Controlled Release Polymeric Formulations

8 Controlled Release of Biologically Active Agents SEYMOUR YOLLES, THOMAS L E A F E , MARIO SARTORI, MARY TORKELSON, and LAIRD WARD Department of Chemistry, University of Delaware, Newark, Del. 19711

Downloaded by FUDAN UNIV on November 20, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0033.ch008

FRED BOETTNER Rohm and Haas Co., Springhouse, Pa. 19477

Previous papers from t h i s laboratory have dealt with the development o f a system f o r d e l i v e r y of drugs at c o n t r o l l e d rates, over a long period of time (1,2), and with the release rates of n a r c o t i c antagonists (2,4,5) and anticancer agents (3) from composites with polymers. The influence of the (a) nature of the polymer, (b) molecular weight of the polymer and (c) form of the composites on the release rates was also investigated (2,6). This paper reports the r e s u l t s of recent investigations r e l a t i v e t o in vivo and in v i t r o release rates of progesterone (Δ'-pregnene-3,20dione), β-estradiol (estra-1,3,5 (10)-triene-3,17d i o l ) and dexamethasone (9-fluoro-11 β, 17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione) from p o l y ( l a c t i c acid) (PLA) composites. Experimental All countings were performed with l i q u i d s c i n tillation spectrometers . The counting s o l u t i o n consisted of a mixture of 2,5-diphenyloxazole (22.0 g), 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene (0.4 g ) , and T r i t o n X 100 (a nonionic wetting agent) (1000 ml) d i l u t e d to 4000 ml with toluene in the i n vivo experiments. A q u a s o l was used as a 1

® 2

®3

1. Packard Tri-Carb model 3003 (Packard Instruments, Downers Grove, IL) was used i n the i n vivo experi ments, and a Beckman LS-100 (Beckman Instruments, F u l l e r t o n , CA) was used i n the i n v i t r o experiments. 2. Rohm and Haas, P h i l a d e l p h i a , PA 3. New England Nuclear Corp., Boston, MA

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

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s c i n t i l l a t i o n l i q u i d i n the i n v i t r o experiments. Preparation of Progesterone-PLA Chips. Unlabeled progesterone^ (0.61 g) , ^-^C-progesterone^ (3.5 ml benzene s o l u t i o n ) , p o l y ( l a c t i c a c i d ) , prepared as i n réf. (2) (1.05 g) and t r i b u t y l c i t r a t e (0.09 g) were dissolved i n dichloromethane (150 ml). The solvent was flashed o f f under reduced pressure and the residue, wrapped i n aluminum f o i l , was melt-pressed at 170° under a t o t a l load of 3 metric tons f o r 30 sec. (shims 0.90 mm thick were used) to produce f i l m s of uniform thickness i n which no imperfection due to a i r or gas was observed.^--x The films were ground i n a cooled CRC Micro-MillCE^ The p a r t i c l e s obtained were screened, and f r a c t i o n s of an average s i z e of 0.45-0.25 mm were c o l l e c t e d . The s p e c i f i c r a d i o a c t i v i t y was 0.052 mCi/g. Unlabeled composites were prepared i n the same way, except that l^c-progesterone was not added. 5


6

Preparation of Progesterone-PLA Beads. Solution A. To Acrysol& A-5^ (24% solution) (16 σ) was added a s o l u t i o n of CM-cellulose 9M31F® ' (1.7% solution) (41 g) i n 550 ml d i s t i l l e d water, prepared the night before. A f t e r adjusting pH to 10.3-10.4 with a 50% sodium hydroxide s o l u t i o n , a s o l u t i o n of Pharmagel® (0.4 g) i n 50 ml water, 2 drops of T r i t o n X 155^ and 10 g of dichloromethane were added and s t i r r e d at 50°C j u s t before use. Solution B. PLA (9 g) [prepared as i n r e f . (2)] was added to a mixture of methanol (8 ml) and dichloromethane (125 g) while s t i r r i n g , and j u s t before mixing with s o l u t i o n A, a s o l u t i o n of progesterone ( p a r t i a l l y tagged as i n the preparation of the chips) i n methanol and dichloromethane was added. Solution A was placed i n a one l i t e r , round bottom, 3-neck-flask, equipped with paddle s t i r r e r . Solution Β was added while s t i r r i n g (120-130 rpm) and passing nitrogen over the surface of the l i q u i d . The reaction mixture was heated to 60-65° over 1-2 hr. period. (If agglomerates are noticed, a g i t a t i o n i s increased to 170-175 rpm.) A f t e r cooling to r . t . , 8

4. 5. 6. 7. 8.

Sigma Chem. Co., St. Louis, MO Carver laboratory press model C The Chemical Rubber Co., Cleveland, OH Hercules Inc., Wilmington, DE Kind and Knox G e l a t i n Co., Cherry H i l l , NJ


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the beads were c o l l e c t e d by f i l t r a t i o n and washed with water. A y i e l d of about 9 g of beads was obtained. The beads obtained were screened and f r a c t i o n s of an average s i z e 0.42-0.25 mm were c o l l e c t e d . The s p e c i f i c a c t i v i t y of the composite was 0.0358 mCi/g. Annealed beads were obtained by heating beads prepared as above at 150-160° f o r 10 sees, i n a r o t a t i n g tube. Preparation of Estradiol-PLA Chips. A mixture of 3 - e s t r a d i o l (3.01 g ) , t r i b u t y l c i t r a t e (0.76 g) , p o l y ( l a c t i c acid) (11.25 g) and dichloromethane (350 ml) was s t i r r e d at r . t . Solvent was evaporated under vacuum at 60° and the residue worked up as described above f o r the preparation of progesteronePLA chips.


4

Preparation of Dexamethasone-PLA Chips. A mixture of dexamethasone (3.01 g), t r i b u t y l c i t r a t e (0.76 g), p o l y ( l a c t i c acid) (11.26 g) and d i c h l o r o methane (250 ml) was s t i r r e d at r . t . Solvent was evaporated under vacuum at 60° and the residue worked up as previously described f o r the preparation of progesterone-PLA chips. A 10% composite was prepared s i m i l a r l y . 4

Experiments In Vivo. A) Implantation Method. These experiments Were performed on groups of three or f i v e beagle type mongrels, weighing approximately 10 Kg, by implanting suspensions i n methylcellulose of 28.6 mg of progesterone-PLA (A.I. 35%) per K i l o animal when non-annealed beads were used, and 68.5 mg of progesterone-PLA (A.I. 35%) per Kilo-animal when chips were used. The following implanting procedure was used: progesterone-PLA composite (beads or chips) was placed i n the b a r r e l of a 5 ml p l a s t i c syringe and methylcellulose s o l u t i o n i n p h y s i o l o g i c a l s a l i n e added while shaking u n t i l an even d i s t r i b u t i o n of the composite p a r t i c l e s throughout the methylcellulose was obtained. This suspension was i n j e c t e d subcutaneously p o s t e r i o r to the shoulder i n the back region of e i t h e r side. Urine samples were c o l l e c t e d and radioassayed by standard l i q u i d s c i n t i l l a t i o n counting technique (see réf. (2)).



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B) Control Experiments on urinary Excretion R a d i o a c t i v i t y . In order to determine the r e l a t i o n ship of urinary excretion r a d i o a c t i v i t y to actual release of progesterone from implanted composites with PLA, c o n t r o l experiments were performed. A s o l u t i o n of 0.1 + 0.02 g of tagged progesterone, sans PLA, i n methylcellulose was implanted subcuta neous l y into 5 dogs and the r a d i o a c t i v i t y of urine monitored d a i l y . An average of 29% of the implanted drug was recovered i n urine during the 30 day t e s t . In the supposition that the remaining 71% consisted of material released through other ways, the values of the progesterone released i n urine have been corrected by multiplying them by the factor 3.45 (= 100/29). C) Blood Level of Drug by Radioimmunoassay. The Radioimmunoassay (RIA) technique described i n ref. (7) and (IB) was used f o r the determination of the blood l e v e l of progesterone and dexamethasone i n dogs and of e s t r a d i o l i n wethers. In the t e s t s with progesterone, composites containing 15% and 30% A.I., as a 30% suspension i n methylcellulose, were i n j e c t e d i n t o bitches. Doses of 15, 60 and 240 mg of progesterone f o r animal were used and the blood l e v e l s of progesterone were determined d a i l y by RIA techniques. In the t e s t s with e s t r a d i o l , composites of PLA containing 20% e s t r a d i o l were injected as a 30% suspension i n methylcellulose. Doses of 16 mg of e s t r a d i o l were used and the blood levels measured d a i l y by RIA techniques. In the t e s t s with dexamethasone, composites of PLA containing 10% and 20% A.I. were i n j e c t e d as sus pension i n methylcellulose. Doses of 16 mg of dexamethasone per animal were used and the blood l e v e l s measured d a i l y as above. Experiments In V i t r o . The release rates of progesterone-PLA composites were also determined i n v i t r o by d i a l y z i n g samples of composite with Ringer's s o l u t i o n . The d i a l y s i s c e l l ^ consisted of two P l e x i g l a s s blocks 5 χ 5 χ 1.5 cm with hemi s p h e r i c a l chambers separated by a lamb skin membrane. The following procedure was used: the assembled d i a l y s i s c e l l was placed on a magnetic s t i r r e r and one of the needle i n l e t s i n the lower h a l f of the c e l l was connected to a pump ( p e r i s t a l t i c ) capable 9.

National S c i e n t i f i c Co., Cleveland, OH



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of d e l i v e r i n g 10 ml/hour of Ringer's s o l u t i o n (pH 7.0). The other side of the lower h a l f of the c e l l (other e x i t needle) was connected to a tube leading to a 500 ml graduated receiver f o r sample c o l l e c t i o n . The pump was started and the lower h a l f of the c e l l was f i l l e d with d i a l y z i n g f l u i d (Ringer's s o l u t i o n ) , being c a r e f u l to remove a l l a i r bubbles from below the surface of the membrane. (This insures contact between d i a l y z i n g f l u i d and the whole membrane surface.) Dialyzing f l u i d (1-2 ml), which remained s t a t i c through the experiment, was placed i n the upper h a l f of the c e l l . Composite sample, such as beads or chips, was added through a 5 mm hole i n the top of the c e l l and while s t i r r i n g the upper chamber was f i l l e d to capacity by means of a f i n e tipped eyedropper (Pasteur dropper). Samples of d i a l y s i s e f f l u e n t were c o l l e c t e d every 24 hrs. (250 ml/day) and the r a d i o a c t i v i t y determined by standard technique. Counting solutions were prepared by d i s s o l v i n g one ml of d i a l y s i s e f f l u e n t i n 10 ml of s c i n t i l l a t i o n s o l u t i o n . The counts found were corrected f o r (a.) background (subtract 14,875 dpm/ml; previously determined by counting four d i f f e r e n t 1 ml samples of the Ringer's s o l u t i o n ) ; (b.) f o r recorded counting e f f i c i e n c y . Dividing counts found minus background by f r a c t i o n a l e f f i c i e n c y gave the corrected dpm/ml. Total dpm/day was obtained by multiplying dpm/ml by the t o t a l sample volume. The sample s i z e and type are given i n Table I. Calculations f o r %/day were made as follows: %/day Table I.

T o t a l dpm/day χ 100 T o t a l dpm i n c e l l

Type of Composite

and Sample Size

Type of Composites

Sample S i z e

chips chips film film non-annealed beads non-annealed beads annealed beads annealed beads

.0345 .0399 .0653 .0669 .0364

3,069,150 3,100,230 5,073,810 5,198,130 2,706,822

.0433 .0414

3,441,200 3,290,200

#

g

dpm

in cell


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Results and Discussion 14 The i n vivo t e s t s with C-progesterone-PLA chips, i n j e c t e d as methylcellulose suspensions i n dogs, show that the cumulative amount of drug released during an 82 day period averaged 79% of the administered dose. Of t h i s amount, only an average of 23% was recovered i n urine. The d a i l y amount of progesterone released (see F i g . 1) averaged 2.5% of the dose during the f i r s t 10 days a f t e r implantation, decreased gradually to about 1% of dose during the following 30 days and then remained r e l a t i v e l y constant at about 0.6% f o r the duration of the t e s t . When non-annealed beads instead of chips were used, the release rates were s i m i l a r t o those obtained by implanting a suspension of neat ^- Cprogesterone. Almost 100% of the administered dose was released from the composites i n about 45 days, and of t h i s amount only about 30% was found i n the urine. The d a i l y amount of progesterone released a f t e r reaching a maximum of about 13% of the dose during the f i r s t two days a f t e r implantation decreased gradually to 1% of dose during the following 18 days and then remained at 0.2-0.5% of the dose during the following 23 days (Fig. 1). This behavior indicates that the non-annealed beads, due to t h e i r macror e t i c u l a r type of structure, provide the drug with ample opportunity to d i f f u s e r a p i d l y through the polymer matrix. In the i n vivo tests by RIA with progesterone chips (30% A. I..), blood l e v e l s of progesterone of 3 ng/ml f o r a period of 21 days (Fig. 2) were obtained when doses of 240 mg i n PLA chips were i n j e c t e d i n b i t c h e s . The blood l e v e l then decreased to 0.7 ng/ml during the following days and remained f a i r l y constant at about t h i s l e v e l f o r the duration of the tests (60 days). These concentrations are above the normal progesterone l e v e l s found i n c o n t r o l t e s t s . Similar release p r o f i l e s were obtained i n t e s t s performed by i n j e c t i n g 15 and 60 mg of progesterone i n PLA chips of 30% A.I. Blood l e v e l s of about 0.8 ng/ml were found during the f i r s t 25 days a f t e r i n j e c t i o n , decreasing to lower than 0.6 during the following 30 days of the t e s t . In t e s t s with progesterone composites (15% A . I . ) , a blood l e v e l of 2.8 ng/ml was reached i n the f i r s t day when doses of 240 mg were i n j e c t e d ; then the blood l e v e l 4


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1

1

1

DAYS Figure 1.

14

In vivo daily release of C-progesterone

DAYS Figure 2.

Daily level of progesterone in blood


1


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decreased to about 0.5 ng/ml i n 25 days following a slope s i m i l a r to that obtained with a 30% composite. As expected, the higher loading dose afforded the higher blood l e v e l . The i n v i t r o releases of progesterone from Cprogesterone-PLA composites, as beads, chips and f i l m s , were determined by the membrane d i a l y s i s technique described above. The r e s u l t s reported i n F i g . 3 show that progesterone i s released f a s t e r from PLA composites i n form of non-annealed beads than from composites as chips or f i l m s . The d a i l y release from non-annealed beads began at a rate of about 4% during the f i r s t 2 days, then decreased gradually and remained at about 3% f o r the duration of the t e s t (20 days). The d a i l y release from chips started at about 2.5% i n the f i r s t 3 days and decreased to about 1.1% during the following 7 days. Composites i n form of films released progesterone at the very low rates of about 1%/day i n the f i r s t day and 0.3%/day f o r the duration of the t e s t (13 days). A considerably reduced release of progesterone from beads was obtained by heating the beads at 150-160° f o r 10 seconds ( F i g . 4). By t h i s treatment the release rate of progesterone became s i m i l a r to that from composites i n form of chips. Comparison of these i n v i t r o r e s u l t s with those obtained i n the above described i n vivo tests by the implantation method show ( F i g . 5) that the release rates of C-progesterone f chips, determined by the d i a l y s i s technique, are s i m i l a r to those obtained i n experiments with dogs and that the d i a l y s i s technique i s a u s e f u l method f o r estimating the in vivo release rates of progesterone from PLA chips. Previously reported (2) i n v i t r o techniques, e.g., with a Raab extractor, always gave poor c o r r e l a t i o n s with i n vivo experiments. The i n "vivo t e s t s with e s t r a d i o l composites i n wethers by RIA show that e f f i c a c i o u s blood l e v e l of e s t r a d i o l can be obtained f o r a period of about 55 days ( F i g . 6). Weight gain was s i g n i f i c a n t l y above the normally expected (9). I t should be noted that for some unknown reasons, the tests showed rather s i g n i f i c a n t increases of endogeneous blood l e v e l f o r day 21. The i n vivo t e s t s by RIA with dexamethasone composites (20% A.I.) show that blood l e v e l s of the drug reached an average of 6.8 ng/ml during the f i r s t 3 days a f t e r i n j e c t i o n ( F i g . 7), decreased gradually 14

r

o

m



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DAYS Figure 3.

In vitro release of progesterone by dialysis

DAYS Figure 4. In vitro daily release of progesterone from beads


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20 Figure 5.

In vivo vs. in vitro release of progesterone

0.35 r — 1 6 mg A.I. CONTROLS

60

Figure 6.

Daily level of estradiol in blood


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7.0F


—

0' 0

10

Figure 7.

20

30 DAYS

CONTROL 16 mg AI ,20% COMPOSITE 16mg A I , 10% COMPOSITE

40

50

60

Daily level of dexamethasone in blood

to about 1 ng/ml during the following 27 days and then remained constant at about 0.1 ng/ml f o r the duration of the t e s t (60 days). Similar p r o f i l e s were obtained i n tests with composites containing 10% A.I. A maximum blood l e v e l of 5 ng/ml was obtained during the f i r s t 2 days. Also i n t h i s case, the higher loading dose showed the higher blood level. In a previous paper (2_) i t was shown that d i f f e r e n t p r o f i l e s f o r the i n vivo rates were obtained i n c o n t r o l l e d release of narcotic antagoni s t s from films and chips. Rates f o r films followed a f i r s t order decay while chips gave a zero order p r o f i l e . The difference i n behavior may be a t t r i b uted to the bodies inflammatory processes triggered by the implant; the one f o r chips presumably being more e f f i c i e n t and creating a more e f f e c t i v e b a r r i e r . In the blood l e v e l work reported i n t h i s paper, lengthy f l a t p r o f i l e s are again seen with progesterone and e s t r a d i o l (Fig. 2 and F i g . 6). However, the rate curve f o r dexamethasone i s t y p i c a l l y f i r s t order (Fig. 7). This behavior supports the hypothes i s because dexamethasone i s a well-known a n t i flaramatory agent. We g r a t e f u l l y acknowledge the contributions t o t h i s work of Drs. Lloyd Faulkner and Gordon Niswender of Colorado State u n i v e r s i t y , the guidance o f Drs. E. D. Weiler and M. C. Seidel of Rohm and Haas and the assistance of Dr. Shayam Hirwe and Robert A l l a n Barker of the u n i v e r s i t y of Delaware. This work was l i b e r a l l y supported by a grant from the Rohm and Haas Company of P h i l a d e l p h i a , Pennsylvania.


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L i t e r a t u r e Cited 1. 2. 3.


4.

5. 6.

7. 8. 9.

Y o l l e s , Seymour; Eldridge, John E.; and Woodland, James H. R.; Polymer News (1971), 1, 9. Woodland, James H. R.; Y o l l e s , Seymour; Blake, David Α.; H e l r i c h , Martin; and Meyer, Francis J . ; J . Med. Chem. (1973), 16, 897. Y o l l e s , Seymour; Leafe, Thomas D. and Meyer, Francis J . ; J . Pharm S c i . (1975), 64, 115. Leafe, Thomas D.; Sarner, Stanley; Woodland, James H. R.; Y o l l e s , Seymour; Blake, David Α.; and Meyer, Francis J . ; Narcotic Antagonists Advances in Biochemical Psycopharmacology, V o l . 8, p. 569, Raven Press, New York, NY, 1973. Y o l l e s , Seymour; Leafe, Thomas D.; Woodland, James H. R.; and Meyer, Francis J . ; J. Pharm. S c i . (1975), 64, 348. Y o l l e s , Seymour; E l d r i d g e , John E.; Leafe, Thomas D.; Woodland, James H. R.; Blake, David R.; and Meyer, Francis J . ; Controlled Release of B i o l o g i c a l l y A c t i v e Agents - Advances i n Experimental Medicine and Biology, V o l . 47, p. 177, Plenum Press, New York, NY, 1973. Niswender, Gordon D.; Steroids (1973), 22, 413. England, B. G.; Niswender, G. D.; and Midgley, A. R.; J . Clin. Endocrinol Metab. (1974), 38, 42. Faulkner, Lloyd C.; Niswender, Gordon D.; Colorado State University, Fort C o l l i n s . Private Communication.


Controlled Release Polymeric Formulations

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