy a t 278 nanometers. The b i o l o g i c a l l y a c t i v e form i s le'vorotatory. Chloramphenicol i s reasonably s t a b l e i n n e u t r a l or moderately a c i d i c s o l u t i o n s but degrades r a p i d l y i n a l k a l i n e s o l u t i o n . I t has been r e p o r t e d t h a t no s i g n i f i c a n t d e g r a d a t i o n occurs when aqueous s o l u t i o n s are b o i l e d f o r f i v e hours or when s o l u t i o n s i n the. pH range of 2 to 9 are s t o r e d a t room temperature f o r 24 hours ( E h r l i c k , e t a l (1947)). The k i n e t i c s o f degr a d a t i o n o f chloramphenicol i n s o l u t i o n has been s t u d i e d by. H i g u c h i and B i a s (1953) . These r e s e a r c h e r s s t u d i e d the formation o f c h l o r i d e i o n s i n aqueous s o l u t i o n s s t o r e d a t 87.0° and 91.7° C. T h e i r r e s u l t s indicated'.Jthfat the over a l l r a t e o f c h l o r i d e p r o d u c t i o n i s the summation of the r a t e s o f a t l e a s t t hree f i r s t o r d e r r e a c t i o n s - d i r e c t u n c a t a l y z e d h y d r o l y t i c cleavage o f c h l o r i d e i o n s from the whole chloramphenicol molecule; d i r e c t h y d r o x y l i o n c a t a l y z e d cleavage o f c h l o r i d e ions from th'e whole chloramphenicol molecule; and h y d r o l y s i s o f chloramphenicol a t the amide l i n k a g e f o l l o w e d by h y d r o l y s i s of the d i c h l o r o a c e t a t e i o n s formed t o y i e l d c h l o r i d e i o n s and other d e g r a d a t i o n p r o d u c t s . In s p i t e o f such o b s e r v a t i o n s , the a n t i b i o t i c i s co n s i d e r e d t o be very s t a b l e a t room temperature. S a t u r a t e d aqueous s o l u t i o n s (0.25%) may be s t o r e d , f o r s e v e r a l months, i n a r e f r i g e r a t o r and show no evidence o f d e g r a d a t i o n . However, weaker s o l u t i o n s tend t o degrade a f t e r days or weeks of storage when s t o r e d under s i m i l a r c o n d i t i o n . (The Pharmacological B a s i s o f T h e r a p e u t i c , 4th E d i t i o n ) . Chloramphenicol has a wide spectrum o f a n t i b a c t e r i a l a c t i v i t y i For example, i t can be used t o t r e a t Rocky Mountain s p o t t e d f e v e r , Q-fever, and many b a c t e r i a l i n f e c t i o n s . However, because o f major s i d e e f f e c t s which, i n some i n s t a n c e s , have caused death, the use o f the a n t i b i o t i c should be r e s t r i c t e d . I t i s s t i l l the drug o f c h o i c e f o r the treatment o f t y p h o i d f e v e r , except i n c h i l d r e n (The Pharmacological B a s i s o f T h e r a p e u t i c s , 4th E d i t i o n ) . Chloramphenicol i s r a p i d l y absorbed from the g a s t r o -i n t e s t i n a l t r a c t . S i g n i f i c a n t plasma l e v e l s are o b t a i n e d w i t h 30 minutes a f t e r a d m i n i s t r a t i o n . Peak plasma c o n c e n t r a t i o n s are reached i n approximately two hours. Such l e v e l s range from 20 t o 40 meg./ml. a f t e r an o r a l dose of two gm. and from 40 to 60 meg./ml. a f t e r an o r a l dose o f fo u r gm. The b i o l o g i c a l h a l f - l i f e o f the drug i s about 1 1 l - j t o 3^ - hours. Chloramphenicol i s the drug o f choice f o r the treatment of S a l m o n e l l a . - T h e . u s u a l . d o s e l i s 2-sgml • i n i t i a l l y , c f o l l o w e d by 1 gm. every s i x hours f o r fo u r weeks. - 36 -(e) A m p i c i l l i n Trihydrate The chemical name for t h i s a n t i b i o t i c i s 6-(D-2-Amino-2 phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo-[3.2 heptane-2-carboxylic Acid; (D-cc1- Aminobenzyl ) p e n i c i l l i n . I t occurs as white, needle-like c r y s t a l , or as a white, c r y s t a l l i n e powder. The cry s t a l s are bi r e f r i n g e n t and show extinction under polarized l i g h t . I t i s odorless, or has the f a i n t odor c h a r a c t e r i s t i c of the p e n i c i l l i n s . I t occurs as the trihydrate, which i s stable at room temperature. Its molecular weight i s 40 3.41. Melting points (with decomposition) of 214.5° -215.5°C. and 202° - 204°C. have been reported (Analytical P r o f i l e s of Drug Substances, Volume 2). I t contains not less than 90.0% ci6 Hi9 N3°4 S> calculated on the anhydrous basis. I t i s soluble i n water (1 gm./90 ml. E^O) , soluble i n methanol; and insoluble i n benzene, i n carbon tetr a c h l o r i d e , and i n chloroform. The k i n e t i c s of degradation of a m p i c i l l i n i n solution have been investigated at 35°C. and at a pH range of 0.8 to 10 (Hou and Poole (1969)). The observed rates were f i r s t order and were s i g n i f i c a n t l y influenced by acid and base c a t a l y s i s . The apparent heats of ac t i v a t i o n for a m p i c i l l i n degradation i n solution were 16.4, 18.3, and 9.2 kcal./mole i n buffers of pH 1.35, 4.93, and 9.78, respectively. The pH-rate p r o f i l e i n buffer solutions showed a minimum at a pH of 5.85. A m p i c i l l i n was found to be stable i n solutions - 37 -w i t h pH v a l u e s r a n g i n g f r o m 3 t o 9 when s t o r e d f o r 24 h o u r s a t 5 ° C . a n d 2 5 ° C . . The a n t i b i o t i c was u n s t a b l e i n pH 10 s o l u t i o n s w h e r e s t o r e d u n d e r s i m i l a r c o n d i t i o n s ( A l l e n a n d Sommers ( 1 9 7 1 ) ) . W e i s s a n d P a l m e r (1964) s h o w e d t h a t a m p i c i l l i n p o w d e r was s t a b l e (when s t o r e d i n a c l o s e d s y s t e m a t r o o m t e m p e r a t u r e a n d a t r e l a t i v e h u m i d i t y v a l u e s o f 43% and.81%) f o r s i x w e e k s . A m p i c i l l i n i s a l s o s t a b l e f o r n i n e w e e k s when s t o r e d a t 3 5 ° C . i n t h e same' c l o s e d s y s t e m . A m p i c i l l i n i n a q u e o u s s o l u t i o n a t a c o n c e n t r a t i o n o f a b o u t 500 m e g . / m l . h a s b e e n q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y - t h e a b s o r b a n c e b e i n g m e a s u r e d a t 25 8 n a n o m e t e r s ( S t e w a r t ( 1 9 6 7 ) ) . A m p i c i l l i n was t h e f i r s t c o m m e r c i a l l y a v a i l a b l e s e m i s y n t h e t i c p e n i c i l l i n t o p r o v i d e i n c r e a s e d a c t i v i t y a g a i n s t G r a m - n e g a t i v e b a c t e r i a . I t i s u s e f u l i n t h e t r e a t m e n t o f i n f e c t i o n s due t o s e n s i t i v e s t r a i n s o f S h i g e l l a , S a l m o n e l l a , E . c o l i , H i n f l u e n z a e , A e r o b a c t e r , a n d P . n i r a b i l i s . I t i s a l s o u s e d t o t r e a t u u r i n a i r y i E t r a c t i i n f e e . t i o n s x : G a u s e C 9 b y ; ; E . , ^ c o l i , P . n i r a b i l i s , n o n h e m o l y t i c s t r e p t o c o c c i , a n d p e n i c i l l i n G - r e s i s t a n t e n t e r o c o c c i . S i n c e t h e a n t i b i o t i c i s a c i d s t a b l e , i t i s w e l l a b s o r b e d a f t e r o r a l a d m i n i s t r a t i o n . A m p i c i l l i n m a y - b e u s e d i n t h e a n h y d r o u s f o r m o r as t h e t r i h y d r a t e . T h e d o s e v a r i e s w i t h t h e t y p e a n d t h e s e v e r i t y o f t h e i n f e c t i o n b e i n g t r e a t e d . F o r m i l d - - H r t o - m o d e r a t e l y s e v e r e - 38 -i n f e c t i o n s , the o r a l dose f o r a d u l t s i s two to f o u r gm. per day, d i v i d e d i n t o e q u a l p o r t i o n s and given every s i x hours. For severe i n f e c t i o n s , i t i s b e s t t o administer the drug p a r e n t e r a l l y in. doses ranging from f o u r t o e i g h t gm. per day. A m p i c i l l i n i s the drug of c h o i c e f o r the treatment.of S a l m o n e l l a and S h i g e l l a . The o r a l dose i s 0 . 5 t o 1 gm. every s i x hours. - 39 -I I I . EXPERIMENTAL Apparatus (a) Constant Temperature Tank A diagram o f the water.bath i s shown i n F i g u r e 5. I t c o n s i s t s of a motor-driven tumbler (Barber Colman Company), equipped w i t h e i g h t heads, f o u r on e i t h e r s i d e o f a c e n t r a l s h a f t . The spa c i n g between adjacent heads i s such t h a t e i t h e r e i g h t 250 ml. p l a s t i c r e a c t i o n b o t t l e s o r e i g h t 50 ml. c e n t r i f u g e tubes can be p l a c e d i n the tank. The tumbler r o t a t e s a t 39 r.p.m.. The r o t a t i n g p a r t o f the tumbler i s t o t a l l y immersed i n water c o n t a i n e d i n a g l a s s tank w i t h i n t e r n a l dimensions of 59 x 30 x 30 cm.. The tank i s covered w i t h b l a c k paper and f i t t e d w i t h a hard l i g h t - r e s i s t a n t cover. The temperature of the tank i s c o n t r o l l e d t o 0.5°C. of the d e s i r e d v alue w i t h a Haake E51.Thermoregulater. (b) Beckman Model DU Spectrometer . (c) Bausch and Lomb S p e c t r o n i c 505 Recording Spectrophotometer, (d) F i s h e r Accumet 220 pH meter . (e) Damon / IEC B 20-A C e n t r i f u g e . (f) Thomas Hoover C a p i l l a r y M e l t i n g P o i n t Apparatus, A.H. Thomas Company, P h i l a d e l p h i a , Pa.. (g) C e n t r i f u g e tubes, 50 ml., 28.6 x 104 mm.. (h) R e a c t i o n B o t t l e s , 250 ml., 8-250 Nalge . - 40 -F i g u r e 5. A water bath w i t h a tumbler used to determine a d s o r p t i o n of drug by k a o l i n - 41 -(i) Wang Model 600 Programmable C a l c u l a t o r . 2. Chemicals and Reagents (a) T e t r a c y c l i n e H y d r o c h l o r i d e , U.S.P. (TC-HC1). The drug was obtained from C h a r l e s E. F r o s s t and Company, Montr e a l , Canada. The drug decomposed a t from 170 - 180°C. (b) Neomycin S u l f a t e (NC-S0 4). The drug was o b t a i n e d from E l i L i l l y and Company, I n d i a n a p o l i s , Ind. The drug decomposed a t from 200 - 220°C. (c) Lincomycin.Hydrochloride Monohydrate,.U.S.P. (LC-HC1). The m e l t i n g p o i n t o f t h i s drug was found t o be '.JAY 144.8 - 150.5°C.. The a n t i b i o t i c was ob t a i n e d from the Upjohn Company, Kalamazoo, Michigan. (d) Chloramphenicol, (CM). The m e l t i n g p o i n t of the drug was found t o be 149 .4 - 1 5 0 . 5 ° C . The drug was obtained from Park, Davis and Company, L t d . , B r o c k v i l l e , O n t a r i o . (e) A m p i c i l l i n T r i h y d r a t e (AAMf). The drug was o b t a i n e d from B r i s t o l L a b o r a t o r i e s o f Canada, Montreal, Quebec. The drug decomposed a t from-180 -' ?200 oC. (f) K a o l i n N.F. T h i s substance was purchased from M a l l i n c k r o d t Chemical Works, S t . L o u i s , Miss. I t complied w i t h the t e s t s g i v e n i n the N.F. (g) H y d r o c h l o r i c A c i d , Reagent Grade. (h) S u l f u r i c A c i d , A n a l y t i c a l Grade. - 42 -(i) Sodium Hydroxide, Reagent Grade, (j) CuS0 4. 5H 20, Reagent Grade. (k) E t h a n o l , Reagent Grade. 3. P r e p a r a t i o n of The S o l u t i o n s Use i n The Experiment (a) pH 1.2 S o l u t i o n . Add 24 ml. of h y d r o c h l o r i c a c i d t o two l i t e r s o f d i s t i l l e d water. Mix w e l l . A d j u s t the pH of the s o l u t i o n t o 1.2 by f u r t h e r a d d i t i o n , dropwise, of h y d r o c h l o r i c a c i d . (b) pH 3.0 S o l u t i o n . Add h y d r o c h l o r i c a c i d , dropwise w i t h s t i r r i n g , to two l i t e r s o f d i s t i l l e d water u n t i l the pH i s 3.0. (c) pH 5.0 S o l u t i o n . Prepare a 0.1 N h y d r o c h l o r i c a c i d s o l u t i o n by d i l u t i n g 8.5 ml. of h y d r o c h l o r i c a c i d t o 1 l i t e r . Add t h i s s o l u t i o n , dropwise w i t h s t i r r i n g , to two l i t e r s o f d i s t i l l e d water u n t i l the pH reaches 5.0. (d) Stock Copper S u l f a t e S o l u t i o n . D i s s o l v e 0.400 gm. o f CuS0 4. 5H 20 i n 1000.0 ml. o f d i s t i l l e d water. (e) Sodium Hydroxide S o l u t i o n . D i s s o l v e 52.0 gm. of sodium hydroxide i n 500 ml. of d i s t i l l e d water. C o o l . Add d i s t i l l e d water t o make one l i t e r o f s o l u t i o n . - 43 -(f) A l k a l i n e Copper S u l f a t e S o l u t i o n . Mix equal volumes of stock copper s u l f a t e s o l u t i o n and sodium hydroxide s o l u t i o n . Such a s o l u t i o n c o n t a i n s 0.02% w/v copper, s u l f a t e pentahydrate and i s 0.65 M., w i t h r e s p e c t t o sodium hydroxide. T h i s s o l u t i o n must be f r e s h y prepared because, on s t a n d i n g , a copper h y d r o x i d e ' p r e c i p i t a t e may form. T h i s s o l u t i o n i s used f o r the a n a l y s i s of Lincomycin h y d r o c h l o r i d e . (g) 50% v/v S u l f u r i c A c i d S o l u t i o n . Add 500 ml. of s u l f u r i c a c i d t o 500 ml. of d i s t i l l e d water. 4. A n a l y s i s and S t a b i l i l y C h a r a c t e r i s t i c of The T e s t Drugs (a) T e t r a c y c l i n e H y d r o c h l o r i d e The p u r i t y of the drug was determined by u s i n g the absorbance r a t i o method d e s c r i b e d by Pernarowski (1969). The drug was analyzed by a s p e c t r o p h o t o m e t r y method.. ( l ) i . S p e c t r a l C h a r a c t e r i s t i c s of TC-HC1 A c c u r a t e l y weigh 100 mg. of TC-HC1. T r a n s f e r to a 100 ml. v o l u m e t r i c f l a s k w i t h the a i d of about 50 ml. of d i s t i l l e d water. D i l u t e t o 100.0 ml. w i t h d i s t i l l e d water. D i l u t e 25.0 ml. of t h i s s o l u t i o n t o 1000.0 ml. w i t h d i s t i l l e d water. Record the spectrum of t h i s s o l u t i o n u s i n g a B.a.us;qh&afidL'B@in^ - 44 -photometer and d i s t i l l e d water as the blank: See F i g u r e 6. An absorbance maxima occured a t 357 nm.. T h i s value agreed w i t h t h a t determined by u s i n g a Beckman Model DU Spectrometer. Repeat the procedure d e s c r i b e d above w i t h pH 1.2, 3.0 and 5.0 s o l u t i o n s . The wavelength of maximum a b s o r p t i o n f o r the drug i n these s o l v e n t s was found t o be 357 nm. Subsequent analyses were c a r r i e d out a t t h i s wavelength. (2) A b s o r p t i v i t y Value o f TC-HC1. A c c u r a t e l y weigh 100 mg. o f TC-HC1. D i s s o l v e and d i l u t e to 100.0 ml. w i t h d i s t i l l e d water (stock s o l u t i o n ) . D i l u t e 10.0, 15.0, 20.0, 25.0, and 30.0 ml. of the stock s o l u t i o n t o one l i t e r w i t h water. T h i s y i e l d s a s e r i e s o f s o l u t i o n s o f c o n c e n t r a t i o n 10, 15, 20, 25 and 30 mg. TC-HC1 / 1., r e s p e c t i v e l y . Record the absorbance o f these s o l u t i o n s at 357 nm. u s i n g a Beckman Model DU Spectrometer and d i s t i l l e d water as the blank. C a l c u l a t e the a b s o r p t i v i t y v a l u e by u s i n g l e a s t square a n a l y s i s . On the b a s i s of 10 d e t e r m i n a t i o n s , the a b s o r p t i v i t y v alue f o r TC-HC1 was 31.70-':-(standard e r r o r o f estimate i s equal t o 0.002). Repeat the above procedure w i t h pH 1.2, 3.0 and 5.0 s o l u t i o n s . The a b s o r p t i v i t y v a l u e s of the drug i n pH 1.2, - 45 -u U O 0.8 U 0.7 0.6 U 0.5 U 0.4 I-380 370 360 Wavelength, nm. 350 F i g u r e 6. S p e c t r a l C h a r a c t e r i s t i c s of T e t r a c y c l i n e H y d r o c h l o r i d e ( 25.0 mg./lOOO ml. ). -. 46 -3.0, and 5.0 s o l u t i o n s were found t o be w i t h i n the l i m i t s o f the above va l u e . T h i s a b s o r p t i v i t y value (31.70) was chosen f o r a l l subsequent a n a l y s e s . The c o n c e n t r a t i o n s of TC-HC1 were determined by u s i n g Beer's Law. A G = ^ T F ( E q - 5 ) Where, c = C o n c e n t r a t i o n (gm./l^ A = Absorbance s a g = A b s o r p t i v i t y value b = C e l l l e n g t h (1 cm.) (3) S t a b i l i t y o f TC-HC1 i n Aqueous S o l u t i o n . The s t a b i l i t y o f the drug i n aqueous s o l u t i o n over a p e r i o d o f 10 hours, a t 3 7 . 0 ° C , was i n v e s t i g a t e d . P i p e t 20.0 ml. of the stock s o l u t i o n ( c o n c e n t r a t i o n = 1 mg./ml.) i n t o a 50 ml. c e n t r i f u g e tube. Add 20.0 ml. of d i s t i l l e d water to y i e l d a f i n a l c o n c e n t r a t i o n o f 0.5 mg./ml.. Stopper the c e n t r i f u g e tube w i t h a p l a s t i c cap and p l a c e the tube i n t o the constant temperature tank ( 3 7 . 0 ° C ) . Tumble f o r 10 hours. D i l u t e a s u i t a b l e a l i g u o t o f the s o l u t i o n t o the working range f o r TC-HC1 and r e c o r d the spectrum of the s o l u t i o n oh the lausjch aaridLLomb SSpect r o n i c 5505 SSpe'ctrppho tome t e r . The spectrum of t h i s s o l u t i o n was found t o be the same - 47 -as t h a t o b t a i n e d f o r f r e s h l y prepared aqueous s o l u t i o n , and f o r s o l u t i o n s s t o r e d at room temperature f o r 10 hours. No changes i n a b s o r p t i o n maximum or a b s o r p t i v i t y v a l u e were observed. (b) Neomycin S u l f a t e An a n a l y t i c a l procedure f o r t o t a l neomycin. B and C, proposed by Dutcher and h i s coworkers (1953), was used f o r the d e t e r m i n a t i o n o f NC-S0 4. (1) P r e p a r a t i o n o f a C a l i b r a t i o n Curve A c c u r a t e l y weigh 150.37 mg. of NC-SO^((equivalent to 100 mg. of neomycin base) and d i s s o l v e i n 100.0 ml. of d i s t i l l e d water. T h i s i s the stock s o l u t i o n . P i p e t 0.5, 0.6, 0.7,0.8 and 0.9 ml. a l i q u o t s o f the stock s o l u t i o n i n t o each o f f i v e 15 x 150 nm. t e s t tubes and d i l u t e to 10.0 ml. wi t h 50% v/v s u l f u r i c a c i d s o l u t i o n . Mix the s o l u t i o n s w e l l . P l a c e the tubes v e r t i c a l l y i n t o a b o i l i n g water bath f o r 1.5 hours. Cool to room temperature. Record.the spectrum o f the 0.9 mg. neomycin / 10.0 ml. s o l u t i o n , o v e r a r wavelength range of 200 t o 350 nm. u s i n g the S p e c t r o n i c 505 Spectrophotometer and 50% v/v s u l f u r i c a c i d s o l u t i o n as the blank. The wavelength o f maximum a b s o r p t i o n occuired a t from 284,to 285 nm. See F i g u r e 9. Maximum a b s o r p t i o n occured a t 285 nm. when determined - 48 -0.8 L 0.7 U CD U J 0.6 -u o cn 0.5 I-0.4 I-300 290 280 270 Wavelength, nm. F i g u r e 7. S p e c t r a l C h a r a c t e r i s t i c s of Neomycin S u l f a t e . - 49 -by u s i n g the Beckman Model DU Spectrometer. The a b s o r p t i v i t y v alue o f NC-S0 4 was determined at 285 nm. Record the absorbance of each o f the above s o l u t i o n a t the wavelength o f maximum a b s o r p t i o n . C a l c u l a t e the a b s o r p t i v i t y value by u s i n g l e a s t square a n a l y s i s . The above procedure was repeated three times. Beer's Law was obeyed. On the b a s i s o f 15 d e t e r m i n a t i o n s , the a b s o r p t i v i t y v alue f o r t h i s drug was 8.20 (standard e r r o r of estimate i s equal to 0.006). On the b a s i s of p r e l i m i n a r y experiments, i t was found t h a t 50% v/v s u l f u r i c a c i d s o l u t i o n was the o p t i m a l a c i d c o n c e n t r a t i o n f o r the purposes o f t h i s i n v e s t i g a t i o n . The p u b l i s h e d method recommended the use of a 40% v/v s u l f u r i c a c i d s o l u t i o n (Dutcher (1953)). Repeat the procedure d e s c r i b e d above w i t h pH 1.2, 3.0. and 5.0 s o l u t i o n s . The maximum a b s o r p t i o n f o r the drug i n such s o l u t i o n occured a t 2 85 nm.. The a b s o r p t i v i t y v a l u e s were e x p e r i m e n t a l l y s i m i l a r t o the value given above,. (2) S t a b i l i t y o f NC-S0 4 i n Aqueous S o l u t i o n The s t a b i l i t y o f the drug i n aqueous s o l u t i o n , over a p e r i o d of ten hours, was i n v e s t i g a t e d . P i p e t 40.0 ml. o f the stock s o l u t i o n i n t o a 50.0 ml. c e n t r i f u g e tube. Place the stoppered c e n t r i f u g e tube i n t o the constant temperature - 50 -tank (37.0°C.) and tumble f o r ten hours. Remove the tube from the tank. Pipet. 0.9 ml. i n t o a 15 x 150 nm. t e s t tube. Add 9.1 ml. o f 50% v/v s u l f u r i c a c i d s o l u t i o n . Mix w e l l . P l a c e v e r t i c a l l y i n . a b o i l i n g water bath f o r 1.5 hours. Cool t o room temperature. Record the spectrum of t h i s s o l u t i o n u s i n g the S p e c t r o n i c 505 Spectrophotometer. The spectrum;, the wavelength of maximum a b s o r p t i o n , and the a b s o r p t i v i t y v alue f o r the drug i n s o l u t i o n were the same as t h a t observed at room temperature, (c) Lincomycin H y d r o c h l o r i d e The a l k a l i n e copper s u l f a t e assay method was used f o r the a n a l y s i s o f LC-HC1. T h i s method depends upon the formation o f a "U.V.-Chromophore" by u s i n g a l k a l i n e copper s u l f a t e s o l u t i o n . The method was developed by C h u l s k i ( ( 1 9 6 2 ) . (1) S p e c t r a l C h a r a c t e r i s t i c s f o r and A b s o r p t i v i t y Value of LC-HC1 Weigh a c c u r a t e l y 125 mg. o f LC-HC1. D i s s o l v e and d i l u t e t o 250.0 ml. wi t h d i s t i l l e d water. T h i s i s the stock s o l u t i o n . Add 3.0 ml. of the stock s o l u t i o n to 10.0 ml. o f the a l k a l i n e copper s u l f a t e s o l u t i o n . Record the spectrum over the 200 t o 350 nm. wavelength range u s i n g the Bausch and Lomb S p e c t r o n i c 505 Spectrophotometer. The blank s o l u t i o n i s prepared by s u b s t i t u t i n g 3.0 ml. o f water f o r the a n t i b i o t i c s o l u t i o n . - 51 -An absorbance maximum occured a t 277 nm. See F i g u r e 8. T h i s value was confirmed by u s i n g the Beckman Model DU S p e c t r o n i c . Subsequent analyses were c a r r i e d out a t t h i s wavelength. The a b s o r p t i v i t y v alue was determined at t h i s wavelength. P i p e t 1.0, 1.5, 2.0, 2.5 and 3.0 ml. o f the stock s o l u t i o n i n t o f i v e 25 ml. v o l u m e t r i c f l a s k s . Add 2.0, 1.5, 1.0 and 0.5 ml. of d i s t i l l e d water t o a s e r i e s o f v o l u m e t r i c f l a s k c o n t a i n i n g 1.0, 1.5, 2.0 and 2.5 ml. of LC-HC1 s o l u t i o n , r e s p e c t i v e l y . P i p e t 10.0 ml. o f a l k a l i n e copper s u l f a t e to each o f the f i v e v o l u m e t r i c f l a s k s . a r i d mixed w e l l . Record the absorbance value of these s o l u t i o n u s i n g the Beckman Model DU Spectrometer. The a b s o r p t i v i t y v alue was determined by u s i n g l e a s t square a n a l y s i s ; On the b a s i s of 10 d e t e r m i n a t i o n s , the a b s o r p t i v i t y value islequalst0q\7s.;39tc('S'tan;dardter-rordodrestimateqislequal to 0.004) . Repeat the above procedures w i t h pH 1.2, 3.0 and 5.0 s o l u t i o n s . The wavelength of maximum a b s o r p t i o n f o r such s o l u t i o n s was 277 nm.. The a b s o r p t i v i t y values f o r the drug i n pH 1.2, 3.0, and 5.0 s o l u t i o n s were found to be w i t h i n the l i m i t s of the above v a l u e . (2) S t a b i l i t y o f LC-HC1 i n Aqueous S o l u t i o n The s t a b i l i t y of the drug i n aqueous s o l u t i o n , over a - 52 -F i g u r e 8. S p e c t r a l C h a r a c t e r i s t i c s o f L i n c o m y c i n H y d r o c h l o r i d e . - 53 -p e r i o d o f s i x hours, was i n v e s t i g a t e d . P i p e t 20.0 ml. o f the stock s o l u t i o n t o a 50,.ml;..:.. c e n t r i f u g e tube. Add 20.0 ml. of d i s t i l l e d water to t h i s tube.and stopper w i t h a p l a s t i c cap. Tumble i n the constant temperature tank (37.0°C.) f o r s i x hours. Remove the tube from the tank. Mix 3.0 ml. of t h i s s o l u t i o n w i t h 10.0 ml. of a l k a l i n e copper s u l f a t e s o l u t i o n . Record the spectrum of t h i s s o l u t i o n i n the manner as d e s c r i b e d above. The r e s u l t s o f the s t a b i l i t y study i n d i c a t e d no change - i n the wavelength.of maximum a b s o r p t i o n or a b s o r p t i v i t y value of LC-HC1 over a p e r i o d o f s i x hours a t 37.0°C. (d) Chloramphenicol A spectrophotometrie method was used f o r the d e t e r m i n a t i o n o f CM. (1) S p e c t r a l C h a r a c t e r i s t i c s f o r and A b s o r p t i v i t y Value of CM A c c u r a t e l y weigh 250 mg. o f CM. D i s s o l v e i n 5.0 ml. o f e t h a n o l and d i l u t e t o 250.0 ml. w i t h d i s t i l l e d water. T h i s i s the stock s o l u t i o n . D i l u t e 20.0 ml. of the stock s o l u t i o n t o one l i t e r w i t h d i s t i l l e d water. Record the spectrum of t h i s s o l u t i o n u s i n g the BausbhaandLLomb S p e c t r o n i c 505 Spectrophotometer and d i s t i l l e d water as the blank. - 54 -An. absorbance maxima occured a t 278 nm. See F i g u r e 9. T h i s wavelength o f maximum a b s o r p t i o n was confirmed by u s i n g the Beckman Model DU Spectrometer. The . a b s o r p t i v i t y value was determined a t 278 nm. D i l u t e 10.0, 15.0, 20.0, 25.0 and 30.0 ml. a l i q u o t s of the stock s o l u t i o n to one l i t e r w i t h d i s t i l l e d water. T h i s y i e l d s a s e r i e s o f s o l u t i o n of c o n c e n t r a t i o n 10, 15, 20, 25 and 30 mg. CM/1., r e s p e c t i v e l y . Record the absorbance o f these solutionsuusdingtifeheBBe'ckmanMModelDDUS Spectrometer and d i s t i l l e d water as the blank. C a l c u l a t e the a b s o r p t i v i t y value by u s i n g l e a s t square a n a l y s i s . On the b a s i s o f ten de t e r m i n a t i o n s , . t h e a b s o r p t i v i t y value o f CM a t 278 nm. was found to be 29.23 (standard e r r o r of estimate i s equal t o 0.00 3). Repeat the procedures d e s c r i b e d above w i t h pH 1.2, 3.0, and 5.0 s o l u t i o n s . The wavelength o f maximum a b s o r p t i o n and a b s o r p t i v i t y . . value f o r the drug were e x p e r i m e n t a l l y s i m i l a r to the value g i v e n above. (2) S t a b i l i t y o f CM i n Aqueous S o l u t i o n The s t a b i l i t y o f the drug i n aqueous s o l u t i o n , over a p e r i o d of e i g h t hours, was s t u d i e d . P i p e t 20.0 ml. of the stock s o l u t i o n to a 50 ml. c e n t r i f u g e tube. Add 20.0 ml. of d i s t i l l e d water - 55 -cm - 56 -to produce a f i n a l c o n c e n t r a t i o n o f 0.5 mg. GM/ml. Stopper the tube w i t h a p l a s t i c cap and tumble i n the constant temperature tank (37.0°C.) f o r e i g h t hours. Remove the tube from the tank and d i l u t e a 4.0 ml. a l i q u o t o f the s o l u t i o n to 100.0 ml. wit h d i s t i l l e d water. Record the spectrum of the s o l u t i o n on the Baus'chaand Lomb S p e c t r o n i c 505 Spectrophotometer. The spectrum, wavelength o f maximum a b s o r p t i o n , and the a b s o r p t i v i t y v alue a t 278 nm. were unchanged a f t e r e i g h t hours. (e) A m p i c i l l i n T r i h y d r a t e A m p i c i l l i n t r i h y d r a t e was assayed s p e e t r o p h o t o m e t r i c a l l y . (1) A b s o r p t i v i t y Value o f AM Weigh a c c u r a t e l y 500 mg. of AM. D i s s o l v e i n 250.0 ml. o f d i s t i l l e d water. T h i s i s the stock . s o l u t i o n . D i l u t e 25.0 ml. of the stock s o l u t i o n to 100.0 ml. with d i s t i l l e d water. Record the spectrum of the s o l u t i o n u s i n g the Bausjeh, and / Lomb S p e c t r o n i c 505 Spectrophotometer and d i s t i l l e d water as the blank. Maximum a b s o r p t i o n s occured at 256, 261 and 267 nm;. See F i g u r e 10. The 256 nm. wavelength was s e l e c t e d f o r a l l subsequent a n a l y s e s . T h i s maximum was confirmed by u s i n g the Beckman Model DU Spectrometer. The a b s o r p t i v i t y v alue was determined a t t h i s wavelength. - 57 -0.8 0.7 1-u •S 0.6 o t n 0.5 0.4 270 260 250 Wavelength, nm. 240 F i g u r e 10. S p e c t r a l C h a r a c t e r i s t i c s of-..-. A m p i c i l l i n T r i h y d r a t e . - 58 -D i l u t e 20.0, 25.0, 30.0, 35.0, 40.0 and 50.0 ml. a l i q u o t s of the stock s o l u t i o n to 100.0 ml. w i t h d i s t i l l e d water.. F i n a l drug c o n c e n t r a t i o n s are 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0 gm. AM/1., r e s p e c t i v e l y . Record the absorbance of these X -s o l u t i o n s u s i n g the Beckman Model DU Spectrometer and d i s t i l l e d water as the blank. C a l c u l a t e the a b s o r p t i v i t y v alue by u s i n g l e a s t square a n a l y s i s . On the b a s i s of 12 d e t e r m i n a t i o n s , the a b s o r p t i v i t y value o f AM at 256 nm. was 0.83 (standard e r r o r o f estimate' i s equal to 0.002). Repeat the above procedure w i t h pH 1.2, 3.0, and 5.0 s o l u t i o n s . R e s u l t s , s i m i l a r to those g i v e n above, were ob t a i n e d . (2) S t a b i l i t y o f AM i n Aqueous S o l u t i o n The s t a b i l i t y of the drug i n aqueous s o l u t i o n , over a p e r i o d of s i x hours, was i n v e s t i g a t e d . P i p e t 40.0 ml. of the stock s o l u t i o n i n t o a 50 ml. c e n t r i f u g e tube. P l a c e i n the r e a c t i o n tank (37.0°C.) and tumble the stoppered tube f o r s i x hours. Record the spectrum of t h i s s o l u t i o n u s i n g the s p e c t r o n i c 505 Spectrophotometer. The spectrum of t h i s s o l u t i o n was found to be the same as t h a t shown i n F i g u r e 10. The a b s o r p t i v i t y v a l u e , a t 256 nm., was unchanged a f t e r s i x hours. - 59 -5. A d s o r p t i o n C h a r a c t e r i s t i c s of The.Test Drugs (a) Determination of E q u i l i b r i u m Times The method.used to determine e q u i l i b r i u m c o n d i t i o n s f o r TC-HC1 i s o u t l i n e d below and i s i l l u s t r a t i v e ; o f ;.that used f o r a l l t e s t drugs. Data f o r such d r u g s ( i . e . , e q u i l i b r u i m times, drug c o n c e n t r a t i o n s , etc.) w i l l be i n c l u d e d i n the s e c t i o n on r e s u l t s . A c c u r a t e l y weigh 500 mg. o f TC-HC1. D i s s o l v e i n 500.0 ml. of d i s t i l l e d water. T r a n s f e r 40.0 ml. of TC-HC1 s o l u t i o n i n t o a 50 ml. c e n t r i f u g e tube c o n t a i n i n g 500.0 mg. of k a o l i n . Stopper the tube, p l a c e i n the r e a c t i o n tank (37.0°C.) and tumble f o r 15 minutes. Remove the tube from the tank and c e n t r i f u g e (10,000 r.p.m. f o r 10 minutes). Analyse f o r TC-HC1 i n the supernatant s o l u t i o n . C a l c u l a t e the amount of drug adsorbed by k a o l i n by s u b t r a c t i n g the amount remaining i n s o l u t i o n from the i n i t i a l c o n c e n t r a t i o n . Repeat the procedure d e s c r i b e d above but leave tubes i n the r e a c t i o n tank f o r 30 minutes, 1, 2, 3 , 4 , and 5 hours, r e s p e c t i v e l y . I t was found t h a t e q u i l i b r i u m c o n d i t i o n s were reached a f t e r f o u r hours i n the r e a c t i o n tank. (b) A d s o r p t i o n Isotherms i n Water a t 37.0 PC. Weigh a c c u r a t e l y 500 mg. of TC-HC1. D i s s o l v e - 60 -i n 500.0 ml. of d i s t i l l e d water. T r a n s f e r 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0 and 40.0 ml. of the drug s o l u t i o n i n t o each o f e i g h t 50 ml. c e n t r i f u g e tubes, each tube c o n t a i n i n g 500.0 mg. of k a o l i n . To each tube add s u f f i c i e n t d i s t i l l e d water t o g i v e a f i n a l volume o f 40.0 ml. Stopper the tubes and tumble i n the constant temperature tank (37.0°C.) f o r f o u r hours. Remove the tubes from the tank and c e n t r i f u g e the suspensions (10,000 r.p.m. f o r 10 minutes). P i p e t an a l i q u o t of the c l e a r supernatant s o l u t i o n i n t o a v o l u m e t r i c f l a s k and d i l u t e w i t h s u f f i c i e n t d i s t i l l e d water to g i v e a s o l u t i o n with a TC-HCl c o n c e n t r a t i o n w i t h i n the optimum range f o r s p e c t r o p h o t o m e t r i c a n a l y s i s . C a l c u l a t e the amount of TC-HCl adsorbed on k a o l i n by s u b t r a c t i n g the amount of drug remaining i n the s o l u t i o n from the i n i t i a l c o n c e n t r a t i o n . Graph the a d s o r p t i o n isotherm, (c) A d s o r p t i o n S t u d i e s i n pH 1.'2, 3.0 and 5.0 S o l u t i o n s A c c u r a t e l y weigh 500 mg. of TC-HCl and d i s s o l v e i n 100.0 ml. of pH 1.2 s o l u t i o n . Measure the pH o f the s o l u t i o n w i t h the F i s h e r Accumet 220 pH meter. T r a n s f e r the s o l u t i o n q u a n t i l a t i v e l y to a 500 ml. v o l u m e t r i c f l a s k and make to volume wit h pH 1.2 s o l u t i o n . T r a n s f e r 5.0, 10.0, 15.0, - 61 -20.0, 25.0, 30.0, 35.0, and 40.0 ml. o f the drug s o l u t i o n t o each of e i g h t 50 ml. c e n t r i f u g e tubes, each tube c o n t a i n i n g 500.0 mg. of k a o l i n . A d j u s t the volume of each tube to 40.0 ml. with pH 1.2 s o l u t i o n . Stopper and tumble the tubes i n the constant temperature tank (37.0°C.) f o r f o u r hours. C e n t r i f u g e the tubes and t r a n s f e r the c l e a r supernatant l i q u i d s i n t o e i g h t beakers. Measure the pH value of each s o l u t i o n . P i p e t a n . a l i q u o t o f the s o l u t i o n i n t o a v o l u m e t r i c f l a s k and d i l u t e with pH 1.2 s o l u t i o n to the optimum c o n c e n t r a t i o n range f o r s p e c t r o p h o t o m e t r i c a n a l y s i s and determine the c o n c e n t r a t i o n of TC-HC1. C a l c u l a t e the amount of drug adsorbed on k a o l i n . Graph the a d s o r p t i o n isotherm. Repeat the above procedure u s i n g pH 3.0 and 5.0 s o l u t i o n s . 6. D e s o r p t i o n C h a r a c t e r i s t i c s o f The T e s t Drugs Desorpt i o n data was obtained f o r t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin s u l f a t e and l i n c o m y c i n h y d r o c h l o r i d e . R e s u l t s i n d i c a t e d t h a t no chloramphenicol and a m p i c i l l i n t r i h y d r a t e were adsorbed onto k a o l i n . T h e r e f o r e , d e s o r p t i o n experiments f o r these two a n t i b i o t i c s were not necessary. A g e n e r a l procedure i s given below. (1) Prepare a sample f o r the d e s o r p t i o n study by e q u i l i b r a t i n g the drug s o l u t i o n and k a o l i n a t 37.0° C . C o n c e n t r a t i o n s used are given i n the l a t t e r p a r t . o f t h i s s e c t i o n . (2) T r a n s f e r the e q u i l i b r a t e d sample i n t o a 250 ml. r e a c t i o n b o t t l e . Add a c a l c u l a t e d volume of d i s t i l l e d water and tumble i n the r e a c t i o n tank f o r a s p e c i f i e d p e r i o d of time. C e n t r i f u g e . C a l c u l a t e the amount of drug remaining on k a o l i n . S i m i l a r procedures were used f o r each of the t e s t drugs. D i f f e r e n c e s w i t h r e s p e c t to i n i t i a l c o n c e n t r a t i o n s , d e s o r p t i o n times, and time f o r e q u i l i b r i u m a d s o r p t i o n are i n c l u d e d i n the s e c t i o n on r e s u l t s . A s p e c i f i c procedure f o r TC-HCl i s given below. (1) P r e p a r a t i o n of A Sample f o r The D e s o r p t i o n S t u d i e s A c c u r a t e l y weigh 250 mg. of TC-HCl and d i s s o l v e i n 250.0 ml. of d i s t i l l e d water. T r a n s f e r 40.0 ml of the ::c i-'-'..-the s o l u t i o n i n t o a 50 ml. c e n t r i f u g e tube c o n t a i n i n g 500.0 mg. of k a o l i n . Stopper the tube and tumble i n the r e a c t i o n tank (37.0°C.) f o r four hours. Prepare f o u r samples. (2) D e s o r p t i o n S t u d i e s T r a n s f e r f o u r samples to f o u r 250 ml. r e a c t i o n b o t t l e s . Add 160.0 ml. of d i s t i l l e d water t o each b o t t l e . L a b e l the b o t t l e s , 1, 2, 3, and 4. A f t e r d i l u t i o n , c e n t r i f u g e b o t t l e no. 1. immediately. Analyze the supernatant l i q u i d . Determine the - 63 -amount of TOHC1 remaining on k a o l i n . T h i s i s the amount of TC-HC1 a t time zero. B o t t l e s no. 2, 3, and 4 were tumbled i n the constant temperature tank (37.0°C.) f o r one, t h r e e , and s i x hours r e s p e c t i v e l y . At the end of each time p e r i o d , continue the d e s o r p t i o n procedure as d e s c r i b e d above. - 6 4 -IV. RESULTS AND DISCUSSION 1. Preliminary Evaluation of Kaolin The following procedure was used to determine the presence or absence of impurities i n kaolin which may absorb radiant energy. Weigh accurately 500.0 mg. of kaolin and suspend i n 40.0 ml. of d i s t i l l e d water i n a 50 ml. centrifuge tube. Tumble the stoppered tube i n a constant temperature tank (37.0°C.) for four hours. Remove the tube from the tank and centrifuge at 10,000 r.p^m. for 10 minutes. Record the spectrum of the clear supernatant solution using the Bausch and Lomb Spectronic 505 Spectrophotometer over the 200 - ; " " \ 450 nm. wavelength range. Use d i s t i l l e d water as the blank. No absorption of radiant energy occured at any of the above wavelengths. However, for experimental purpose, the supernatant solution prepared i n the manner indicated above was used as a spectrophotometric blank for adsorption studies i n water. For studies i n pH 1.2, 3.0, and 5.0 solutions, blank solutions were prepared i n an analogous manner. 2. Determination of Equilibrium Times Preliminary studies showed that chloramphenicol and a m p i c i l l i n trihydrate were not adsorbed by kaolin; Therefore, equilibrium time studies were not carried out for these two drugs.-(a) Equilibrium Time for TC-HC1 - 65 -The amount, of. TC-HCl adsorbed by one gm. of kaolin with respect to time i s shown i n Table 1.. These r e s u l t s show- that equilibrium conditions are reached after four hours i n the constant temperature tank. Two TC-HCl concentrations - 1.0 mg./ml.and 0.5 mg./ml. - were used i n these investigations. No. concentration e f f e c t was. observed and t h i s time period was, therefore, used for a l l TC-HCl adsorption studies. (b) Equilibrium Time for NC-S04 I t was found that equilibrium conditions are reached afte r four hours i n the.constant temperature tank. See Table 2. This equilibrium time period was used for a l l NC-SO^ adsorption studies. (c) Equilibrium Time for LC-HC1 Similar equilibrium time studies were carr i e d out for LC-HC1. Results are shown i n Table 3 and indicate that equilibrium conditions are reached within 30 minutes. However, an experimentally convenient two hours time period was .us.ed-Lf.6SCa 1 JbdEC*H€lbaas©r^.ti>©n studies. Equilibrium times for TC-HCl, NC-S04 and LC-HC1 i n pH 1.2, 3.0 and 5.0 solutions were the same as those i n water i 3. The Adsorption and Desorption Characteristics of The Test Drugs . " (a) Tetracycline Hydrochloride (1) Adsorption Isotherms i n Water at 37.0°C. - 66 -T a b l e 1 . T h e E f f e c t o f T i m e o n T h e A m o u n t o f T e t r a c y c l i n e H y d r o c h l o r i d e A d s o r b e d p e r Gm. o f K a o l i n T i m e o f T u m b l i n g ( m i n . ) M g . A d s o r b e d p e r Gm. K a o l i n * 0 1 5 . 5 9 -15 1 7 . 3 6 30 1 7 . 7 3 60 1 7 . 7 5 120 1 8 . 9 9 180 2 0 . 8 5 240 2 0 . 5 2 300 2 0 . 3 7 T a b l e 2 . T h e E f f e c t o f T i m e o n T h e A m o u n t o f N e o m y c i n S u l f a t e * * A d s o r b e d p e r G M . o f K a o l i n T i m e o f T u m b l i n g ( m i n . ) M g . A d s o r b e d p e r Gm. K a o l i n * 15 1 5 . 6 1 30 1 6 . 1 0 60 1 6 . 4 9 120 1 6 . 1 0 180 1 6 . 4 9 240 1 6 . 5 9 300 1 6 . 5 9 * C a l c u l a t e d o n t h e b a s i s o f two. d e t e r m i n a t i o n s ? . .**.*.. C a l c u l a t e d w i t h r e s p e c t t o n e o m y c i n b a s e . - 67 -Table 3. The E f f e c t o f Time on The Amount o f Lincomycin H y d r o c h l o r i d e * Adsorbed per Gm. of K a o l i n Time of Tumbling (min.) Mg. Adsorbed per Gm. K a o l i n * * 15 ~< 6.64 30 7.06 60 7.12 90 7.06 120 7.16 180 7.20 240 7.07 * I n i t i a l c o n c e n t r a t i o n : 0.5 mg./ml. ** C a l c u l a t e d on the b a s i s o f two d e t e r m i n a t i o n s . - 68 -The data f o r the adsorption..of =TC-HCl~by k a o l i n i n water are shown i n Table 4. The amount of TC-HC1 adsorbed by k a o l i n was c a l c u l a t e d by s u b t r a c t i n g the amount remaining i n s o l u t i o n a t e q u i l i b r i u m from the i n i t i a l c o n c e n t r a t i o n . For example -The i n i t i a l amount of TC-HC1 i n 40.0 ml. H 20 i s equal to 25.0 mg. A f t e r tumbled f o r fou r hours, the tube was c e n t r i f u g e d . A 2.0 ml. a l i q u o t o f the c l e a r supernatant s o l u t i o n was d i l u t e d to 100.0 ml. wit h d i s t i l l e d water. T h e r e f o r e , the d i l u t i o n f a c t o r i s equal t o 50. The -absorbance v a l u e , a t 357 nm., f o r t h i s s o l u t i o n i s 0.499, t h i s v a l u e being the average o f two d e t e r m i n a t i o n s . The e q u i l i b r i u m c o n c e n t r a t i o n (c) of t h i s s o l u t i o n i s c a l c u l a t e d by s u b s t i t u t i o n the absorbance value (0.499) and the a b s o r p t i v i t y value o f TC-HC1 a t 357 nm. (31.70) i n t o Equation 5 and m u l t i p l y i n g by the d i l u t i o n f a c t o r (50). T h e r e f o r e , c, the e q u i l i b r i u m c o n c e n t r a t i o n was equal t o 392.3 mg./1000ml.. The amount of drug remaining i n the s o l u t i o n was equal t o 392.3 x 0.0 4 or 15.69 mg. The amount of drug adsorbed by 500.0 mg. of k a o l i n was equal t o 9.31 mg. T h e r e f o r e , x, the amount of drug adsorbed by one gm. of m k a o l i n , was equal t o 18.62 mg. A p l o t of x versus c i s shown i n F i g u r e 11. T h i s m graph i n d i c a t e s t h a t the a d s o r p t i o n i s of the Langmuir or L2 type (see F i g u r e 2 ) , a c c o r d i n g t o the c l a s s i f i c a t i o n of G i l e s and his-coworkers (1960). A s t r a i g h t l i n e was Table 4. Adsorption.Data f o r T e t r a c y c l i n e Hydrochloride;by K a o l i n * I n i t i a l C o n c e n t r a t i o n E q u i l i b r i u m Mg. Adsorbed per c/(x/m) (mg./lOOO ml.) C o n c e n t r a t i o n (c) Gm. (x/m) £ range (mg./lOOO ml.) 125 26.73 7.86 + 0.04 3.40 .•."SO ^'-"t^ <*•"+"' ft . n^-250 75.79 13.94 + 0.05, 5.44 375 159.43 17.25 + 0.20 9 .25 500 271.23 18 . 3 0 ±:0.63 14. 82 625 392.30 18.62 +0.19 21.07 750 512.58 18.99 + 0.38 26.99 875 639.94 18.81+0.14 34.03 1000 762.58 18.99+0.14 40.15 * 500 mg. of k a o l i n were suspended i n 40.0 ml. of drug s o l u t i o n i n water a t a temperature o f 37°C. C a l c u l a t e d values are'based on two. determinations. •H rH o 200 400 600 E q u i l i b r i u m C o n c e n t r a t ion (mg./1000 ml.) F i g u r e 11. A d s o r p t i o n isotherm at 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n water. - 71 -o b t a i n e d b y p l o t t i n g c v e r s u s c ( c o r r e l a t i o n f a c t o r = 0.999^6) . x / m See F i g u r e 1 2 . T h e b e s t s t r a i g h t l i n e t h r o u g h t h e e x p e r i m e n t a l d a t a was c a l c u l a t e d b y t h e m e t h o d o f l e a s t s q u a r e s . T h e v a l u e s o f t h e L a n g m u i r c o n s t a n t s , a a n d b , w e r e o b t a i n e d f r o m t h e r e c i p r o c a l s o f t h e i n t e r c e p t a n d s l o p e v a l u e s ( t h a t i s , _ 1 a n d 1) o f t h i s s t r a i g h t l i n e . T h e s e v a l u e s w e r e ab b 0 . 0 5 0 4 a n d 1 . 5 1 5 5 , r e s p e c t i v e l y . T h e r e f o r e , t h e a d s o r b e n t c a p a c i t y , b , i s e q u a l t o 1 9 . 8 4 m g . / g m . a n d t h e a d s o r p t i o n c o e f f i c i e n t , a , i s e q u a l t o 0 . 0 3 3 3 l i t e r / m g . (See T a b l e 5 . ) (2) A d s o r p t i o n I s o t h e r m s i n pH 1 . 2 , 3 . 0 , a n d 5 . 0 S o l u t i o n s T h e s e s o l u t i o n ' s . . w e r e s p r e p a r e d a b y r M s i n g h y d r o c h l o r i c a c i d . No b u f f e r s w e r e u s e d t o m a i n t a i n t h e pH o f t h e s e s o l u t i o n s b e c a u s e t h e b u f f e r i n g r e d i e n t s may i n t e r f e r e w i t h t h e a d s o r p t i o n o f d r u g s b y k a o l i n . F u r t h e r , t h e pH v a l u e s c h a n g e d d u r i n g t h e c o u r s e o f a n e x p e r i m e n t . T h e r e f o r e , t h e v a l u e s r e p o r t e d i n t h e s e a d s o r p t i o n s t u d i e s w e r e t h o s e o b s e r v e d b y r e c o r d i n g t h e pH o f t h e c l e a r s u p e r n a t a n t s o l u t i o n s a f t e r e q u i l i b r i u m c o n d i t i o n s h a d b e e n a t t a i n e d ( i . e . , f o u r h o u r s ) . A r m s t r o n g a n d C l a r k e (19 73) r e p o r t e d t h a t c h l o r i d e , s u l f a t e a n d c i t r a t e i o n s i n t e r f e r e d w i t h t h e u p t a k e o f c r y s t a l v i o l e t b y k a o l i n . T h e y n o t e d t h a t t h e d e g r e e o f i n t e r f e r e n c e d e c r e a s e d w i t h t h e v a l e n c y o f t h e i n t e r f e r i n g i o n . T h e r e f o r e , i n pH 1 . 2 , 3 . 0 , a n d 5 . 0 s o l u t i o n s , c h l o r i d e i o n s may i n t e r f e r e w i t h t h e a d s o r p t i o n o f t h e t e s t d r u g s 200 400 600 E q u i l i b r i u m C o n c e n t r a t i o n (mg./lOOO ml.) F i g u r e 12. Langmuir isotherm at 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n water. Table 5. A d s o r p t i o n o f T e t r a c y c l i n e H y d r ochloride by K a o l i n at 37°C. A d s o r p t i o n Medium E q u i l i b r i u m pH A d s o r p t i o n Isotherm Langmuir Constants a b 1 i t er/mg. mg. /gm. F r e u n d l i c h Constants k" ' 1/n mg./gm. water 3.4 Langmuir 0.0333 19.84 pH 1.2 s o l u t i o n 1.2 F r e u n d l i c h 1.05 0.53 pH 3.0 s o l u t i o n 3.2 Langmuir 0.0198 20.02 pH 5.0 s o l u t i o n 5.4 Langmuir 0.0367 20.05 - 74 -but such, i n t e r f e r e n c e should be. minimal. The a d s o r p t i o n isotherms f o r TC-HC1 i n pH 1.2, 3.0, and 5.0 are shown i n F i g u r e 13. In pH 3.0 and. 5.0 s o l u t i o n s , the a d s o r p t i o n o f TC-HC1 by k a o l i n i s s i m i l a r t o t h a t i n water and the isotherms are of the Langmuir type. T h e r e f o r e , there i s a l i n e a r r e l a t i o n s h i p ^ between c and c. See x/m F i g u r e 14. The Langmuir c o n s t a n t s , a and b, were c a l c u l a t e d and are giv e n i n Table 5. From F i g u r e 14, the a d s o r p t i o n i s o t h e r m i n pH 1.2 s o l u t i o n i s o f the F r e u n d l i c h type.. A p l o t of l o g x versus l o g c m g i v e s a s t r a i g h t l i n e (Figure 15). The c o r r e l a t i o n f a c t o r f o r t h i s s t r a i g h t l i n e i s equal to 0.999.) The c o n s t a n t s , k and 1, i n the Equation 2, are 1.049 and 0.527, r e s p e c t i v e l y , n These isotherms (see F i g u r e 14) i n d i c a t e t h a t the a d s o r p t i o n of TC-HC1 by k a o l i n decreased i n pH 1.2 j and 3.0 s o l u t i o n s and i n c r e a s e d i n pH 5.OS/ The a d s o r p t i o n i s o t h e r m i n pH 5.01 ;'solution i s s i m i l a r t o t h a t i n water. The a d s o r p t i o n o f TC-HCl i n pH 1.2 s o l u t i o n i s lower than t h a t i n water a t low drug c o n c e n t r a t i o n s but h i g h e r a t h i g h e r drug c o n c e n t r a t i o n s . Sorby, e t a l (1966) r e p o r t e d t h a t the a d s o r p t i o n o f p h e n o t h i a z i n e : d e r i v a t i v e s , such as promazine h y d r o c h l o r i d e j by k a o l i n depends upon the pH o f the medium. They found t h a t the a d s o r p t i o n of promazine h y d r o c h l o r i d e i s g r e a t e r a t pH 6.5 than a t pH 2.5. T h e i r e x p l a n a t i o n s f o r t h i s pH e f f e c t are -200 400 600 E q u i l i b r i u m C o n c e n t r a t i o n (mg./lOOO ml.) F i g u r e 13. A d s o r p t i o n isotherm at 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n pH 1.2 (A ), pH 3.0 ( • ), and pH 5.0 ( O ) s o l u t i o n s . E q u i l i b r i u m pH of suspensions: 1.2, 3.2, and 5.4, r e s p e c t i v e l y . F i g u r e 14. Langmuir isotherm a t 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n pH 3.0 ( • ) , and pH 5.0 ( O ) s o l u t i o n s . 2.0 2.5 l o g ( E q u i l i b r i u m C o n c e n t r a t i o n i n mg./lOOO, ml.) F i g u r e 15. F r e u n d l i c h isotherm at 37°C. f o r t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n suspended i n pH 1.2 s o l u t i o n . CD Changes i n pH imply changes i n the r e l a t i v e amounts- of protonated and nonprotonated amine pr e s e n t i n the s o l u t i o n . I f the nonprotonated form of the drug i s being adsorbed, then a d s o r p t i o n would be expected t o be g r e a t e r a t h i g h e r pH v a l u e s , ( i i ) At lower pH v a l u e s , hydrogen i o n s and the protonated amine may compete f o r a n i o n i c s i t e s on the k a o l i n s u r f a c e or i n the e l e c t r i c a l double l a y e r e x i s t i n g around such p a r t i c l e s . T h e i r r e a s o n i n g may be used, i n p a r t , to e x p l a i n TC-HCl a d s o r p t i o n by k a o l i n i n pH 3 .;0'i:an"d, Sl,0;i'S.i^lui:«i.6ns. TC-HCl has three pKa v a l u e s - 3.30, 7.68, and 9.69 (Leeson, e t _ a l ( 1 9 6 3 ) ) . C o l a i z z i and K l i n k (1969) r e p o r t e d t h a t , a t pH 5 to 6, TC-HCl e x i s t s i n the z w i t t e r i o n i c form; at pH 3.3, i n both the c a t i o n i c c and z w i t t e r i o n i c forms;and a t pH 2, i n the c a t i o n i c form. T h e r e f o r e , the i n c r e a s e i n a d s o r p t i o n of TC-HCl by k a o l i n w i t h an: i n c r e a s e i n pH may be due to an i n c r e a s e i n the n o n c a t i o n i c or z w i t t e r i o n i c forms of TC-HCl. Based on the expanation of Sorby, e t a l (1966), the p o s i t i v e l y charge s p e c i e s are adsorbed but other forms may a l s o be a t t r a c t e d to the k a o l i n s u r f a c e . On the other hand, i n pH l.V2j£,. the a d s o r p t i o n of TC-HCl by k a o l i n i s g r e a t e r than i n pH 3 . 0 , 5 . 0 1 , s o l u t i o n s or i n water. A t t h i s pH values',;. TC-HCl i s i n the c a t i o n i c form. T h e r e f o r e , the c a t i o n i c form i s b e t t e r adsorbed on the n e g a t i v e l y charge surfaces: of k a o l i n . The a d s o r p t i o n of TC-HCl i n the - 79 -pH 172;,'-solution i s of F r e u n d l i c h . isotherm, which (implies v multilayer;;;' a d s o r p t i o n . T h e r e f o r e , the a d s o r p t i o n o f TC-HC1 by k a o l i n i s complicated and i s probably mediated s i m u l t a n e o u s l y through s e v e r a l . d i f f e r e n t mechanisms., Mechanism based on simple e l e c t r o s t a t i c charge alone cannot f u l l y e x p l a i n the a d s o r p t i o n p r o c e s s . (3) D e s o r p t i o n C h a r a c t e r i s t i c s of TC-HG1 The h i g h d i l u t i o n method used i n t h i s i n v e s t i g a t i o n i s a m o d i f i c a t i o n of the procedure recommended by Sorby (1965) and i s d e s c r i b e d i n d e t a i l . i n the p r e v i o u s s e c t i o n (See Chapter I I I , P a r t 6.). The i n i t i a l ^gcincentration of TC-HC1 i n the d e s o r p t i o n study was equal to 30 mg./ 40 ml. of water. T h i s c o n c e n t r a t i o n r e p r e s e n t s an amount of TC-HC1 adsorbed by 500.0 mg. of k a o l i n on the p l a t e a u o f the a d s o r p t i o n isotherm. (See F i g u r e 11). When such a sample i s d i l u t e d t o 200 ml. w i t h d i s t i l l e d water, the e q u i l i b r i u m c o n c e n t r a t i o n should on the s l o p e of the curve i f d e s o r p t i o n does occur. The amount of TC-HC1 t h a t i s bound to k a o l i n w i t h r e s p e c t to time i s given, i n T a b l e 6. These r e s u l t s show t h a t d e s o r p t i o n reached e q u i l i b r i u m w i t h i n one hour. The amount o f drug r e t a i n e d by the k a o l i n a t one, t h r e e , and s i x hours i s 15.03 mg./ gm.. I f the a d s o r p t i o n process of TC-HC1 by k a o l i n i s r e v e r s i b l e , x • • — can be c a l c u l a t e d by u s i n g Equation 6. See appendix f o r d e r i v a t i o n of t h i s e q u a t i o n . - 80 -Table 6. Desorption- of T e t r a c y c l i n e Hydro c h l o r i d e "from. .•Kaolin * Time (hour) Mg. Remain Adsorbed on One Gm. of K a o l i n * * 0 17.23 1 15.03 3 15.03 6 15.03 *See t e x t f o r method. **Values r e p o r t e d are the average of the d u p l i c a t e d e t e r m i n a t i o n s . - 81 -0...5a(^)2 - t 0.5ab + 0.2 '.+ aT )• ~ ' + abT = 0 (Eq. 6) Where a and b are the Langmuir constants f o r the t e s t drug and T i s the i n i t i a l amount of drug i n mg. i n the suspension. S u b s t i t u t i n g the v a l u e s a = 0.0333, b = 19.84, and T = 3 0 i n t o Equation 6, y i e l d an ^ v a l u e of 15.61 mg./gm. Comparing t h i s v a l u e . t o t h a t o b t a i n e d from the d e s o r p t i o n experiment i n d i c a t e s t h a t the a d s o r p t i o n of TC-HCl i s a r e v e r s i b l e p r o c e s s . (4) A d s o r p t i o n I n t e r a c t i o n s between T h e r a p e u t i c Doses of TC-HCl and K a o l i n In t h i s i n v e s t i g a t i o n , 19.84 mg. of TC-HCl were adsorbed by one gm. of k a o l i n . T h i s may have no t h e r a p e u t i c s i g n i f i c a n c e i f a t h e r a p e u t i c dose (250 mg.) of the drug i s a d m i n i s t e r e d along w i t h o n l y one gm. o f k a o l i n . However, the dose of k a o l i n used i n the treatment o f d i a r r h e a i s l a r g e , b e i n g equal to i about s i x gm. I f 250' mg. of the a n t i b i o t i c and s i x gm. of k a o l i n are a d m i n i s t e r e d c o n c u r r e n t l y (e.g., i n the treatment o f d y s e n t r y ) , a s i g n i f i c a n t d r u g - k a o l i n i n t e r a c t i o n may occur. The per cent of the TC-HCl dose (P) t h a t may be adsorbed by s i x gm. of k a o l i n i n 40.0 ml. s o l u t i o n a t e q u i l i b r i u m can be c a l c u l a t e d u s i n g Equation 7. See appendix f o r d e r i v a t i o n of t h i s e q uation. aSP 2 - ( 6ab + 0.04 + aS ) P + 6ab = 0 (Eq. 7) Where S i s the dose of drug i n mg. and a and b a r e the Langmuir constants,. - 82 -C a l c u l a t i o n s i n d i c a t e d t h a t 47.21. % of a 250 mg. dose of TC-HC1 would. be. adsorbed, by six.gm..of k a o l i n . However, d e s o r p t i o n s t u d i e s showed t h a t TC-HC1 i s r a p i d l y desorbed. T h i s may imply t h a t k a o l i n w i l l not decrease'the i n v i v o a v a i l a b i l i t y of the a n t i b i o t i c . However, Sorby (1965) claimed t h a t i n v i t r o d e s o r p t i o n cannot always be used as a guide t o i n v i v o e f f e c t . S i m i l a r l y , El-Nakeeb and Yousef (1968) r e p o r t e d t h a t the a n t i b a c t e r i a l a c t i v i t y o f TC-HC1 was reduced to 55 % i n the presence of k a o l i n . I f - t h i s v a l u e i s compared wit h t h a t r e p o r t e d h e r e i n (47.21 % ) , i t becomes apparent t h a t i n v i t r o a d s o r p t i o n isotherms have some s i g n i f i c a n c e . As -with most i n v i t r o i n v e s t i g a t i o n s , d e f i n i t i v e c o n c l u s i o n s are unwarranted and must be based on plasma l e v e l s o f a n t i b i o t i c i n the presence or absence of k a o l i n . (b) Neomycin S u l f a t e (1) A d s o r p t i o n Isotherm i n Water at 3 7 . 0 ° C -A d s o r p t i o n data f o r NC-S0 4 i n water a t 37.0°C. are g i v e n i n Table 7. The curve obtained by p l o t t i n g — y v e r s u s c i s shown i n F i g u r e 16. The a d s o r p t i o n isotherm i s o f the Langmuir type. Based on the l i n e a r form of the Langmuir c equation (Equation 4), a p l o t of —j— a g a i n s t c, y i e l d s a s t r a i g h t l i n e (See F i g u r e 17). The v a l u e s o f the Langmuir c o n s t a n t s , a and b, were ob t a i n e d from the i n t e r c e p t and s l o p e v a l u e s of the above s t r a i g h t l i n e ( c o r r e l a t i o n f a c t e r = 0.979). These v a l u e s are r e p o r t e d i n Table 8. Table 7. A d s o r p t i o n Data, f o r Neomycin S u l f a t e ' b y K a o l i n * I n i t i a l C o n c e n t r a t i o n E q u i l i b r i u m Mg. Adsorbed per c/(x/m) (mg./lOOO ml.) Co n c e n t r a t i o n , ( c ) Gm. (x/m) +range (mg./lOOO ml.) 375 252.44 9.80 + 0.39 25.75 500 350.00 12.00 ± 0.97 29.17 625 470.73 12.34 +0.78 38.14 750 563.41 .14.93+0.11 37.75 875 678.05 15.76 + 0.97 43.02 1000 787.80 16.98 + 0.78 46.41 * 500 mg-. of k a o l i n were suspended i n 40.0 ml. of drug s o l u t i o n i n water at a temperature of 37°C. C a l c u l a t e d values are based on two dete r m i n a t i o n s . I I I I 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./1000 ml.) F i g u r e 16. A d s o r p t i o n isotherm a t 37°C. f o r neomycin s u l f a t e by kaolinp.suspended^in water. • • • • • • 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./1000 ml.) F i g u r e 17. Langmuir isotherm at 37°C. f o r neomycin s u l f a t e by k a o l i n suspended i n water. (2) Adsorption Isotherms i n pH 1.2,3.0, and 5.0 Solutions The isotherms obtained at these pH values are shown i n Figure 18. Results obtained indicate that the adsorption of NC-SO^ from pH 1.2, 3.0, and 5.0 solutions are of the c Langmuir type. The s t r a i g h t . l i n e s obtained by-plotting versus c (Equation 4) are shown i n Figure 19. The co r r e l a t i o n factors for these l i n e s are 0.968, 0.930, and 0.937,respectively. The corresponding values for the Langmuir constants are reported i n Table 8. These r e s u l t s show that the adsorption of NC-S04 from pH 5.0 solution i s higher than from pH 3.0 and 1.2 solutions but i s si m i l a r to that observed i n water. This r e l a t i o n s h i p between pH and the extent of adsorption of NC-SO^ by kaolin appears to conform to the adsorption process described by Sorby, et a l (1966). As stated previously, these researchers attribute changes i n adsorption to changes'in amount of free base present with an increase i n the pH of the system. However, i t i s not possible to conclude, on the basis of information obtained from these pH studies,that the observed e f f e c t s are due to one s p e c i f i c adsorption mechanism. The fact that considerable adsorption occurs at pH 1.2 supports the hypothesis that adsorption of the protonated form also occurs to a considerable extent. Therefore, adsorption of NC-SG>4 by kaolin i s probably mediated through a combination of mechanisms. / ' t _L 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./1000 ml.) F i g u r e 18. A d s o r p t i o n isotherm a t 37°C. f o r neomycin s u l f a t e by k a o l i n suspended i n pH 1.2 • ( A ) , pH 3.0 ( • ) , and pH 5.0 ( O ) s o l u t i o n s . E q u i l i b r i u m pH of suspensions: 1.3, 3.4, and 6.3, r e s p e c t i v e l y . 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./lOOO ml.) F i g u r e 19. Langmuir isotherm a t 37°C. f o r neomycin s u l f a t e by k a o l i n suspended i n pH 1.2 (A), pH 3.0 (• ) , and pH 5.0 (O) s o l u t i o n s . Table 8. A d s o r p t i o n o f Neomycin S u l f a t e by K a o l i n . a t 37°C. A d s o r p t i o n Medium. E q u i l i b r i u m pH A d s o r p t i o n Isotherm Langmuir Constants a b l i t e r / m g . mg./gm.r water pH. 1. 2 s o l u t i o n pH 3.0 s o l u t i o n pH 5.0 s o l u t i o n 6.4 1.3 3.4 6.3 Langmuir Langmuir Langmuir Langmuir 0.0023 25.88 0.0026 14.88 0.0018 15.86 0.0030 16.82 oo V O - 90 -(3) D e s o r p t i o n C h a r a c t e r i s t i c s of NC-SO^ The procedures used i n t h i s study are s i m i l a r t o those p r e v i o u s l y d e s c r i b e d . . The i n i t i a l c o n c e n t r a t i o n used to prepare the e q u i l i b r a t e d sample f o r the d e s o r p t i o n s t u d i e s was equal to 40 mg. neomycin base/ 40 ml. of water. The amount of neomycin base remaining on k a o l i n w i t h r e s p e c t to time i s r e p o r t e d i n Table 9. The r e s u l t s i n d i c a t e d t h a t d e s o r p t i o n reached e q u i l i b r i u m w i t h i n one hour. T h i s amount . of drug remaining on one gm. of k a o l i n i s equal t o 17.81 mg. I f the a d s o r p t i o n of the drug i s : a e r e y e r s i b l e r p r o c e s s / the amount of drug adsorbed on one gm. of k a o l i n can be c a l c u l a t e d by u s i n g Equation 6. T h i s t h e o r e t i c a l amount of drug, i s , t h e r e f o r e , 6.93 mg. Comparison of these two values (that i s , 6.93 mg. and 17.81 mg.) i n d i c a t e s t h a t NC-S0 4 d i d desorb from k a o l i n but t h a t the e x t e n t of d e s o r p t i o n i s much s m a l l e r than the e x t e n t of a d s o r p t i o n . (4) A d s o r p t i o n I n t e r a c t i o n s between T h e r a p e u t i c s / Doses of NC-S0 4 and K a o l i n Neomycin s u l f a t e i s i n c o r p o r a t e d i n t o k a o l i n c o n t a i n i n g a n t i d i a r r h e a l p r e p a r a t i o n s . For example, Kaomycin (The Upjohn Company) c o n t a i n s neomycin s u l f a t e , k a o l i n and o t h e r p h a r m a c e u t i c a l i n g r e d i e n t s . The u s u a l dose of Kaomycin co n t a i n s 220.5 mg. o f neomycin base and 6 gm. of k a o l i n . The per cent of NC-S0 4 dose ( c a l c u l a t e d w i t h r e s p e c t to neomycin base) t h a t w i l l be adsorbed by s i x gm. of k a o l i n , a t the e q u i l i b r i u m , i n 40 ml. o f aqueous s o l u t i o n can be - 91 -Tab l e 9. D e s o r p t i o n of Neomycin S u l f a t e from K a o l i n * Time (hour) Mg.. Remain Adsorbed on. One Gm. of K a o l i n * * 0 17.56 1 17. 81 3 17.81 6 17.81 * See t e x t f o r method. ** Values r e p o r t e d are the average o f d u p l i c a t e d e t e r m i n a t i o n s . - 92 -calculated by using Equation- 7. It was found that 59.05% Of neomycin base w i l l be adsorbed by s i x gm. of kaolin i f the system reached equilibrium conditions. The corresponding values for pH 1.2, 3.0, and 5.0 solutions were equal to 37.31%, 37.28%, and 41.48%, respectively. I t i s obvious that the amount of neomycin adsorbed increases with an increase i n pH. Further, the process i s only p a r t i a l l y r e v e r s i b l e and, on this basis, can be compared, i n part, with the r e s u l t s obtained for lincomycin hydrochloride. Neomycin sulfate has been incorporated into kaolin containing a n t i d i a r r h e a l preparations on the assumption that, because of i t s low absorbability, i t w i l l act d i r e c t l y on the bacteria i n the g a s t r o i n t e s t i n a l t r a c t . If 60% of the dose i s adsorbed by kaolin, the a n t i b a c t e r i a l a c t i v i t y of the a n t i b i o t i c w i l l be decreased'accordingly. This value may be compared with the decrease i n a n t i b a c t e r i a l a c t i v i t y reported for t e t r a c y c l i n e and neomycin /^,( 55% and 59%, respectively) ( El-Nakeeb and Yousef (1968)). Even i f such combined i n vitro-data cannot be f u l l y extrapolated to i n vivo conditions, i t i s probably preferable to not incorporate the a n t i b i o t i c into the kaolin containing a n t i d i a r r h e a l preparations. As with lincomycin hydrochloride, i f t h i s a n t i b i o t i c i s indicated, i t should be administered;/as such and, i f Wagner's assessment i s correct, Kaopectate ( or i t s equivalent ) could be administered two hours l a t e r . Unlike lincomycin hydrochloride, serum leve l s would have l i t t l e s i g n i f i c a n c e . Only c a r e f u l l y c o n t r o l l e d c l i n i c a l t r i a l s would p r o v i d e the evidence necessary t o f u l l y a s s e s s t h e . r e l a t i v e e f f i c a c y o f Kaopectate and Kaomycin or e q u i v a l e n t p r o d u c t s . (c) Lincomycin H y d r o c h l o r i d e (1) A d s o r p t i o n Isotherm in.Water a t 37.0°C. The data f o r the a d s o r p t i o n of LC-HC1 by k a o l i n i n water are shown i n Table 10. A p l o t of the amount of drug adsorbed by k a o l i n (—) versus the e q u i l i b r i u m c o n c e n t r a t i o n (c) i s m • s shown i n F i g u r e 20. T h i s graph i n d i c a t e s t h a t the isotherm i s o f the Langmuir or L2 type.(See F i g u r e 2 ). T h e r e f o r e , a p l o t o f x / m versus c (See ^ Equation 4) y i e l d s a s t r a i g h t l i n e (Figure 21). The slope and i n t e r c e p t v a l u e s of t h i s s t r a i g h t l i n e ( c o r r e l a t i o n f a c t o r = 0.989) were c a l c u l a t e d by the method of l e a s t squares. The Langmuir cons t a n t , a and b, were c a l c u l a t e d from the r e c i p r o c a l o f the i n t e r c e p t and slope v a l u e s . The a d s o r p t i o n c o e f f i c i e n t , a , . i s equal t o 0.0083 l i t e r / m g . , and the monolayer c a p a c i t y , b, i s equal to 9.6936 mg./gm.. (2) A d s o r p t i o n Isotherms i n pH 1.2, 3.0, and 5.0 S o l u t i o n s The a d s o r p t i o n isotherms f o r LC-HC1 i n pH 1.2, 3.0 and 5.0 s o l u t i o n s are shown i n Figure,22. The adsorption, isotherms f o r t h i s a n t i b i o t i c in.pH 3.0 and 5.0 s o l u t i o n s are s i m i l a r to t h a t i n water, t h a t i s , they are o f the Langmuir type. T h e r e f o r e , : a s t r a i g h t , l i n e was o b t a i n e d when was p l o t t e d ^ versus c (See F i g u r e 23 and F i g u r e 24). The Langmuir Table 10.. A d s o r p t i o n Data f o r Lincomycin. H y d r o c h l o r i d e by • K a o l i n * I n i t i a l C o n c e n t r a t i o n E q u l i b r i u m Mg. Adsorbed per c/(x/m) (mg./lOOO ml.) C o n c e n t r a t i o n (c) Gm. (x/m)+range (mg./lOOO ml.) 125 72.71 4.18 ± 0.14 17.38 250 178.85 5.69 ± 0.05 31.42 375 293.78 6.-48 + 0.23 45. 21 500 406.36 7.49 ± 0.70• 54.24 625 534.78 7.21 + 0.94 74.09 750 642.67 8.59+0.47 74.84 875 762.29 9.02 ± 0.47 • 84.54 1000 897.16 8.23 +0.97 109.05 * 500 mg. o f k a o l i n were suspended i n 40.0 ml. of drug s o l u t i o n i n water a t -a temperature of 37°C. C a l c u l a t e d values are based on. two determinations. CD XI U O w TJ < tn S F i g u r e 20 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./lOOO ml.) A d s o r p t i o n isotherm at 37°C. f o r l i n c o m y c i n h y d r o c h l o r i d e by k a o l i n suspended i n water. 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t ion (mg./1000 ml.) F i g u r e 22. A d s o r p t i o n isotherm at 37°C. f o r li n c o m y c i n h y d r o c h l o r i d e by k a o l i n suspended i n pH 1.2 ( A ), pH 3.0 ( • ), and pH 5.0 ( O ) s o l u t i o n s . E q u i l i b r i u m pH of suspensions: 1.2,3.2, and 6.5, r e s p e c t i v e l y . 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./lOOO ml.) F i g u r e 23. Langmuir isotherm a t 37°C. f o r lincomycin, h y d r o c h l o r i d e by k a o l i n suspended in" pH 3.0 s o l u t i o n . • 200 400 600 800 E q u i l i b r i u m C o n c e n t r a t i o n (mg./lOOO ml.) F i g u r e 24. Langmuir isotherm at 37°C. f o r l i n c o m y c i n h y d r o c h l o r i d e by k a o l i n suspended :irf .pH 5;-0;solution. - 100 -c o n s t a n t s , a and b, were c a l c u l a t e d i n the manner d e s c r i b e d above and are t a b u l a t e d i n Table 11. The a d s o r p t i o n o f LC-HC1 by k a o l i n i n pH 1.2 s o l u t i o n obeys n e i t h e r the Langmuir nor the F r e u n d l i c h e q u a t i o n s . A d s o r p t i o n o f LC-HC1 by k a o l i n i s pH dependent. A d s o r p t i o n i s g r e a t e r a t pH 6.5 than e i t h e r pH 3.2 or pH 1.2. A p o s s i b l e e x p l a n a t i o n f o r t h i s pH e f f e c t can be based on the a d s o r p t i o n processes o u t l i n e d by Sorby, e t a l (1966) and Ridout (1968 b ) . These authors r e p o r t e d t h a t unchanged or f r e e base i s p r e f e r e n t i a l l y adsorbed by k a o l i n . Since the pKa value of l i n c o m y c i n base i s equal t o 7.6, the amount of nonprotonated l i n c o m y c i n , a t pH 6.5, i s h i g h e r than a t the lower pH v a l u e s . T h e r e f o r e , the a d s o r p t i o n o f L C - H C l by k a o l i n should i n c r e a s e w i t h an i n c r e a s e i n pH. However, the a d s o r p t i o n of LC-HCl by k a o l i n i n pH 1.2 s o l u t i o n (see F i g u r e 22) i s of the S type, based on the c l a s s i f i c a t i o n o f G'ijles and h i s co-workers (1960). The isotherm i s i n i t i a l l y concave a t low c o n c e n t r a t i o n s of the drug. A c c o r d i n g to these authors,, S-type a d s o r p t i o n occurs when th r e e c o n d i t i o n s are f u l f i l l e d . The molecule -(i) should be monofunctional, ( i i ) should a t t r a c t l i k e molecules, causing i t to pack v e r t i c a l l y , i n r e g u l a r a r r a y i n the adsorbed l a y e r , and ( i i i ) should compete s t r o n g l y , f o r s u b s t r a t e s i t e s , w i t h molecules of the s o l v e n t or of another adsorbed s p e c i e s . Table 11. A d s o r p t i o n o f Lincomycin H y d r o c h l o r i d e b y - K a o l i n . a t 37°C. A d s o r p t i o n Medium E q u i l i b r i u m pH Ad s o r p t i o n Isotherm Langmuir Constants a b l i t e r / m g . mg./gm. water pH 1.2. s o l u t i o n pH. 3.0 s o l u t i o n pH 5.0 s o l u t i o n 6.4 1.2 3.2 6.5 Langmuir S type.. . ...! Langmuir Langmuir 0.0083 9.69 0.0028 5.45 0.0070 10.25 - 102 -These c r i t e r i a do not apply t o , i n t o t a l , t o LC-HC1 a d s o r p t i o n by k a o l i n . A d s o r p t i o n isotherms, i n pH 3.2 and 6.5 s o l u t i o n s are o f the Langmuir type and, t h e r e f o r e , the ad s o r p t i o n mechanism a t lower pH val u e must be due to other f a c t o r s . (3) D e s o r p t i o n C h a r a c t e r i s t i c s o f LC-HC1 De s o r p t i o n s t u d i e s f o r LC-HC1 were c a r r i e d out i n the manner o u t l i n e d f o r TC-HC1. Becaused o f a n a l y t i c a l problems, the lowest i n i t i a l c o n c e n t r a t i o n o f LC-HC1 t h a t would s a t u r a t e k a o l i n * c o u l d not be used t o prepare the sample f o r d e s o r p t i o n . T h e r e f o r e , 30.0 mg. of drug / 40.0 ml. of water was used to prepare the e q u i l i b r a t e d sample f o r d e s o r p t i o n s t u d i e s . • The amount o f LC-HCl remaining on k a o l i n w i t h r e s p e c t to time i s shown i n Table 12. These r e s u l t s show t h a t d e s o r p t i o n reached e q u i l i b r i u m a t three hours. The amount of drug remaining on k a o l i n a t the end of t h i s time p e r i o d i s 6.05 mg./gm.. I f the a d s o r p t i o n o f LC-HCl i s a r e v e r s i b l e p r o c e s s , the amount of LC-HCl adsorbed on one gm. of k a o l i n can be c a l c u l a t e d by u s i n g Equation 6 ( See S e c t i o n on T e t r a c y c l i n e H y d r o c h l o r i d e ). T h i s c a l c u l a t e d amount of drug adsorbed on one gm. of k a o l i n i s equal t o 5.59 mg. Comparison of t h i s 'rtheo|?eTtiea'l value to t h a t o b t a i n e d e x p e r i m e n t a l l y ( 6.05 mg./gm.) i n d i c a t e s t h a t the a d s o r p t i o n o f LC-HCl by k a o l i n i s not completely r e v e r s i b l e . - 103 -Table 12. D e s o r p t i o n o f Lincomycin H y d r o c h l o r i d e from K a o l i n * . Time (hour) Mg. Remain Adsorbed on One Gm. of K a o l i n * * 0 7;26 1 7.36 3 6.05 6 6.05 * See t e x t f o r method. ** Values r e p o r t e d are the average o f d u p l i c a t e d e t e r m i n a t i o n s . - 104 -(4) A d s o r p t i o n I n t e r a c t i o n s between T h e r a p e u t i c Doses of LC-HCl and K a o l i n Wagner (1966) r e p o r t e d t h a t LC-HCl serum c o n c e n t r a t i o n s decreased i f Kaopectate ( The Upjohn Company Brand of K a o l i n Mixture w i t h P e c t i n NF) was admi n i s t e r e d w i t h the a n t i b i o t i c . More s p e c i f i c a l l y , i f three f l u i d ounces of Kaopectate ( i . e . , 17.76 gm. o f k a o l i n ) are a d m i n i s t e r e d a t the same time as the LC-HCl capsule (500 mg.), the l i n c o m y c i n serum c o n c e n t r a t i o n was onl y one-tenth of t h a t observed when the a n t i b i o t i c was admin i s t e r e d alone. The serum l e v e l s o f l i n c o m y c i n were reduced by 50% when Kaopectate was admi n i s t e r e d two hours a f t e r the a n t i b i o t i c c a p s u l e , but serum l e v e l s d i d not change when Kaopectate was admi n i s t e r e d two hours p r i o r t o the a d m i n i s t r a t i o n o f the c a p s u l e . On the b a s i s o f data r e p o r t e d h e r e i n , the per cent o f LC-HCl dose t h a t w i l l be adsorbed by s i x gm. of k a o l i n , a t e q u i l i b r i u m , i n 40 ml. of aqueous s o l u t i o n can be c a l c u l a t e d by u s i n g Equation 7. Such c a l c u l a t i o n s showed t h a t 11.44% of the LC-HCl dose (500 mg.) w i l l be adsorbed by s i x gm. of k a o l i n . The per cent o f LC-HCl dose adsorbed by s i x gm.of k a o l i n e q u i l i b r a t e d i n pH 3.2 and 6H5 s o l u t i o n s were a l s o c a l c u l a t e d and are equal to 6.35% and 12.14%, r e s p e c t i v e l y . In water, the percentages adsorbed by 17.76 gm. of k a o l i n e q u i l i b r a t e d i n 40 ml. and 120 ml. are 33.94% and 33.01%, r e s p e c t i v e l y . The d i f f e r e n c e i n percentage v a l u e s i s equal to 0.93% and i n d i c a t e s t h a t the e f f e c t o f volume change i s - 105 -minimal. These r e s u l t s can now be compared w i t h t h e - i n v i v o v a l u e s obtained by Wagner (1966). I f t h e r a p e u t i c doses-of Kaopectate and LC-HCl are administered to the p a t i e n t a t the same time, l i n c o m y c i n serum l e v e l s w i l l be decreased by 90%. However, the v a l u e s c a l c u l a t e d h e r e i n i n d i c a t e t h a t only 33.94% o f the LC-HCl dose i s bound by k a o l i n . The l a t t e r v a l u e i s o n l y o n e - t h i r d o f t h a t observed by,Wagner. T h i s may imply t h a t i n . v i v o e f f e c t s are f a r g r e a t e r than those observed i n v i t r o . Such d i f f e r e n c e s are" o f t e n observed, f o r example, when i n V i t r o d i s s o l u t i o n data i s compared t o serum drug l e v e l s . More important, Kaopectate c o n t a i n s p e c t i n and other adjuvants which may a l s o a f f e c t LC-HCl a d s o r p t i o n . Cyclamates, f o r example, decrease LC-HCl serum l e v e l s (Wagner (1970)). Lincomycin h y d r o c h l o r i d e i s not i n c o r p o r a t e d i n t o k a o l i n c o n t a i n i n g p r e p a r a t i o n s . The drug i t s e l f may cause d i a r r h e a and, t h e r e f o r e , i t may be necessary to a d m i n i s t e r Kaopectate to t h e . p a t i e n t . I f continued use of LC-HCl i s i n d i c a t e d , the i n t e r a c t i o n between the a n t i b i o t i c and k a o l i n should always be taken i n t o c o n s i d e r a t i o n . A d s o r p t i o n can be minimized i f the k a o l i n c o n t a i n i n g p r e p a r a t i o n i s a d m i n i s t e r e d two hours p r i o r t o the a d m i n i s t r a t i o n of the LC-HCl dose.. (d) Chloramphenicol S t u d i e s i n water and i n pH 1.2, 3.0, and 5.0 s o l u t i o n s ' i n d i c a t e d t h a t chloramphenicol was not adsorbed by k a o l i n . - 106 -Chloramphenicol i s a n e u t r a l substance. I f the mechanism o f a d s o r p t i o n by k a o l i n i s based on charge, the a n t i b i o t i c should not be adsorbed onto the n e g a t i v e l y charged k a o l i n s u r f a c e . Chloramphenicol i s the drug of c h o i c e (The' P h a r m a c o l o g i c a l B a s i s o f T h e r a p e u t i c s , 4th E d i t i o n ) f o r the treatment of b a c i l l a r y dysentry (e.g.; s a l m o n e l l a ) . The use o f the drug i s u s u a l l y c o n t r a i n d i c a t e d because of major and o f t e n f a t a l e f f e c t s f o l l o w i n g repeated and l a r g e doses of the a n t i b i o t i c . However, i f i t must be used, i t c o u l d be, on t h e . b a s i s of the data h e r e i n , a d m i n i s t e r e d to the p a t i e n t along w i t h a k a o l i n c o n t a i n i n g p r e p a r a t i o n . (e) A m p i c i l l i n T r i h y d r a t e A m p i c i l l i n i s an amphoteric substance. Hou and Poole (1969) r e p o r t e d a pK^ value of 2.66 and a pK 2 value of 7.24. However, the - i n t r i n s i c a c i d i t y o f the molecule:.: i s much g r e a t e r than i t s b a s i c i t y and, t h e r e f o r e , o n l y the sodium or'potassium s a l t s are r e a d i l y formed. S t u d i e s in.water and i n pH 1.2, 3.0, and 5.0 s o l u t i o n s i n d i c a t e d t h a t a m p i c i l l i n i s not adsorbed by k a o l i n . I f the c a t i o n i c form i s p r e s e n t i n s o l u t i o n s o f low pH, i t would be expected t h a t the a n t i b i o t i c would be adsorbed by k a o l i n . However, s i n c e such a d s o r p t i o n does not occur, t h e r e i s probably no r e a c t i o n between the h y d r o c h l o r i c a c i d p r e s e n t i n . the s o l u t i o n and the b a s i c p o r t i o n s of the molecule. A t h i g h e r - 107 -pH v a l u e s , the molecule would be expected t o be n e g a t i v e l y charged and would not, t h e r e f o r e , be a t t r a c t e d to the k a o l i n s u r f a c e . A m p i c i l l i n i s l i s t e d as the drug of choice ( The PJiarJmacp'lqg&ealBBas^^ ,ti4thEEdition) f o r t h e treatment of b a c i l l a r y d ysentry. I t s s i d e e f f e c t s are of l e s s e r s i g n i f i c a n c e thari those r e p o r t e d f o r chloramphenicol and, on the b a s i s of the data o b t a i n e d h e r e i n , t h i s a n t i b i o t i c c o u l d be adm i n i s t e r e d t o the p a t i e n t along w i t h k a o l i n c o n t a i n i n g p r e p a r a t i o n s . - 108 -V. SUMMARY AND CONCLUSIONS Di a r r h e a i s u s u a l l y a symptom of a b a c t e r i a l or v i r a l i n f e c t i o n . However, l a c k of adequate d i g e s t i v e enzymes, p a r a s i t i c i n f e c t i o n s , v a r i o u s m e t a b o l i c and hormonal d i s t u r b a n c e s , i n c r e a s e d g a s t r o i n t e s t i n a l m o t i l i t y r e s u l t i n g i n decreased t r a n s i t time, p a t h o l o g i c a l c o n d i t i o n s of the i n t e s t i n a l mucosa, and v a r i o u s s u r g i c a l o p e r a t i o n s upon the d i g e s t i v e t r a c t may cause d i a r r h e a ( F e d e r a l R e g i s t e r (1975)). K a o l i n Mixture w i t h Pectin.NF (or an e q u i v a l e n t k a o l i n c o n t a i n i n g p r e p a r a t i o n ) has been used f o r over 200 years f o r the treatment of d i a r r h e a . In more r e c e n t y e a r s , neomycin s u l f a t e has been i n c o r p o r a t e d i n t o such p r e p a r a t i o n s . I t i s assumed t h a t t h i s a n t i b i o t i c a c t s d i r e c t l y on the b a c t e r i a i n the g a s t r o i n t e s t i n a l t r a c t . I f the .diagnosed c o n d i t i o n a l s a b a c i l l a r y dysentry (e.g.;*, s a l m o n e l l a ) , the a d m i n i s t r a t i o n of chloramphenicol or a m p i c i l l i n has been recommended (The P h a r m a c o l o g i c a l B a s i s of T h e r a p e u t i c s / 4th E d i t i o n ) . The concurrent a d m i n i s t r a t i o n of a k a o l i n c o n t a i n i n g p r e p a r a t i o n i s the r u l e r a t h e r than the e x c e p t i o n . In a d d i t i o n t o above drugs, opium and b e l l a d o n n a a l k a l o i d s are o f t e n i n c o r p o r a t e d i n t o p r e p a r a t i o n s c o n t a i n i n g k a o l i n . - 109 -The.Food and Drug A d m i n i s t r a t i o n has, over,the p a s t few y e a r s , appointed. A d v i s o r y Review P a n e l s whose b a s i c f u n c t i o n i s to review a wide v a r i e t y of o v e r - t h e - c o u n t e r products (Federal R e g i s t e r (1975)). Although one of these Panels has c l a s s i f i e d some of the above drugs as e f f e c t i v e , no evidence was p r e s e n t e d t o i n d i c a t e t h a t combinations of-such drugs w i t h k a o l i n are more e f f e c t i v e than, f o r example. K a o l i n Mixture w i t h P e c t i n NF. They do, however, r e f e r e n c e the i n t e r a c t i o n between l i n c o m y c i n h y d r o c h l o r i d e and Kaopectate (the Upjohn Company brand o f K a o l i n Mixture w i t h P e c t i n NF) b u t do not emphasize the p o s s i b i l i t y t h a t drug substances may be adsorbed by k a o l i n . Because of t h e i r i n a b i l i t y to p r o p e r l y c l a s s i f y some of the commercially a v a i l a b l e a n t i d i a r r h e a l p r e p a r a t i o n s , they do suggest t h a t f u r t h e r r e s e a r c h i s r e q u i r e d b e f o r e a s c i e n t i f i c a l l y v a l i d assessment of the v a r i o u s products can be made. In t h i s i n v e s t i g a t i o n , the i n v i t r o a d s o r p t i o n of t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin s u l f a t e , l i n c o m y c i n h y d r o c h l o r i d e , chloramphenicol, and a m p i c i l l i n t r i h y d r a t e by k a o l i n were s t u d i e d . A d s o r p t i o n s t u d i e s were c a r r i e d out a t 37.0°C. i n water and i n pH 1.2, 3.0, and 5.0 s o l u t i o n s . Chloramphenicol and a m p i c i l l i n t r i h y d r a t e are not adsorbed by k a o l i n * - 110 -The a d s o r p t i o n isotherms f o r t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin sulfate', and l i n c o m y c i n h y d r o c h l o r i d e i n aqueous s o l u t i o n were of. the Langmuir type, w i t h the f o l l o w i n g e x c e p t i o n s . (a) A F r e u n d l i c h a d s o r p t i o n i s o t h e r m was o b t a i n e d f o r t e t r a c y c l i n e h y d r o c h l o r i d e i n pH 1.2 s o l u t i o n s . The- c o n s t a n t s , k and 1/n, were found t o be 1.049 and 0.527, r e s p e c t i v e l y . (b) An S type a d s o r p t i o n i s o t h e r m (see F i g u r e 2) was obtained f o r l i n c o m y c i n h y d r o c h l o r i d e i n pH 1.2 s o l u t i o n s . The Langmuir c o n s t a n t s , a and b, f o r t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin s u l f a t e , and lincomycin. h y d r o c h l o r i d e were c a l c u l a t e d . The constant 'b' i s expressed i n terms of mg. of drug adsorbed per gm. of k a o l i n . T h i s value i s a good measure of the a d s o r p t i o n c a p a c i t y of k a o l i n . (a) In water, 19.84 mg. o f t e t r a c y c l i n e h y d r o c h l o r i d e are adsorbed by one gm. of k a o l i n . T h i s value i s s i m i l a r t o t h a t i n pH 3.0 s o l u t i o n s (20.12 mg.) and i n pH 6.5 s o l u t i o n s (20.05 mg.). (b) In water, 25.88 mg. of neomycin base are adsorbed by one gm.. o f k a o l i n . The amounts adsorbed from pH 1.2, 3.0, and 5.0 s o l u t i o n s are 14.88 mg.,. 15.86 mg., and 16.82 mg., r e s p e c t i v e l y . - I l l -Cc) In water> 9.69 mg.. of l i n c o m y c i n h y d r o c h l o r i d e are adsorbed by one gm. of k a o l i n . The v a l u e s , i n pH 3.2 and 6.5 s o l u t i o n s , are equal t o 5.45 mg., and 10.25 mg., r e s p e c t i v e l y . 12. D e s o r p t i o n s t u d i e s f o r t e t r a c y c l i n e h y d r o c h l o r i d e , neomycin s u l f a t e , and l i n c o m y c i n h y d r o c h l o r i d e were c a r r i e d out i n water. R e s u l t s showed t h a t -(a) The a d s o r p t i o n of t e t r a c y c l i n e h y d r o c h l o r i d e by k a o l i n i s a r e v e r s i b l e p r o c e s s . , (b) The a d s o r p t i o n , o f neomycin s u l f a t e by k a o l i n i s an i r r e v e r s i b l e p r o c e s s . (c) The a d s o r p t i o n of l i n c o m y c i n h y d r o c h l o r i d e i s a p a r t i a l l y r e v e r s i b l e p r o c e s s . 13; Sorby (1968) claimed t h a t p r e e q u i l i b r a t i o n between the drug and adsorbent i s an important f a c t o r w i t h r e s p e c t to drug a b s o r p t i o n from the g a s t r o i n t e s t i n a l t r a c t . He a l s o suggested t h a t t o t a l .quantityoofaadsorbeht a d m i n i s t e r e d i s of more s i g n i f i c a n c e than any tendency f o r the drug t o desorb under i n v i t r o c o n d i t i o n s . 14. The percentage of drug dose t h a t w i l l be adsorbed by s i x gm. o f k a o l i n (the u s u a l dose of k a o l i n ) , a t e q u l i b r i u m , i n 40 ml. o f aqueous s o l u t i o n was c a l c u l a t e d . The v a l u e s are' -(a) On the b a s i s of a 250 mg. dose o f t e t r a c y c l i n e h y d r o c h l o r i d e , 47.21% would be expected-to be adsorbedvby s i x gm. of k a o l i n . - 112 -(b) On the b a s i s of a 220.5 mg. dose of neomycin base (the amount pr e s e n t i n Kaomycin (the Upjohn Company brand of K a o l i n Mixture w i t h P e c t i n and Neomycin S u l f a t e ) ) , 57.98% would be expected to be adsorbed by s i x gm. o f k a o l i n . (c) On the b a s i s of a 500 mg. dose of l i n c o m y c i n h y d r o c h l o r i d e , 11.44% would be expected t o be adsorbed by s i x gm. of k a o l i n . T h i s value i n c r e a s e s to 33.94% i f 17.76 gm. of k a o l i n are a d m i n i s t e r e d t o the p a t i e n t . I f the above two v a l u e s afeacbmpared to the i n v i v o data r e p o r t e d by Wagner (1966), i t becomes e v i d e n t t h a t the decrease i n drug plasma l e v e l s (by about 90%) i s much g r e a t e r than t h a t which c o u l d be p r e d i c t e d on the b a s i s of i n v i t r o a d s o r p t i o n s t u d i e s . However, Wagner (1966) s t u d i e d a commercial p r e p a r a t i o n (kaopectate) and i t may be t h a t other i n g r e d i e n t s c o n t r i b u t e to the dramatic decrease i n blood l e v e l s . The k a o l i n s u r f a c e i s n e g a t i v e l y charged. T h e r e f o r e , a d s o r p t i o n should be based on the a t t r a c t i o n of p o s i t i v e l y charged molecules to the s u r f a c e . T h i s simple assumption cannot be used t o e x p l a i n the a d s o r p t i o n isotherms o b t a i n e d i n t h i s i n v e s t i g a t i o n . I t appears, t h e r e f o r e , t h a t the a d s o r p t i o n of these a n t i b i o t i c s by k a o l i n are mediated through s e v e r a l d i f f e r e n t mechanisms sim u l t a n e o u s l y - 113 -Chloramphenicol i s the drug of choice f o r the treatment of Salmonella i n f e c t i o n s (The Pharmacological B a s i s o f T h e r a p e u t i c s , 4th E d i t i o n ) . Since the drug i s not adsorbed by k a o l i n , c o n current a d m i n i s t r a t i o n o f the a n t i b i o t i c , i f i n d i c a t i o n s so warrant, and k a o l i n c o n t a i n i n g p r e p a r a t i o n s should not, on the b a s i s o f i n v i t r o data, i n t e r f e r e w i t h the t h e r a p e u t i c a c t i v i t y o f the drug substance. A m p i c i l l i n i s the drug o f c h o i c e f o r the treatment o f Salmonella and S h i g e l l a i n f e c t i o n s (The Pharmacological B a s i s o f T h e r a p e u t i c s , 4th E d i t i o n ) . Since the drug i s not adsorbed by k a o l i n , i t c o u l d be i n c o r p o r a t e d i n t o k a o l i n c o n t a i n i n g p r e p a r a t i o n s . However, s i n c e such p r e p a r a t i o n s would allow f o r no adjustment of a n t i b i o t i c dose, i t i s probably p r e f e r a b l e t o a d m i n i s t e r the two s u b s t a n c e s ^ s e p a r a t e l y . 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Agents Chemother., 355. - 119 -APPENDIX D e r i v a t i o n o f Equation 6 and Equation 7 In a d r u g - k a o l i n suspension, the f o l l o w i n g r e l a t i o n a p p l i e s : T = F + A (i) where T = t o t a l amount of drug p r e s e n t i n the system; F = amount of drug remaining d i s s o l v e d i n the a d s o r p t i o n medium; and A = amount o f drug adsorbed by k a o l i n . A l l t h r e e q u a n t i t i e s are measured i n mg.: At a d s o r p t i o n e q u i l i b r i u m : F = V.C ( i i ) A = W.- ( i i i ) m i n these equations, V i s the volume, i n l i t e r s , o f the. a d s o r p t i o n medium; C being the e q u i l i b r i u m c o n c e n t r a t i o n , i n m g . / l i t e r , o f drug i n the a d s o r p t i o n medium; W b e i n g the amount of k a o l i n p r e s e n t i n gm.; and — i s the mg. of drug adsorbed per gm. o f k a o l i n . S u b s t i t u t i n g equations ( i i ) and ( i i i ) i n t o (i) y i e l d s : T = V.C + W.-m (iv) - 120 -r e a r r a n g i n g (iv) g i v e s : . T - W.-„ m c =  V When t h i s v a l u e of C i s s u b s t i t u t e d i n t o the Langmuir a d s o r p t i o n equation (Eq. 3 ) , the f o l l o w i n g i s o b t a i n e d : . . T - W.-ab ( •• m ) x = ' V • • m T, - W - -1 + a ( v m ) or v . . ab .(. T - W.|) . . - ^ ' — ( v i ) V + ,fa ( T - W.- ) m Expanding (vi) and r e a r r a n g i n g terms give; aW (^) 2 ~ ( abW + V + aT ) + abT = , 0 ( v i i ) In the a d s o r p t i o n studies> W = 0.5, Vi= 0.2. S u b s t i t u t i o n of these q u a n t i t i e s i n t o ( v i i ) y i e l d s : 0.5a ( £ ) 2 - (0.5ab + 0.2 + a'T)-'+ abT = 0 m 'm which i s Equation 6. Consider the case i n which a normal dose of a drug (S mg.) is'suspended w i t h a normal dose of k a o l i n (W gm.) - 121 -i n 40 ml. of a d s o r p t i o n medium, then: T = S V = 0.04 With these s u b s t i t u t i o n s ' , e quation (vi) i s r e w r i t t e n as: . . . ab( S -. W.- ) :. x m , ..... = : ~ ( V l l l ) 0.04 + a( S - W.- ) m The percentage o f dose (P) adsorbed by W gm. of kaolin-, i s expressed as: . . W.*-P = m or ?-x'. •'.•= PS (ix) ^ifr- W s u b s t i t u t i n g t h i s v alue of ~ i n t o ( v i i i ) y i e l d s : P = ab ( 1 - P ) (x) W 0.04 +• aS ( 1 - P) Expanding (x) and r e a r r a n g i n g terms g i v e : aSP 2 - ( Wab + 0.04 + aS ) P + Wab = 0 (xi) When W i s equal t o 6 gm. S u b s t i t u t i o n o f t h i s value i n the above equation (xi) y i e l d s : aSP 2 - ( 6ab + 0.04 + aS ) P + 6ab = 0 which i s Equation 7. When W i s equal t o 17.76 gm. S u b s t i t u t i o n of t h i s v a l u e i n (xi) y i e l d s : . aSP 2 - ; (17.76ab + 0.04 + aS)p: + 17.76ab = 0 

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