Controlled Release Polymeric Formulations - American Chemical

10 Fallopian Tube Cauterization by Silver Ion-Polymer Gels HARRY P. GREGOR, H . T. HSIA, and SHEILA PALEVSKY

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Department of Chemical Engineering and Applied Chemistry, Columbia University, New York, N.Y. 10027 R. S. NEUWIRTH St. Luke's Hospital Center, New York, N.Y. 10025 R. M . RICHART College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032

The s p e c i f i c stimulus for t h i s study was the observation o f Neuwirth and Richart (1) that permanent female s t e r i l i z a t i o n could be effected by F a l l o p i a n tube closure i n a non-surgical manner by i n j e c t i o n o f a s i l v e r n i t r a t e s o l u t i o n retrograde into the tube. However, solutions were much too f l u i d and could not be l o c a l i z e d e f f e c t i v e l y . The use o f a hydrophilic o i n t ment as the c a r r i e r for s i l v e r n i t r a t e was studied. Closure with rabbits, monkeys and also a few humans was observed. However, t h i s d e l i v e r y system was difficult to control although closure did occur with the r e q u i s i t e reliability. In addition, there was the problem of material being introduced into non-desirable parts o f the body c a v i t y . It was then postulated that if one could e f f e c t the release o f s i l v e r ions by a s o l i d , r o d - l i k e device which can be inserted into the F a l l o p i a n tube with the use of a hysteroscope, a p r a c t i c a l s o l u t i o n could r e s u l t . The hysteroscope i s a f i b e r o p t i c endoscope which allows transuterine d e l i v e r y of an agent without the necessity o f surgery. The purpose o f t h i s study was to examine the employment o f various non-toxic polymers which can form a matrix for the release of s i l v e r ions at desirable r a t e s . Use was made o f alginates and Pluronics, using s i l v e r n i t r a t e as the active agent. A model i n v i t r o system was developed and the rate o f s i l v e r release investigated. Materials

American Chemical Society Library 147 1155Paul16th St. N. W. and Harris; Controlled Release Polymeric Formulations ACS Symposium Series; Washington, D. C.American 20036Chemical Society: Washington, DC, 1976.

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The Pluronics (BASF Wyandotte) are block copolymers o f ethylene oxide and propylene oxide with the empirical formula

HO[ CH CH 0] [ C (CH ) HCH 0] [ CH^CI^O] H 2

2

a

3

2

b

c

where a and c are approximately equal. Pluronics are highly r e s i s t a n t to e i t h e r acid or a l k a l i attack, not p r e c i p i t a t e d by most m e t a l l i c ions and, although most are soluble i n water, they are r e l a t i v e l y non-hygroscopic. The Pluronic polyols are non-ionic surfactants with unusually low t o x i c i t y . T y p i c a l t o x i c o l o g i c a l r e s u l t s which have been reported (.2,3.) include: i n t r a venous i n j e c t i o n of a Pluronic at 0.1 g/kg body weight into dogs and 1.0 g/kg body weight into r a b b i t s caused no t o x i c symptoms; Pluronics are neither metabolized by the body nor absorbed by i t ; the acute o r a l t o x i c i t y of the Pluronic compound used here (F127) was 15 g/kg; the intravenous acute t o x i c i t y was 2.25 g/kg. A l g i n i c a c i d i s a l i n e a r block copolymer of Dmannuronic (M) acid and L-guluronic (G) a c i d , with 1,4 linkage r e s u l t i n g i n one free carboxylic and two free hydroxyl groups per uronic acid residue; the aldehydes are blocked(Fig.1). The d i s t r i b u t i o n of the uronic acid residues i n the polymer molecules and t h e i r sequence are not accurately known, and a l g i n i c acid i s known t o be chemically heterogenous(4) . The l i n e a r alginate molecule consists of blocks, i n which one or the other uronate predominates. These have a number average of 20-30 monomer u n i t s and are separated by regions which contain a l t e r n a t i n g mannuronic and gulur o n i c a c i d residues (J3,6.). The use o f IR techniques gives an i n d i c a t i o n of the uronate composition (7.). While absorption bands at 10.8 and 12.4jum were stronger for samples r i c h i n mannuronic residues, the bands at 10.6 and 12.7jum were stronger for those with higher contents o f guluronic a c i d . The r a t i o o f mannuronic to guluronic monomer u n i t s i s dependent upon the exact algae, i t s age and i t s l o c a t i o n . The d i s s o c i a t i o n constant of a l g i n i c acid i s a function o f t h i s r a t i o . The pK values o f mannuronic acid and guluronic a c i d are 3.38 and 3.65, respectively. a

Paul and Harris; Controlled Release Polymeric Formulations ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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-G-G-G-G Figure 1.

Structure of alginic acid

Data obtained from t h i n - l a y e r chromatography, pH and v i s c o s i t y measurements indicate that ions are held by a l g i n i c a c i d v i a ion exchange as w e l l as chelating mechanisms. Mechanisms not involving ion-exchange are due p r i m a r i l y to the presence o f the two v i n c i n a l hydroxyl groups i n each uronic acid residue. Chelation with Ca i s strong (8) and c r o s s - l i n k i n g occurs. Mg does not react or form g e l - l i k e complexes as does Ca (9., 10). The intermolecular forces and mechanisms i n volved i n i o n i c c r o s s - l i n k i n g are not w e l l understood. The sodium alginate used i n t h i s study was Kelcos o l (Kelco Co.), a Na alginate o f high molecular weight. The t o x i c i t y of the alginates has been investigated ext e n s i v e l y over many years. Some of the r e s u l t s obtained are: incorporation of sodium alginate into d i e t of r a t s , mice, chicks, cats and guinea pigs for prolonged periods caused no deleterious e f f e c t s (11,12); the LD50 of alginates i s above 5 g/kg body weight (13_) 7 the im-


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FORMULATIONS

p l a n t a t i o n of calcium alginate around the femoral shaft i n the guinea p i g showed complete absorption w i t h i n a period of two weeks (14). S i l v e r alginate was prepared from a 2% (w/v) s o l u t i o n of Kelgin-Gel LV (low molecular weight sodium font) which was added to a 3% (w/v) s i l v e r n i t r a t e s o l u t i o n . Vigorous a g i t a t i o n , followed by washing, f i l t r a t i o n and a i r - d r y i n g , then ground to 50-100 microns with a MicroM i l l gave a tough and hard m a t e r i a l . Experimental S i l v e r impregnated s o l i d plugs were prepared either by compressing a powder or by melting a compressed powder mixture i n a mold. Excessive exposure to l i g h t caused photodecomposition. Aluminum blocks were used as molds, with holes having diameters of 0.9 or 1.4 mm d r i l l e d and t h e i r tops reamed. A piece of Lucite was bolted to the bottom of the metal mold. The molding process required repeatedly f i l l i n g the d i s h l i k e hole on top of the mold with powdered m a t e r i a l and then forcing t h i s into the hole with a s t e e l rod having the same diameter. Force was exerted by the use of a small d r i l l press with a lever arm of about 8 inches, applying the normal pressure that can be exerted manua l l y by the operators. The l i m i t of pressure used was determined by the stress f a i l u r e of the rods. The sol i d plug was then ejected from the die a f t e r removing the Lucite p l a t e . I t could be used "as i s " or be subjected to a coating treatment. When s o l i d Pluronics were used, the mold was placed on a hot plate a f t e r the removal of the Lucite block. The same powder was placed i n the reamed out cone. When the powders l i q u i f i e d and f i l l e d the hole, the assembly was removed from the heater, cooled to room temperature and the i n s e r t s ejected. With the sodium alginate-based formulations, a paste of the mixed powders was prepared by the dropwise addition of a small quantity of d i s t i l l e d water, f o l lowed by rapid mixing, and then the plugs were formed i n the mold. Powdered mixtures that were too dry were d i f f i c u l t to dislodge from the mold and did not remain coherent afterwards; those containing too much water were soft and lacking i n mechanical strength.


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Some of the i n s e r t s were coated by dipping i n a 1% high MW sodium alginate s o l u t i o n and then into 10% calcium n i t r a t e s o l u t i o n . The coating immediately hardened with s l i g h t shrinkage. With t h i s treatment the suppositories became much stronger and easier to handle. Upon exposure to aqueous media, a l l treated i n s e r t s retained t h e i r s t r u c t u r a l i n t e g r i t y much longer than t h e i r uncoated counterparts. Small tubes of regenerated c e l l u l o s e were used as model F a l l o p i a n tubes. A casting s o l u t i o n of 3% c e l l u lose t r i a c e t a t e (acetyl content 39.4% Eastman Kodak) i n acetone was used. Glass tubes having a uniform diameter of 1.4-1.6 mm (melting point c a p i l l a r y tubes) were dipped into t h i s solution, rotated h o r i z o n t a l l y i n ambient a i r for drying, dipped and dried two more times to form a f i l m of the desired thickness. The l a s t coat was not allowed to dry completely; as soon as i t began to become opaque, the tube was placed i n a hydrolysis s o l u t i o n of 0.1 M sodium carbonate, 0.1 M sodium bicarbonate at pH 10 for 24 hours at 65° C. These tubelets were then rinsed and stripped o f f the glass tubes. Depending upon the mode of preparation, these tubes had a w a l l thickness of 20 t 6 microns and had a water content of about 70%. In v i t r o rate studies were c a r r i e d out by placing i n s e r t s into the moist c e l l u l o s e tubelets. A f t e r the ends were t i e d o f f with thread, each set was placed i n to a 10 ml beaker and 5 ml of a saline s o l u t i o n was added. The composition of t h i s s o l u t i o n was: 145 mM i n NaN(>3, 4.6 mM i n KNO3, and 3.5 mM i n Ca(N03)2# approximating the c a t i o n i c composition i n the F a l l o p i a n tube of the rabbit (15,16). At appropriate time i n t e r v a l s , a l i q u o t s were removed and analyzed for t h e i r s i l v e r content by t i t r a t i o n . For the animal experiments, the s u r g i c a l procedure was as follows: a midline abdominal i n c i s i o n was made and the f i m b r i a l end of the F a l l o p i a n tube i d e n t i f i e d , followed by i n s e r t i o n of the plug. This i n s e r t was then squeezed down (toward the i n t e r s t i t i a l portion) as far as possible, preferably to a point about 4 cm from the proximal end of the tube. Table I l i s t s some of the i n s e r t s and t h e i r gross composition.



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FORMULATIONS

TABLE I COMPOSITION (WEIGHT %) OF INSERT PREPARATION Insert NaAlg CaAlg AgAlg Pluronic CGP (F127) No. HMW LMW LMW AqNO-* 100 1* 2* 50 50 3* 75 25 1 60 7 30 9 3 22 45 8 30 60 9 40 5 11 50 30 15 12 40 60 10 14 30 60 10 40 17 50 1 18 10 80 9 2 15 19 13 70 2 18 20 80 21 90 10 22** 30 10 80 23* 20 70 24* 30 2

* Post-coated with 1% NaAlg (HMW), hardened i n 10% Ca(N0 ) 2H 0. * Add 60 parts sucrose. Calcium glycerophosphate. e

3

2

2

Results In i n v i t r o t e s t s , each i n s e r t swelled s l i g h t l y during the course of these studies. At the end of each experiment, the a x i a l expansion was about 15% while r a d i a l swelling amounted to 5-10%. Figure 2 shows the cumulative release of s i l v e r as a function of time. When Pluronic was the only polymer c a r r i e r , most of the s i l v e r was released within the f i r s t f i f t e e n minutes. This rapid release was observed for a l l Pluronic p o l y o l s . When a sodium alginate was used as the polymer c a r r i e r , t h i s rate was slowed down substant i a l l y . The same e f f e c t was obtained when an a d d i t i o n a l Ag source was s i l v e r acetate suspended i n sesame o i L H i s t o l o g i c a l r e s u l t s are i n Table I I . The death of a circumscribed t i s s u e or of an organ i s termed n e c r o s i s . F i b r o s i s r e f e r s to the development i n an


10.

GREGOR E T A L .

0

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Tube Cauterization

2

4

6 Time, hr

153

8

10

Figure 2. Silver release in vitro. Ο AgNO« 30%, pluronic F127 70%; Π AgNO 50%, low MW NaAlg. 50%; Δ AgNO 30%, silver acetate 10%, low MW NaAlg 60%. s

s

organ of excess fibrous connective t i s s u e s , often i n response to necrosis. F i b r o s i s i s a necessary but not s u f f i c i e n t condition leading to tubal blockade. When tubal t i s s u e s are severely damaged, the tube may en large i n diameter and be f i l l e d with f l u i d , a condi t i o n known as hydrosalpinx. It should be noted that b i l a t e r a l tubal obstruc t i o n i s d i f f i c u l t to achieve i n r a b b i t s , a t t r i b u t e d to the morphology of i t s oviduct. Other investigators have found that formulations and d e l i v e r y methods which produced closure i n primates such as p i g t a i l monkeys did not necessarily e f f e c t closure i n r a b b i t s . In our study, there was no s i g n i f i c a n t difference i n t i s s u e damage when the period between i n s e r t i o n and s a c r i f i c e


CONTROLLED

154

A * 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Β 1 1 2 2 3 3 7 7 8 8 9 9 11 11

15 16

12 12

17

14

18

14

19

17

20

17

21 22 23 24

18 18 19 19

25

20

26 27 28 29,30 31,32

20 21 22 23 24

RELEASE POLYMERIC

FORMULATIONS

TABLE II RESULTS OF ANIMAL EXPERIMENTS Ξ D** C Hydrosalpinx 2 4+ 4+ necrosis 4 4+ Minimal damage 2 Minimal damage 4 Minimal necrosis 2 2+ Minimal necrosis 4 Acute inflammation 2+ 3 Hydrosalpinx 4+ 6 Fibrosis 3 3+ Hydrosalpinx 0 6 Acute inflammation 2 3+ Hydrosalpinx 6 4+ necrosis 4+ 3 Hydrosalpinx, 6 1+ 4+ necrosis Focal necrosis deep 3 3+ 6 3+ Hydrosalpinx, 4+ necrosis 3 Hydrosalpinx, 3+ 4+ necrosis 6 4+ 4+ necrosis, i n t o muscularis 3 4+ 4+ necrosis extend ing i n t o f a t 6 Hydrosalpinx, 3+ ne c rο s i s, fibrοs i s Necrosis, thru w a l l 2 3+ 4+ necrosis 6 2 Necrosis, thru w a l l 3+ 4+ necrosis, thru 4+ 6 wall Hydrosalpinx, 3 3+ 4+ necrosis 4+ necrosis 4+ 6 3 No s i g n i f i c a n t alterations 3 Necrosis, epithelium 0 6 Regenerated 6 0

Α-animal no.;B-Insert no.; C-Period to sacrifice(wks); D-Residuum of i n s e r t ; E-Observations. * 1-28, New Zealand rabbits; 29-32, p i g t a i l monkeys. ** Amt. of i n s e r t remaining: 0,1+ to 4+.


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AL.

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was increased from 2-3 weeks to 4-6 weeks. No tubal blockade was achieved e i t h e r i n rabbits or p i g t a i l monkeys. Discussion When s i l v e r alginate was used, i t was so slowly absorbed by the surrounding t i s s u e s i n the rabbit that i t was concluded that, however e f f e c t i v e calcium ions were i n d i s p l a c i n g s i l v e r ions from the a l g i n complex, the oviducal environment did not furnish enough calcium for t h i s purpose. Calcium alginate gels were, on the other hand, completely absorbed within 4 weeks even i n the presence of s i l v e r ions. The s i l v e r n i t r a t e r e l e a s i n g formulations, when placed i n the rabbit F a l l o p i a n tube, produced necrosis of the epithelium and the w a l l at higher concentration^ with no observable e f f e c t at the lower concentrations. Sections of the oviduct not i n contact with the i n s e r t were not a f f e c t e d , i t was apparent that l o c a l i z e d r e lease was obtained, but i t was necessary to c o n t r o l the amount of s i l v e r n i t r a t e present and i t s rate of r e lease so as not to produce so much necrosis that p e r i toneal damage occurred. It was concluded that f a i l u r e to obtain closure was due to two f a c t o r s . F i r s t and more important, the highly invaginated nature of the oviduct was such as to prevent s i l v e r ions from reacting with the e n t i r e e p i t h e l i a l surface over a s u f f i c i e n t length of the tube. Regeneration of the epithelium i s known to proceed r a p i d l y , so islands of non-cauterized t i s s u e grow and prevent the slower i n f i l t r a t i o n of f i b r o b l a s t s from the c e l l s below the epithelium. Second, the duration of chemical c a u t e r i z a t i o n probably was not long enough. Experiments with other chemical c a u t e r i z i n g agents have shown that a severe but very rapid burn does not necess a r i l y produce closure. A subsequent study, one s t i l l i n progress, makes use of p a s t e - l i k e formulations of alginate and s i l v e r s a l t s , which distends the tube as i t i s i n j e c t e d . With t h i s formulation, tubal closure has been observed i n the r a b b i t .


156


FORMULATIONS

Acknowledgment s This work was supported by NICHD contract Nol-HD3-2797. The authors express t h e i r thanks for the t e c h n i c a l assistance of Messrs. George C o t t r a l and Douglas Rosenberg. L i t e r a t u r e Cited 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

16.

Neuwirth, R., Richart, R., and Taylor, H.,Jr., Amer.J.Obstet.Gynec.,(1971),38,51. "The Wonderful World of Pluronic Polyols", BASF Wyandotte Co., 1973, p.2. Private Communications from Wyandotte Co. Haug, Α., Acta Chem.Scand.,(1959),13,601. _______ , ibid.,(1966), 20, 183. _______ , i b i d . , (1967), 21, 691. _______ , Colloques Internationaux du Centre National de la Recherche Scientifique,(1960), No. 103. Cozzi, D., Desideri,P. and L e p r i , L., J . Chromatogr.,(1969),40 (1), 130. Wassermann, A. and cooper, R., Nature, (1957), 180, 1072. T h i e l e , H. and Schact, G., K o l l o i d Z.,(1958), 161, 120. Nilson, H., and Wagner, J . , Pro.Soc.Exp.Bio. Med., (1951), 76, 630. Johnston, J . , Lobdell, B., and Woodard, G., Woodard Research Corp., unpublished data. Johnston, D., SS-3428 and SS-3429, Woodard Research Corp., unpublished data. Blaine, G., Annals of Surgery, (1947),125,102. Mastroianni, L.,Jr., Urzua, M., Avalos, M., and Stambaugh, R., Am.J.Obstet.Gynec., (1969), 103, 703. David, Α., Brackett, B., Garcia, C., and Mastroianni, L.,Jr., J.Reprod.Fert., (1969), 19, 285.


Controlled Release Polymeric Formulations - American Chemical

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