Probing Bioactive Mechanisms - American Chemical Society

Chapter 17

Therapeutic Potential for Parathyroid Hormone Antagonists

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Mark E. Goldman and Michael Rosenblatt Department of Biological Research and Molecular Biology, Merck Sharp and Dohme Research laboratories, West Point, PA 19486

Due to a lack of selective pharmacological agents, therapy of hyperparathyroid hypercalcemia and other hypercalcemic disorders has largely been considered a medical problem managed symptomatically or surgically by clinicians. Based upon recent gains in the understanding of parathyroid hormone (PTH) structure -activity relationships and the pathogenesis of hyperparathyroidism and hypercalcemia, the rational development of drugs for treating these disorders should now be considered. Potent and selective PTH antagonists, when available, may prove useful in treating not only primary hyperparathyroidism and post-renal transplant secondary hyperparathyroidism, but also hypercalcemia of malignancy and osteoporosis. Hypercalcemic d i s o r d e r s caused by i n c r e a s e d c i r c u l a t i n g l e v e l s o f PTH or PTH-like peptides a r e common, a d v e r s e l y a f f e c t most organs and a r e p o t e n t i a l l y l i f e - t h r e a t e n i n g . S e l e c t i v e t h e r a p e u t i c agents, however, are not c u r r e n t l y a v a i l a b l e . To expedite t h e development of such agents, an understanding of p h y s i o l o g i c a l processes r e g u l a t i n g blood c a l c i u m l e v e l s as w e l l a s endocrine changes d u r i n g hypercalcemic s t a t e s i s r e q u i r e d . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s t u d i e s have been i n i t i a t e d based upon knowledge of t h e amino a c i d sequences o f PTH and PTH-like p e p t i d e s . As a r e s u l t of p e p t i d e t r u n c a t i o n , s u b s t i t u t i o n o f n a t u r a l and s y n t h e t i c amino a c i d s and combination of v a r i o u s m o t i f s from PTH and PTH-like p e p t i d e s , novel PTH a n t a g o n i s t s p o s s e s s i n g PTH i n h i b i t o r y a c t i v i t i e s in. v i t r o and i_n v i v o have been developed. NORMAL CALCIUM HOMEOSTASIS The normal maintenance of blood c a l c i u m l e v e l s w i t h i n a narrow range i s r e g u l a t e d by two hormones i n humans: PTH and 1,25 dihydroxy v i t a m i n D ( 1 , 2 5 ( 0 H ) 2 V i t D ) . In t h e absence o f these hormones, blood c a l c i u m l e v e l s f a l l . The i n t e g r a t e d a c t i o n s o f PTH and 1,25(0H) VitD3 a r e r e s p o n s i b l e f o r m a i n t a i n i n g c a l c i u m l e v e l s i n the normal range. C a l c i t o n i n ' s a c t i o n s i n many aspects oppose those 3

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Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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of PTH. Although i t i s r e s p o n s i b l e f o r d e c r e a s i n g c a l c i u m l e v e l s i n lower s p e c i e s , t h e p h y s i o l o g i c a l r o l e o f c a l c i t o n i n i n humans i s uncertain. As blood c a l c i u m l e v e l s f a l l , PTH i s r e l e a s e d from t h e p a r a t h y r o i d gland and a c t s d i r e c t l y upon t h e kidney and bone a s w e l l a s i n d i r e c t l y on t h e i n t e s t i n e s . The r e n a l a c t i o n s o f PTH, mostly mediated by cAMP, a r e summarized i n Table I . These d i r e c t r e n a l PTH a c t i o n s , t h e r e f o r e , e l e v a t e blood c a l c i u m l e v e l s by enhancing c a l c i u m r e a b s o r p t i o n and by lowering blood phosphate c o n c e n t r a t i o n s and pH. The l a t t e r two a c t i o n s o f PTH a l l o w c a l c i u m l e v e l s t o r i s e t o a g r e a t e r e x t e n t without p r e c i p i t a t i n g c a l c i u m phosphate. Table I. Renal Actions

of Parathyroid

Hormone

1. 1 , 2 5 ( 0 H ) V i t D - d e p e n d e n t stimulation o f renal tubular calcium reabsorption 2. I n h i b i t i o n o f phosphate r e a b s o r p t i o n and s t i m u l a t i o n o f proximal t u b u l a r phosphate e x c r e t i o n 3. I n h i b i t i o n o f sodium-hydrogen exchange i n t h e proximal t u b u l e s r e s u l t i n g i n t h e i n h i b i t i o n o f hydrogen i o n s e c r e t i o n and enhanced e x c r e t i o n o f b i c a r b o n a t e . 4. A c t i v a t i o n o f proximal convoluted tubule 25(0H)VitD3 1-a-hydroxylase 2

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Bone i s t h e body's c a l c i u m r e s e r v o i r . PTH s t i m u l a t e s bone r e s o r p t i o n l e a d i n g t o t h e d i s s o l u t i o n o f h y d r o x y a p a t i t e and r e l e a s e of c a l c i u m and phosphate i n t o t h e b l o o d . T h i s a c t i o n o f PTH appears t o be t h e major mechanism f o r t h e r a p i d e l e v a t i o n o f blood c a l c i u m levels. PTH a l s o maintains blood c a l c i u m l e v e l s by promoting c a l c i u m r e a b s o r p t i o n from t h e r e n a l t u b u l e s . I n d i r e c t c a l c i u m l e v e l - i n c r e a s i n g a c t i o n s o f PTH on t h e g u t a r e mediated through t h e v i t a m i n 0 system. F o l l o w i n g s y n t h e s i s i n the s k i n and h y d r o x y l a t i o n by t h e l i v e r , 25(0H)VitD3 i s converted to i t s b i o l o g i c a l l y - a c t i v e form, 1,25(0H)2VitD3, by r e n a l 1-a-hydroxylase ( 1 ) . This i s t h e rate limiting step i n 1,25(0H>2VitD3 s y n t h e s i s and i s mediated by t h e s t i m u l a t i o n o f a d e n y l a t e c y c l a s e - l i n k e d PTH r e c e p t o r s l o c a t e d i n t h e proximal c o n v o l u t e d t u b u l e . 1,25(OH)2VitD a c t s upon t h e i n t e s t i n e s t o i n c r e a s e c a l c i u m a b s o r p t i o n through s t i m u l a t i o n o f a v i t a m i n D-dependent c a l c i u m b i n d i n g p r o t e i n . Although v i t a m i n D p r o d u c t i o n i s n o t d i r e c t l y r e g u l a t e d by acute changes i n c a l c i u m homeostasis, the l , 2 5 ( 0 H ) V i t D and PTH systems a r e i n t e r t w i n e d . 1,25(0H) VUD3 modulates t h e c a l c i u m s e t - p o i n t f o r PTH s e c r e t i o n by s t i m u l a t i n g p a r a t h y r o i d gland v i t a m i n D r e c e p t o r s which decrease PTH gene t r a n s c r i p t i o n and modulate PTH r e l e a s e . PTH i s s y n t h e s i z e d i n t h e p a r a t h y r o i d gland as an 115-amino a c i d gene product (preproPTH) and processed by t h e endoplasmic r e t i c u l u m and g o l g i t o an 84-amino a c i d l i n e a r b i o l o g i c a l l y - a c t i v e p e p t i d e ( f o r a review, s e e (2)). PreproPTH s y n t h e s i s i s r e g u l a t e d a t both t r a n s c r i p t i o n a l and p o s t - t r a n s c r i p t i o n a l l e v e l s by e x t r a c e l l u l a r c a l c i u m and 1,25(0H)2VitD3 ( 1 , 3 , 4 ) . In t h e presence o f e l e v a t e d c o n c e n t r a t i o n s o f c a l c i u m , t h e i n t r a c e l l u l a r d e g r a d a t i o n o f PTH i n 3

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the p a r a t h y r o i d gland i s g r e a t e r than a t low c a l c i u m c o n c e n t r a t i o n s . Release of v a r i o u s endogenous fragments of PTH-(1—84) from the p a r a t h y r o i d gland i s p a r t i a l l y r e s p o n s i b l e f o r the h e t e r o g e n e i t y of c i r c u l a t i n g PTH molecules. PTH i s f u r t h e r metabolized upon r e l e a s e from the p a r a t h y r o i d gland by the kidney and l i v e r . The major c i r c u l a t i n g forms of PTH i n the bloodstream are b i o l o g i c a l l y i n a c t i v e . The c l a s s i c a l b i o l o g i c a l a c t i v i t i e s o f PTH r e s i d e i n the N-terminal 34 amino a c i d s ( 5 ) . The remainder of the PTH molecule may be r e s p o n s i b l e f o r p r o l o n g i n g the h a l f - l i f e of PTH i n v i v o . I n c e r t a i n d i s e a s e s t a t e s , m i d - p o r t i o n and C-terminal fragments may accumulate i n the c i r c u l a t i o n and have been hypothesized t o i n h i b i t e r y t h r o p o i e s i s , c a r d i a c f u n c t i o n , nerve c o n d u c t i o n , red blood c e l l s u r v i v a l time and glucose t o l e r a n c e ( 6 ) . The main s i g n a l t r a n s d u c t i o n mechanism f o r PTH r e c e p t o r s t i m u l a t i o n i s the cAMP system. S t i m u l a t i o n of r e n a l or bone PTH r e c e p t o r s causes a n e l e v a t i o n o f membrane-bound and guanyl nucleotide-dependent adenylate c y c l a s e a c t i v i t y r e s u l t i n g i n the enhanced formation of cAMP from ATP. Recent s t u d i e s suggest t h a t PTH may a l s o s t i m u l a t e i n o s i t o l t r i p h o s p h a t e and d i a c y l g l y c e r o l production in renal t i s s u e (7). PATHOPHYSIOLOGY OF HYPERPARATHYROID HYPERCALCEMIA Symptoms and Side E f f e c t s of Hypercalcemia Hypercalcemia a f f e c t s most organ systems. Depending upon the age of the p a t i e n t , symptoms may e a s i l y be recognized as an i n d i c a t i o n of hypercalcemia or may be confused with o t h e r d i s e a s e s t a t e s . C e n t r a l nervous system symptoms o f hypercalcemia include l e t h a r g y , decreased c o g n i t i v e f u n c t i o n s , d e p r e s s i o n , c o n f u s i o n and i r r i t a b i l i t y . The changes i n c o g n i t i v e a b i l i t i e s can vary from simple memory l o s s t o dementia or p s y c h o s i s . These h y p e r c a l c e m i a induced p e r s o n a l i t y changes may be i n t e r p r e t e d wrongly as normal a g e - r e l a t e d symptoms, e s p e c i a l l y i f they are slow i n appearing. P s y c h i a t r i c symptoms were more f r e q u e n t i n e l d e r l y p a t i e n t s ( 8 ) . During severe hypercalcemic c r i s e s , such as i n advanced m a l i g n a n c i e s , s t u p o r o r coma i s not uncommon. There i s a s t r o n g c o r r e l a t i o n between hypercalcemia and h y p e r t e n s i o n (9,10). Black males may have a h i g h e r r i s k f o r t h i s s i d e e f f e c t (H). Caution must be used when t r e a t i n g h y p e r c a l c e m i a induced h y p e r t e n s i o n s i n c e t h i a z i d e d i u r e t i c s can t r a n s i e n t l y produce hypercalcemia. With l o n g - s t a n d i n g hypercalcemia, c a l c i u m may be d e p o s i t e d on the c a r d i a c v a l v e s and coronary a r t e r i e s (12). Another c a r d i o v a s c u l a r s i d e e f f e c t of hypercalcemia i s l e n g t h e n i n g of the ECG QT i n t e r v a l . D e m i n e r a l i z a t i o n of bone accompanies hypercalcemia, producing o s t e o p e n i c s k e l e t a l changes. Manifestations include osteoporosis, osteomalacia and o s t e i t i s f i b r o s a e t c y s t i c a (13). As a r e s u l t of decreased bone mass, f r a c t u r e s of the femoral neck and spontaneous f r a c t u r e s of the v e r t e b r a e are most f r e q u e n t . Morphometric changes i n metacarpal bones p r o v i d e q u a n t i f i a b l e data f o r the p r o g r e s s i o n and treatment of bone d i s e a s e (11.11). Other s k e l e t a l e f f e c t s i n c l u d e a r t h r a l g i a , p e r i a r t i c u l a r c a l c i f i c a t i o n , gout or pseudogout and loose t e e t h .


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Renal e f f e c t s o f hypercalcemia i n c l u d e reduced glomerular f i l t r a t i o n r a t e (GFR), p o l y u r i a , n e p h r o c a l c i n o s i s , and renal stone d i s e a s e . Hypercalcemia causes renal v a s o c o n s t r i c t i o n which may c o n t r i b u t e t o decreased GFR. The hypercalcemia-induced polyuria r e s u l t s from 1) an impairment o f a c t i v e t r a n s p o r t of NaCl i n t h e loop o f Henle, d i s t a l t u b u l e and c o l l e c t i n g duct and 2) an i n h i b i t i o n o f v a s o p r e s s i n - f a c i l i t a t e d a b s o r p t i o n o f water i n t h e d i s t a l nephron. As a d i r e c t r e s u l t o f t h e p o l y u r i a , many s i d e e f f e c t s i n c l u d i n g p o l y d i p s i a , t h i r s t , n o c t u r i a and dehydration a r e common. P r e c i p i t a t i o n of c a l c i u m s a l t s w i t h i n t h e kidney leads t o c h r o n i c inflammatory r e a c t i o n s ( n e p h r o c a l c i n o s i s ) , f i b r o s i s , renal impairment, n e p h r o l i t h i a s i s and u r o l i t h i a s i s . F u r t h e r renal damage may occur i n d i r e c t l y from h y p e r t e n s i o n . The g a s t r o i n t e s t i n a l m a n i f e s t a t i o n s o f hypercalcemia include abdominal d i s c o m f o r t as a r e s u l t of p e p t i c u l c e r a t i o n o r p a n c r e a t i t i s , p a n c r e a t i c c a l c i f i c a t i o n , vomiting, a n o r e x i a , c o n s t i p a t i o n and weight l o s s . Neuromuscular symptoms vary from f a t i g u e t o hypotonia and a t a x i a . These symptoms may i n c r e a s e t h e r i s k o f bone f r a c t u r e . As with other organ systems such a s the kidney and pancreas, e l e v a t e d c a l c i u m l e v e l s may cause c a l c i u m d e p o s i t i o n i n t h e eye a s demonstrated by c o n j u n c t i v a l c a l c i u m d e p o s i t i o n , c o n j u n c t i v i t i s and band k e r a t i n o p a t h y . Causes o f Hyperparathyroid Hypercalcemia Primary Hyperparathyroidism (1HPT). P r i o r t o t h e r o u t i n e a n a l y s i s of serum c a l c i u m l e v e l s , 1HPT was u s u a l l y diagnosed by t h e r a d i o l o g i c presence of o s t e i t i s f i b r o s a e t c y s t i c a o r t h e presence of renal stones. During t h e l a s t two decades, 1HPT has become recognized as the most common form of hypercalcemia i n t h e general p o p u l a t i o n and i s u s u a l l y diagnosed a t t h e asymptomatic stage. The most common causes o f 1 HPT a r e p a r a t h y r o i d adenomas (80%), h y p e r p l a s i a (15%) and carcinoma (1-5%) (16,12). In t h e h y p e r p a r a t h y r o i d s t a t e , t h e p a r a t h y r o i d gland does n o t become autonomous, although t h e r e g u l a t i o n o f PTH s e c r e t i o n by c a l c i u m i s a l t e r e d . Murray and co-workers (18) demonstrated t h a t modulation o f serum c a l c i u m l e v e l s i n 1HPT p a t i e n t s by EDTA o r c a l c i u m i n f u s i o n s t i l l r e s u l t e d i n t h e a p p r o p r i a t e changes i n PTH secretion. In a d d i t i o n , s t u d i e s with normal and adenomatous p a r a t h y r o i d c e l l s i n c u l t u r e have shown t h a t 1 HPT may cause changes i n t h e c a l c i u m i n h i b i t o r y s e t p o i n t as w e l l as an i n a b i l i t y f o r c a l c i u m t o completely suppress PTH s e c r e t i o n i n s p i t e of reduced PTH mRNA l e v e l s (3,19). M u l t i p l e Endocrine N e o p l a s i a (MEN). Several p o l y g l a n d u l a r d i s o r d e r s are known which r e s u l t i n an autonomous h y p e r f u n c t i o n o f two o r more endocrine glands. These d i s o r d e r s a r e i n h e r i t e d as autosomal dominant t r a i t s . Depending upon which glands a r e d y s f u n c t i o n i n g a t the time of d i a g n o s i s , t h e c l i n i c a l p r e s e n t a t i o n i s v a r i a b l e . In MEN type 1 (MEN!), m u l t i p l e tumors of t h e p a r a t h y r o i d gland, a n t e r i o r p i t u i t a r y gland and p a n c r e a t i c i s l e t s a r e common. Parathy r o i d gland involvement i n MEN! i s t h e most common m a n i f e s t a t i o n and i s seen i n 90-95% of p a t i e n t s .


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MEN types 2 and 3 (MEN2, MEN3) a r e e n t i r e l y d i s t i n c t syndromes from MEN!. MEN2 i s most f r e q u e n t l y c h a r a c t e r i z e d by m e d u l l a r y carcinoma o f t h e t h y r o i d g l a n d . MEN3 resembles MEN2, a l t h o u g h t h e r e are several d i f f e r e n c e s .

Secondary H y p e r p a r a t h y r o i d i s m (2HPT). As w i t h 1HPT, 2HPT i s a m e t a b o l i c d i s o r d e r i n v o l v i n g enhanced s e c r e t i o n o f PTH. In t h e case of 2HPT, however, t h e HPT i s a compensatory p a r a t h y r o i d gland a d a p t a t i o n r e s u l t i n g from prolonged tendency toward hypocalcemia o r r e l a t i v e r e s i s t a n c e t o t h e m e t a b o l i c a c t i o n s o f PTH; i t o c c u r s i n an attempt t o p r o t e c t c a l c i u m homeostasis. Another d i s t i n c t i o n between the 2 forms o f HPT i s t h a t 1HPT i s a p p a r e n t l y i r r e v e r s i b l e whereas 2HPT i s u s u a l l y r e v e r s i b l e ( g i v e n s u f f i c i e n t time) a f t e r removal o f the i n i t i a l s t i m u l u s . Although 2HPT may o c c u r w i t h o s t e o m a l a c i a , v i t a m i n D d e f i c i e n c y or pseudohypoparathyroidism, by f a r , t h e most common cause i s c h r o n i c p r o g r e s s i v e r e n a l d i s e a s e . Loss o f f u n c t i o n a l nephrons i s a common o c c u r r e n c e i n t h e aging process and f o l l o w i n g h y p e r t e n s i v e , inflammatory o r i n f e c t i o u s r e n a l i n j u r y . When t h e GFR f a l l s below 50-75% o f normal, a sequence o f events r e s u l t i n g i n e a r l y 2HPT has been h y p o t h e s i z e d t o o c c u r ( f o r reviews, see 20-22). T h i s sequence can be b r i e f l y summarized as f o l l o w s : 1) decreased GFR causes a reduced e l i m i n a t i o n o f phosphate, 2) a s a r e s u l t o f e l e v a t e d i n t r a c e l l u l a r and e x t r a c e l l u l a r phosphate l e v e l s , t h e r e i s a s t o i c h i o m e t r i c decrease i n serum i o n i z e d c a l c i u m l e v e l s and a decrease i n t h e 1-a-hydroxylation o f 25(0H)VitD , 3) t h i s hypocalcemia and hypovitaminemia 0 then induce t h e enhanced s e c r e t i o n o f PTH which 4) reduces t h e t u b u l a r r e a b s o r p t i o n o f phosphate, thus i n c r e a s i n g phosphate e x c r e t i o n l e a d i n g t o 5) changes towards normal i n serum phosphate, 1,25(0H) VitD3 and c a l c i u m l e v e l s , although m i l d hypocalcemia u s u a l l y p e r s i s t s . As r e n a l d i s e a s e p r o g r e s s e s , t h e r e i s a p r o p o r t i o n a l i n c r e a s e i n serum PTH l e v e l s by s e v e r a l mechanisms. F i r s t , as a d i r e c t response to hypocalcemia, PTH s e c r e t i o n i s enhanced. Second, s i n c e t h e kidney i s t h e s o l e source o f 1 - a - h y d r o x y l a s e , p r o g r e s s i v e r e n a l d i s e a s e reduces t h e f o r m a t i o n o f 1 , 2 5 ( 0 H ) V i t D 3 l e a d i n g t o decreased i n t e s t i n a l a b s o r p t i o n o f c a l c i u m . T h i r d , decreased 1,25(0H) VitD l e v e l s may r e s u l t i n e l e v a t e d PTH s e c r e t i o n by preventing 1,25(0H) VitD3 feedback suppression o f PTH s y n t h e s i s . F o u r t h , s i n c e t h e kidney i s a p r i n c i p a l organ f o r PTH metabolism, r e n a l f a i l u r e may a l s o c o n t r i b u t e t o e l e v a t e d serum PTH l e v e l s by d e c r e a s i n g PTH breakdown (20,21). Recent s t u d i e s suggest t h a t decreased l e v e l s o f 1,25(0H) \MtD3 may be as s i g n i f i c a n t as hypocalcemia f o r i n d u c i n g and m a i n t a i n i n g 2HPT (22,23). Intravenous 1 , 2 5 ( 0 H ) V i t D therapy has been shown t o be b e n e f i c i a l f o r r e d u c i n g t h e bone m a n i f e s t a t i o n s o f 2HPT i n c l u d i n g m e t a s t a t i c c a l c i f i c a t i o n , o s t e i t i s f i b r o s a , r e n a l o s t e o d y s t r o p h y and spontaneous f r a c t u r e s . In advanced 2HPT, however, t h i s treatment may cause v i t a m i n D i n t o x i c a t i o n l e a d i n g t o h y p e r c a l c e m i a , myocardial and pulmonary calcinosis, arterial c a l c i f i c a t i o n , f u r t h e r m e t a s t a t i c c a l c i f i c a t i o n and death. Many e n d o c r i n e t i s s u e s possess t h e a b i l i t y t o s t o r e l a r g e q u a n t i t i e s o f hormone f o r r e l e a s e upon demand. In c o n t r a s t , t h e p a r a t h y r o i d gland s t o r e s l i t t l e PTH. As a r e s u l t , t h e t u r n o v e r r a t e American Chemical Society Library 1 1 5 5 16th St., N.W. Magee et al.; Probing Bioactive Mechanisms Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1989. 3

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PROBING BIOACnVE MECHANISMS

of PTH i s high even under basal c o n d i t i o n s . In 2HPT, t h e r e f o r e , p a r a t h y r o i d h y p e r p l a s i a r e s u l t s as the p a r a t h y r o i d gland attempts t o keep up with demands f o r enhanced hormone p r o d u c t i o n . As the p a r a t h y r o i d t i s s u e e n l a r g e s , changes i n c e l l u l a r c o n t r o l mechanisms have been demonstrated, i n c l u d i n g an i n c r e a s e i n the value of the c a l c i u m s e t - p o i n t and a decreased maximal suppression by c a l c i u m of PTH s e c r e t i o n (21,24). The obvious mechanism to reverse 2HPT i s to remove the s t i m u l i t h a t caused t h e syndrome. Although hemodialysis coupled with c a r e f u l l y c o n t r o l l e d management o f t h e p a t i e n t w i l l provide short-term b e n e f i t , renal t r a n s p l a n t a t i o n i s e v e n t u a l l y r e q u i r e d i n most cases. Following s u c c e s s f u l t r a n s p l a n t a t i o n , serum phosphate and 1,25(0H)2VitD3 l e v e l s w i l l o f t e n r e t u r n t o normal very r a p i d l y . At t h i s stage, however, the h y p e r p l a s t i c p a r a t h y r o i d gland continues to s e c r e t e l a r g e q u a n t i t i e s of PTH. Now the i n c r e a s e d PTH s e c r e t i o n produces frank hypercalcemia which threatens the newly t r a n s p l a n t e d kidney. Since p a r a t h y r o i d gland h y p e r p l a s i a i n 2HPT i s the d i r e c t r e s u l t of i n c r e a s e d and continuous demand f o r PTH s e c r e t i o n , gradual i n v o l u t i o n o f the gland w i l l u s u a l l y occur upon r e s t o r a t i o n o f normal renal f u n c t i o n f o l l o w i n g the t r a n s p l a n t . The h y p o p l a s t i c t r a n s f o r m a t i o n , however, i s a slow process s i n c e p a r a t h y r o i d eel Is do not t u r n o v e r r a p i d l y (2j>). Post-renal t r a n s p l a n t hypercalcemia i s common and i s u s u a l l y r e s o l v e d w i t h i n 1-2 years. I f the hypercalcemia i s mi Id t o moderate and does not cause f u r t h e r s k e l e t a l or renal d e t e r i o r a t i o n , no steps are taken to manage t h i s problem. In cases where the p r e t r a n s p l a n t c o n t r o l of 2HPT was poor, t h e r e i s a g r e a t chance f o r severe hypercalcemia with p o s s i b l e damage t o the renal a l l o g r a f t (25). In t h i s s i t u a t i o n o r when spontaneous r e s o l u t i o n o f the PTH-induced hypercalcemia does not occur w i t h i n 2-3 years, parathyroidectomy must be c o n s i d e r e d . Humoral Hypercalcemia of Malignancy Hypercalcemia of malignancy i s a common occurrence i n s o l i d tumors of the lung and b r e a s t as w e l l as m u l t i p l e myeloma and a d u l t T - c e l l lymphoma/leukemia (26). The hypercalcemia a s s o c i a t e d with b r e a s t cancer i s u s u a l l y seen i n l a t e stages of the d i s e a s e i n p a t i e n t s with e x t e n s i v e bone metastases. In squamous c e l l carcinoma of the lung o r kidney, however, hypercalcemia i s not c o r r e l a t e d with d i s e a s e stage and i s n o t n e c e s s a r i l y a s s o c i a t e d with bone metastases. The hypercalcemia r e s u l t s from i n c r e a s e d bone r e s o r p t i o n , decreased bone formation and increased renal t u b u l a r c a l c i u m r e a b s o r p t i o n . These f i n d i n g s suggest t h a t some tumors may s e c r e t e humoral f a c t o r s with PTH-like a c t i o n s . The humoral hypercalcemia of malignancy hypothesis s t a t e s t h a t an o s t e o l y t i c non-PTH substance i s s e c r e t e d by c e r t a i n tumors and, i n an endocrine manner, i s t r a n s p o r t e d from tumor t o bone through the bloodstream. The evidence f o r t h i s hypothesis i s t h a t 1) bone d e s t r u c t i o n occurs i n p a t i e n t s without bone metastases, 2) serum PTH l e v e l s i n these p a t i e n t s were u s u a l l y normal, 3) PTH mRNA was absent i n tumors (27), and 4) tumor e x t r a c t s from hypercalcemic p a t i e n t s enhanced bone c e l l adenylate c y c l a s e a c t i v i t y and phosphate t r a n s p o r t i n kidney e p i t h e l i a l c e l l s (28-30).


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A 141-amino a c i d tumor-secreted, PTH-like p e p t i d e has r e c e n t l y been i d e n t i f i e d from human carcinomas based upon p a r t i a l sequence a n a l y s i s and cDNA c l o n i n g (31.-33). F u r t h e r s t u d i e s demonstrated t h e presence o f two forms o f mRNA encoding f o r t h i s p r o t e i n which o r i g i n a t e from a s i n g l e gene by a l t e r n a t e s p l i c i n g mechanisms (34) • The 3' u n t r a n s l a t e d r e g i o n i s homologous t o t h e c o r r e s p o n d i n g domain of the c-myc proto-oncogene ( 3 4 ) . Besides tumors, t h e o n l y o t h e r t i s s u e s known t o produce t h i s hypercalcemia f a c t o r (HCF) a r e human k e r a t i n o c y t e s , f e t a l p a r a t h y r o i d g l a n d , p l a n c e n t a and mammary gland (35-38). The mRNA coding f o r t h i s p r o t e i n were expressed i n r a t mammary t i s s u e o n l y d u r i n g l a c t a t i o n and t h e response changed r a p i d l y as a f u n c t i o n o f the s u c k l i n g s t i m u l u s ( 3 5 ) . In t h e mammary g l a n d , HCF may p l a y a p h y s i o l o g i c r o l e i n t h e m o b i l i z a t i o n and/or t r a n s f e r o f c a l c i u m into milk. The N-terminal p o r t i o n o f t h e 141 amino a c i d human HCF (hHCF) bears sequence homology with t h e c o r r e s p o n d i n g r e g i o n s o f human and bovine PTH (hPTH, bPTH): 1

10

15

34

30

25

20

bPTH IA V S E I Q F M H N L G K H L S S M E R V E W L R K K L Q D V H N Y hPTH S V S E I Q L | M H N L G K H L | N | S M E R V E W L R K K L Q D V H N Y hHCF [ A V S E[H|Q L L|H)D KteKJS I Q D L RJ R J R

F

FJLJTTH"

L I A E IH T A

W i t h i n t h e f i r s t 13 amino a c i d s , t h e r e i s 60% sequence homology between PTH and hHCF. A f t e r t h i s r e g i o n , t h e homology i s minimal. To determine i f HCF i s r e s p o n s i b l e f o r mediating some o r a l l o f the components o f t h e humoral hypercalcemia o f malignancy syndrome, an N-terminal fragment o f t h i s p e p t i d e ( h H C F - ( l — 3 4 ) N H ) was s y n t h e s i z e d and i t s b i o l o g i c a l p r o p e r t i e s i n v e s t i g a t e d . The tumor f a c t o r caused a concentration-dependent i n h i b i t i o n o f r a d i o l a b e l e d PTH b i n d i n g t o bovine r e n a l c o r t i c a l membranes, in. v i t r o , w i t h a potency s i m i l a r t o 34-amino a c i d PTH fragments ( 3 9 ) . T h i s p e p t i d e a l s o enhanced a d e n y l a t e c y c l a s e a c t i v i t y i n r e n a l membranes, bone c e l l s and i s o l a t e d , p e r f u s e d kidneys (39,40). Using bone c e l l s , dexamethasone pretreatment modulated t h e a c t i o n s o f both hHCF and PTH s i m i l a r l y ( 4 1 ) . Both p e p t i d e s a l s o i n h i b i t e d bone c e l l a l k a l i n e phosphatase a c t i v i t y in. v i t r o ( 4 1 ) . In. v i v o , u s i n g t h e t h y r o p a r a t h y r o i d e c t o m i z e d r a t model system, hHCF-(l — 3 4 ) N H caused hypercalcemia with an apparent potency 6-10-fold t h a t o f b P T H - ( l ~ 8 4 ) ( 3 9 ) . hHCF-(l — 3 4 ) N H a l s o reduced serum phosphate and e l e v a t e d 1,25(0H) VitD3 l e v e l s . F o l l o w i n g a 48 h r i n f u s i o n o f h H C F - ( l — 3 4 ) N H , h i s t o l o g i c a l changes i n bone c o n s i s t e n t with t h e s t i m u l a t i o n o f bone PTH r e c e p t o r s were e v i d e n t (42). S i m i l a r l y , u s i n g t h e t h y r o p a r a t h y r o i d e c t o m i z e d / n e p h r e c t o m i z e d / low d i e t a r y c a l c i u m r a t bone model, h H C F - ( l — 3 4 ) N H was e q u i p o t e n t with b P T H - ( l — 3 4 ) and more potent than b P T H - ( l — 8 4 ) f o r c a u s i n g hypercalcemia (39.). F u r t h e r support f o r t h e PTH-like a c t i o n s o f hHCF were gained using a PTH a n t a g o n i s t . T h e p r o t o t y p i c a l PTH a n t a g o n i s t , [Tyr ]bPTH-(7—34)NH blocked completely t h e adenylate cyclase-enhancing actions o f hHCF-(l—34)NH on both r e n a l 2

2

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membranes and bone c e l l s i_n v i t r o (43). T h i s PTH a n t a g o n i s t a l s o prevented t h e a c t i o n s o f hHCF-(l--34)NH i n v i v o ( H o r i u c h i et a l . , submitted). In summary, these r e s u l t s suggest t h a t a t l e a s t some and perhaps a l l of the a c t i o n s of hHCF are mediated through s t i m u l a t i o n of the PTH r e c e p t o r . Indeed, recent s t u d i e s using bone c e l l s i n d i c a t e t h a t both hHCF and PTH r e c o g n i z e the same plasma membrane r e c e p t o r (44»i5). As the a c t i o n s of these s t r u c t u r a l l y - d i s t i n c t peptides are compared on o t h e r t i s s u e s and b i o l o g i c a l parameters, i t i s p o s s i b l e t h a t PTH/HCF r e c e p t o r subtypes may be i d e n t i f i e d . Other p o s s i b l e humoral mediators o f bone r e s o r p t i o n f a l l i n t o s e v e r a l c a t e g o r i e s i n c l u d i n g t r a n s f o r m i n g growth f a c t o r s , p r o s t a g l a n d i n E's, c y t o k i n e s , 1,25(0H) VitD3 and colony s t i m u l a t i n g f a c t o r s ( f o r a review, see ( 2 6 ) ) . With the e x c e p t i o n of PTH-like p e p t i d e s , evidence f o r a d i r e c t r o l e o f any i n d i v i d u a l f a c t o r i s l i m i t e d . I t i s p o s s i b l e t h a t s e v e r a l f a c t o r s may a c t t o g e t h e r to cause humoral hypercalcemia of malignancy. 2

2

Osteoporosis and

Hyperparathyroidism

Osteoporosis i s a s p e c i f i c form o f osteopenia i n which the bone h i s t o l o g y i s normal but t h e r e i s a q u a n t i t a t i v e decrease i n the amount o f bone (1_3). O s t e o p o r o t i c r e d u c t i o n o f bone mass, e s p e c i a l l y i n the e l d e r l y , i s a common problem r e s u l t i n g i n an i n c r e a s e d frequency o f p a t h o l o g i c a l f r a c t u r e s and sometimes death from ensuing c o m p l i c a t i o n s . Two o s t e o p o r o t i c syndromes have been hypothesized. Type I or postmenopausal o s t e o p o r o s i s i s 6 - f o l d more common i n women than men and r e s u l t s mainly i n t r a b e c u l a r bone l o s s . Type I o s t e o p o r o s i s i s due p r i m a r i l y t o estrogen d e f i c i e n c y beginning a t menopause. Although type I o s t e o p o r o s i s has not been linked d i r e c t l y t o elevated parathyroid function, i t i s possible t h a t c e r t a i n o s t e o p o r o t i c p a t i e n t s may s e c r e t e f a c t o r s ( s ) which p o t e n t i a t e the a c t i o n s o f PTH (46). Based upon the recent i d e n t i f i c a t i o n o f hHCF (see above), i t i s c o n c e i v a b l e t h a t PTH-like f a c t o r s may be r e l e a s e d which s t i m u l a t e PTH r e c e p t o r s i n c e r t a i n tissues. Type I I , i n v o l u t i o n a l o r s e n i l e o s t e o p o r o s i s occurs l a t e r i n l i f e than type I, has a c l o s e r r a t i o between sexes, r e s u l t s i n both t r a b e c u l a r and c o r t i c a l bone l o s s and i s r e l a t e d t o i n c r e a s e d PTH s e c r e t i o n (26). Under these circumstances, o s t e o p o r o s i s may e s s e n t i a l l y be a s i d e - e f f e c t of 1HPT or 2HPT (15,26,46-48). Type II o s t e o p o r o s i s i s managed, t h e r e f o r e , by c o r r e c t i n g the u n d e r l y i n g cause of the HPT. Incidence of Hypercalcemia and Hyperparathyroid

Disorders

S i n c e t h e i n t r o d u c t i o n o f modern procedures f o r the r o u t i n e automated a n a l y s i s of serum c a l c i u m l e v e l s , hypercalcemia has become recognized as a r e l a t i v e l y common c l i n i c a l problem. 1HPT i s the most common cause o f hypercalcemia i n the general p o p u l a t i o n and the i n c i d e n c e i n c r e a s e s with age (26). In men or women under the age of 40, the annual i n c i d e n c e o f 1HPT was found to be 10 cases/10,000 p o p u l a t i o n (49)- T h i s r a t e rose s t e a d i l y with age t o 92 and 188 cases/10,000 i n men and women, r e s p e c t i v e l y , over 60 years of age.


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In another study of 207 hypercalcemic i n d i v i d u a l s , 111 (54%) were diagnosed as having 1HPT and a g a i n , the g r e a t e s t r i s k group was e l d e r l y women (50). Hypercalcemia i d e n t i f i e d from r o u t i n e serum c a l c i u m m o n i t o r i n g was the i n i t i a l i n d i c a t i o n of 1HPT i n over 50% of the p a t i e n t s (49-51)• The annual age a d j u s t e d i n c i d e n c e of 1HPT was 27.7/100,000 p o p u l a t i o n of the U.S. ( 4 9 ) . In Sweden, the p r e v a l e n c e of 1HPT has been e s t i m a t e d , i n one study, t o be 520 cases/100,000 p o p u l a t i o n ( 5 2 ) . E x t r a p o l a t i o n t o the U.S. p r e d i c t s t h a t over 1 m i l l i o n cases of 1HPT may e x i s t (49). In h o s p i t a l i z e d p a t i e n t s , hypercalcemia of malignancy i s the major cause o f hypercalcemia (26,51,53,54). I n two separate s t u d i e s , 9% of a l l cancer p a t i e n t s had hypercalcemia of malignancy (55,56). Hypercalcemia of malignancy i s most common i n s o l i d tumors such as carcinomas of the lung, b r e a s t , kidney, pancreas and ovary, but a l s o occurs with m u l t i p l e myeloma and a d u l t T - c e l l lymphoma/ leukemia. I t has been estimated t h a t t h e r e i s approximately an equal d i s t r i b u t i o n between humoral f a c t o r s and m e t a s t a t i c bone tumors f o r c a u s i n g hypercalcemia of malignancy (26,57,58). With advanced age, t h e r e i s an i n c r e a s e d impairment of r e n a l f u n c t i o n which has been shown t o c o r r e l a t e with e l e v a t e d PTH l e v e l s and r a d i o l o g i c bone d i s e a s e ( 1 5 ) . When the impairment of f u n c t i o n reaches the r e n a l f a i l u r e stage, 2HPT has been found i n 80-94% of p a t i e n t s (59,60). While some p a t i e n t s are asymptomatic, o s t e o d y s t r o phy i s o f t e n manifested as spontaneous f r a c t u r e s (femoral neck, v e r t e b r a e ) o r bone and j o i n t pains ( 5 9 ) . Chronic h e m o d i a l y s i s may cause an e x a c e r b a t i o n of the 2HPT and i n c r e a s e the r i s k of bone d i s e a s e (61,62). Twenty t o 66% o f p o s t - r e n a l t r a n s p l a n t p a t i e n t s d i s p l a y e d p e r s i s t e n t hypercalcemia and/or 2HPT (14,63-66). In most cases, the p a t i e n t s became normocalcemic and e u p a r a t h y r o i d w i t h i n 1-3 y e a r s . Only a small percentage (3.2-7%) r e q u i r e d parathyroidectomy as a r e s u l t of impaired r e n a l f u n c t i o n o r p r o g r e s s i o n of bone d i s e a s e (li.|0.M.|5»66). F o l l o w i n g i n i t i a t i o n of a n t i h y p e r t e n s i v e therapy with t h i a z i d e d i u r e t i c s , t r a n s i e n t hypercalcemia has been seen i n over o n e - t h i r d of p a t i e n t s ( 6 7 ) . Two p e r c e n t of p a t i e n t s r e c e i v i n g long-term t h i a z i d e d i u r e t i c s a d m i n i s t r a t i o n had p e r s i s t e n t hypercalcemia ( 6 8 ) . In the e l d e r l y ( e s p e c i a l l y women), combined a d m i n i s t r a t i o n of t h i a z i d e s with v i t a m i n 0 supplements ( f o r o s t e o p o r o s i s ) can have s y n e r g i s t i c e f f e c t s on the e l e v a t i o n of serum c a l c i u m l e v e l s r e s u l t i n g i n severe hypercalcemia ( 6 9 ) . S i m i l a r l y , i f the p a t i e n t i s predisposed t o hypercalcemia (1HPT, 2HPT o r i m m o b i l i z a t i o n ) , t h i a z i d e s can p r e c i p i t a t e s i g n i f i c a n t and s u s t a i n e d hypercalcemia (68,70). CURRENT MANAGEMENT OF HYPERCALCEMIA AND HYPERPARATHYROIDISM S u r g i c a l Management The u l t i m a t e t h e r a p e u t i c goal of managing h y p e r p a r a t h y r o i d h y p e r c a l cemia i s r e t u r n i n g blood PTH, c a l c i u m and phosphate l e v e l s t o t h e i r normal range. In 1PTH, when a c t i v e bone, r e n a l o r 61 symptoms are p r e s e n t , surgery i s the most e f f e c t i v e treatment m o d a l i t y when the o f f e n d i n g p a r a t h y r o i d adenoma can be i d e n t i f i e d . In MEN1 o r h y p e r p l a s i a , c o n t r o v e r s y e x i s t s as t o whether tota1 parathyroidectomy


252


with a l l o t r a n s p l a n t a t i o n t o the forearm o r s u b t o t a l parathyroidectomy should be performed. In the case of p a r a t h y r o i d carcinoma, however, attempts should be made t o remove the e n t i r e carcinoma (with c a p s u l e i n t a c t ) , as w e l l as metastases, i f p o s s i b l e . The use of n o n - i n v a s i v e , p r e o p e r a t i v e l o c a l i z a t i o n techniques (high r e s o l u t i o n c e r v i c a l ultrasonography, m e d i a s t i n a l computed tomography, magnetic resonance imaging) may a i d i n t h e i d e n t i f i c a t i o n o f p a r a t h y r o i d t i s s u e , e s p e c i a l l y i n cases of r e c u r r e n t or p e r s i s t e n t 1 HPT (21-21). At present, the b e n e f i t o f p a r a t h y r o i d gland surgery f o r m i l d , asymptomatic 1HPT i s not c l e a r ( H , 7 4 ) . Since t h e r e i s a 50% chance t h a t p a t i e n t s with mild 1HPT w i l l e v e n t u a l l y develop c o m p l i c a t i o n s ( 2 5 , 2 i ) , and s i n c e some o f the renal o r bone impairment may not be r e v e r s i b l e , these p a t i e n t s should be monitored f o r disease p r o g r e s s i o n (]±). However, parathyroidectomy may be c o n t r a i n d i c a t e d i n o l d e r p a t i e n t s , p a t i e n t s with i n c r e a s e d r i s k o f a n e s t h e s i a induced c o m p l i c a t i o n s , and p a t i e n t s with severe concomitant d i s e a s e t h a t i s not exacerbated by 1HPT (75). In c h r o n i c r e n a l f a i l u r e t r e a t e d by r e n a l t r a n s p l a n t a t i o n , h y p e r p a r a t h y r o i d i s m may p e r s i s t , as d i s c u s s e d above. In most cases, the p a r a t h y r o i d glands i n v o l u t e t o normal w i t h i n 3 years (li»5i»&l» 65,66). However, s i n c e severe o r prolonged hypercalcemia impairs r e n a l f u n c t i o n and may cause permanent r e n a l g r a f t i n j u r y , s u b t o t a l parathyroidectomy must be considered f o r p a t i e n t s with p e r s i s t e n t hypercalcemia or acute hypercalcemic c r i s i s . Medical Management The goal o f t h e medical management o f h y p e r p a r a t h y r o i d i s m i s d i r e c t e d a t lowering blood c a l c i u m l e v e l s ( f o r reviews, see 7 7 , 7 8 ) . In 1HPT, o r a l phosphate a d m i n i s t r a t i o n may be b e n e f i c i a l f o r lowering plasma c a l c i u m l e v e l s but the long-term e f f e c t s o f t h i s treatment are u n c e r t a i n . In 2HPT, however, attempts are made t o reduce phosphate a b s o r p t i o n using o r a l phosphate b i n d i n g g e l s (aluminum hydroxide, aluminum carbonate) and d i e t a r y p r o t e i n r e s t r i c t i o n (62). Acutely, t h e use o f s a l i n e i n f u s i o n accompanied by a d m i n i s t r a t i o n of loop d i u r e t i c s enhances u r i n a r y c a l c i u m e x c r e t i o n . C a l c i t o n i n , mithramycin and c o r t i c o s t e r o i d s decrease calcium movement from bone. Reduced i n t a k e o f c a l c i u m and c o r t i c o s t e r o i d s decrease i n t e s t i n a l a b s o r p t i o n o f c a l c i u m . Short-term hemodialysis o r p e r i t o n e a l d i a l y s i s i s e f f e c t i v e f o r the r a p i d removal of c a l c i u m from the blood i n c r i s i s s i t u a t i o n s , e s p e c i a l l y i n p a t i e n t s with renal f a i l u r e o r c o n g e s t i v e h e a r t f a i l u r e . Prolonged h e m o d i a l y s i s , however, i s not a t h e r a p e u t i c s o l u t i o n because of i t s i m p r a c t i c a l i t y and high c o m p l i c a t i o n r a t e . F u r t h e r s u p p o r t i v e measures are e s s e n t i a l f o r p r e v e n t i n g a d d i t i o n a l c o m p l i c a t i o n s o f hypercalcemia. These treatments i n c l u d e normal s a l i n e i n f u s i o n f o r r e s t o r a t i o n of euvolemia and e l e c t r o l y t e i n f u s i o n f o r c o r r e c t i o n o f hypokalemia o r hypomagnesemia. In a d d i t i o n , medications which may exacerbate hypercalcemia ( e x : t h i a z i d e d i u r e t i c s ) should be avoided.


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POTENTIAL CLINICAL USES OF PTH ANTAGONISTS In 1 HPT and MEN, PTH a n t a g o n i s t s may be e f f e c t i v e f o r managing m i l d hypercalcemia, f o r treating acute hypercalcemic crises orf o r t r e a t i n g p a t i e n t s u n f i t f o r surgery (79). Such agents a l s o may be e f f e c t i v e f o r n o r m a l i z i n g blood c a l c i u m l e v e l s i n p r e p a r a t i o n f o r parathyroidectomy, subsequent t o unsuccessful surgery or for t r e a t i n g h y p e r c a l c e m i a caused by m e t a s t a t i c p a r a t h y r o i d carcinoma. S i m i l a r l y , PTH a n t a g o n i s t s may b l o c k t h e h y p e r c a l c e m i c a c t i o n s o f P T H - l i k e p e p t i d e s s e c r e t e d by n o n - p a r a t h y r o i d m a l i g n a n c i e s . PTH a n t a g o n i s t s p r o b a b l y would n o t be e f f e c t i v e i n managing 2HPT p r i o r t o r e n a l t r a n s p l a n t a t i o n , b u t may be q u i t e b e n e f i c i a l f o r t h e t r a n s i e n t and subacute h y p e r p a r a t h y r o i d h y p e r c a l c e m i a p r e s e n t a f t e r t r a n s p l a n t a t i o n . The use of PTH a n t a g o n i s t s a f t e r r e n a l t r a n s p l a n t a t i o n may reduce t h e r i s k o f h y p e r c a l c e m i a - a s s o c i a t e d g r a f t i n j u r y . PTH a n t a g o n i s t s may a l s o have d i a g n o s t i c v a l u e . S i n c e PTH p l a y s a r o l e i n t h e m i n u t e - t o - m i n u t e r e g u l a t i o n o f blood c a l c i u m l e v e l s , s h o r t - t e r m a d m i n i s t r a t i o n o f a PTH a n t a g o n i s t may cause a r a p i d r e d u c t i o n i n blood c a l c i u m l e v e l s i n h y p e r c a l c e m i c p a t i e n t s i f t h e h y p e r c a l c e m i a i s due t o e l e v a t e d PTH l e v e l s . S i m i l a r l y , short-term amelioration of possible symptoms of hypercalcemia (such as l e t h a r g y , c o g n i t i v e d e f e c t s ) may h e l p p i n p o i n t t h e e t i o l o g y o f t h e symptoms ( i . e . h y p e r p a r a t h y r o i d i s m v s . o t h e r d i s o r d e r s ) . If PTH c o n t r i b u t e s t o t h e p a t h o g e n e s i s o f o s t e o p o r o s i s , PTH a n t a g o n i s t s may be e f f e c t i v e f o r i t s t r e a t m e n t . U n t i l such agents a r e t e s t e d i n o s t e o p o r o t i c p a t i e n t s , however, t h i s use i s q u i t e speculative. P o t e n t i a l mechanism-based s i d e e f f e c t s o f PTH a n t a g o n i s t s may be related t o hypocalcemia o r hypovitaminemia D. Under severe conditions, PTH a n t a g o n i s t - i n d u c e d hypocalcemia could lead t o t e t a n y , s e i z u r e s o r death. Since: 1) v i t a m i n D-dependent c a l c i u m absorption from t h e g u t i s e s s e n t i a l f o r maintaining calcium homeostasis, 2) 1 , 2 5 ( 0 H ) V i t D i s r e q u i r e d f o r i n h i b i t i o n o f PTH s y n t h e s i s / s e c r e t i o n and 3. r e n a l and bone t r a n s p o r t o f c a l c i u m i s v i t a m i n D-dependent, v i t a m i n D d e f i c i e n c y would n o t be d e s i r a b l e . Oral and i n j e c t a b l e preparations of vitamin D, however, a r e commercially a v a i l a b l e . 2

3

DESIGNING PTH ANTAGONISTS Methodological

considerations

P r e r e q u i s i t e t o t h e r a p i d e v a l u a t i o n o f drug c a n d i d a t e s i s t h e development o f methods t h a t w i l l a l l o w t h e q u a n t i t a t i v e comparison of a l a r g e s e r i e s o f s t r u c t u r a l l y - r e l a t e d compounds. These assays must be: 1) easy t o e s t a b l i s h , 2) performed r a p i d l y on a r e g u l a r b a s i s , and 3) s i m p l e t o i n t e r p r e t . Secondary assays s h o u l d a l s o be available t o confirm i n i t i a l r e s u l t s and measure both intrinsic a c t i v i t i e s and r e c e p t o r a f f i n i t i e s . The i n h i b i t i o n o f b i n d i n g o f l a b e l e d PTH analogs t o renal membranes jm v i t r o i s a s t a n d a r d method f o r t h e i n i t i a l c h a r a c t e r i z a t i o n o f PTH a g o n i s t s and a n t a g o n i s t s (80-84). T h i s procedure i s advantageous because complete dose-response c u r v e s can be generated w i t h microgram q u a n t i t i e s of compound i n a r a p i d and r o u t i n e manner.


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Using sucrose d e n s i t y g r a d i e n t - p u r i f i e d renal c o r t i c a l membranes, the degradation o f compound (and l i g a n d ) i s minimized thereby p r o v i d i n g a p r e c i s e estimate of the molar a f f i n i t y . Recently, t h i s screening procedure has been optimized by employing HPLC-purified radioligand ([monol I-Tyr ,Nle » ]bPTH-(l--34)NH ) and bovine renal c o r t i c a l t i s s u e (84)• The use of chromatographically-pure radioligand i s advantageous since non-iodinated p e p t i d e , d i - i o d i n a t e d peptide and unreacted l INa can be q u a n t i t a t i v e l y r e s o l v e d from [ m o n o l I - T y r , N l e » l ] bPTH-(l--34)NH which has the best assay c h a r a c t e r i s t i c s . T h i s chromatographic procedure y i e l d s a r a d i o l i g a n d t h a t i s l e s s s u s c e p t i b l e t o n o n - s p e c i f i c r a d i o l y s i s during storage. In a d d i t i o n , by s t o r i n g i n 50 mM T r i s H C l (pH 7.4)/2% BSA a t -70°C, t h e r a d i o l i g a n d remains s t a b l e f o r approximately 2 months. Bovine kidneys were chosen as the t i s s u e source f o r p r e p a r i n g renal c o r t i c a l membranes s i n c e one bovine kidney w i l l y i e l d a s u f f i c i e n t q u a n t i t y of membranes f o r 2-3 months of s c r e e n i n g . Once the a f f i n i t y of a compound i s determined i n the b i n d i n g assay, i n t r i n s i c a c t i v i t y i s q u a n t i f i e d using a renal membrane adenylate c y c l a s e assay (80,84-86). P o t e n t i a l PTH a g o n i s t a c t i v i t y i s evaluated by examining the a b i l i t y of each compound to enhance adenylate c y c l a s e a c t i v i t y i n a PTH a n t a g o n i s t r e v e r s i b l e manner. Antagonist a c t i v i t y i s q u a n t i f i e d by determining the p o t e n t i a l dose-dependent inhibition of [Nle . ,Tyr3 ]bPTH-(l--34)NH s t i m u l a t e d adenylate c y c l a s e a c t i v i t y . Based upon d i f f e r e n c e s between kidney and bone s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s among PTH analogs, compounds should a l s o be evaluated i n a bone-based assay. Bone c e l l l i n e s t h a t c o n t a i n PTH r e c e p t o r s such as ROS 17/2.8 c e l l s or UMR-106 c e l l s , are advantag eous because they c o n s i s t o f a s i n g l e bone c e l l type, can be maintained i n c u l t u r e and have a l s o been used f o r both PTH b i n d i n g and adenylate c y c l a s e s t u d i e s (41,43). Recently, evidence has been provided t h a t the ROS 17/2.8 c e l l adenylate c y c l a s e assay may a l s o be used t o p r e d i c t weak a g o n i s t a c t i v i t y o f PTH analogs ijn v i v o (41,43). Following 1_n v i t r o c h a r a c t e r i z a t i o n , compounds t h a t d i s p l a y potent a c t i v i t y e i t h e r as a g o n i s t s o r a n t a g o n i s t s should be evaluated jjn v i v o t o determine the i n f l u e n c e s o f metabolism, pharmacokinetics e t c . on b i o l o g i c a l a c t i v i t y . Several models using r a t , c h i c k and dog are c u r r e n t l y a v a i l a b l e which provide i n f o r m a t i o n on bone and renal PTH r e c e p t o r e f f e c t s (39,87-90) or s e l e c t i v e bone PTH r e c e p t o r e f f e c t s (87). i n v i v o models are c r i t i c a l s i n c e one potent and promising i n v i t r o PTH a n t a g o n i s t u l t i m a t e l y showed a g o n i s t p r o p e r t i e s in. v i v o (see below). 2 5

3 4

8

1 8

2

2 5

25

34

8

8

2

8

18

4

2

S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of peptide

analogs

As a preface towards the r a t i o n a l design of potent and s e l e c t i v e PTH a n t a g o n i s t s , an understanding o f s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s r e s p o n s i b l e f o r PTH r e c e p t o r occupation and a c t i v a t i o n i s e s s e n t i a l . Since f u l l b i o l o g i c a l a c t i v i t y o f the 84-amino a c i d peptide was shown to r e s i d e i n the N-terminal p o r t i o n ( p o s i t i o n s 1-34) of the bovine PTH molecule ( 5 ) , most s y n t h e t i c e f f o r t s have i n v o l v e d m o d i f i c a t i o n s , s u b s t i t u t i o n s and d e l e t i o n s i n t h i s r e g i o n . F o r


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255

34

example, replacement of the P h e r e s i d u e of b P T H - ( l — 3 4 ) 0 H with a T y r and s u b s t i t u t i o n of a carboxyamide f o r the C-terminal c a r b o x y l i c a c i d r e s u l t e d i n enhanced b i o l o g i c a l a c t i v i t y (91,92). In a d d i t i o n , s u b s t i t u t i o n s of the o x i d a t i o n - s e n s i t i v e methionine r e s i d u e s i n p o s i t i o n s 8 and 18 of t h i s analog with s u l f u r - f r e e n o r l e u c i n e s (Nle) yielded a stable molecule, [Nle . ,Tyr ]bPTH-(1—34) NH , p o s s e s s i n g f u l l PTH a g o n i s t a c t i v i t i e s both i n v i t r o and In v i v o (87,92,93). To understand the regions o f b P T H - ( l — 3 4 ) r e s p o n s i b l e f o r b i o l o g i c a l a c t i v i t y , the e f f e c t s o f both C- and N-terminal t r u n c a t i o n s were s t u d i e d . Stepwise d e l e t i o n s of C-terminal amino a c i d s t o p o s i t i o n 25 r e s u l t e d i n a steady d e c l i n e i n b i o l o g i c a l a c t i v i t y (94,915). F u r t h e r C-terminal t r u n c a t i o n s prevented the demonstration of b i o l o g i c a l a c t i v i t y o r b i n d i n g a f f i n i t y . S i m i l a r t r u n c a t i o n s from the N-terminus reduced a c t i v i t y but much more d r a m a t i c a l l y (80,95). Removal of A l a caused a r a p i d decrease i n a c t i v i t y . One f u r t h e r d e l e t i o n y i e l d i n g bPTH-(3~34) r e s u l t e d i n a l o s s of a g o n i s t a c t i v i t y . As a r e s u l t of these s t u d i e s , the minimum sequence f o r a g o n i s t a c t i v i t y i s c o n s i d e r e d t o be the 2-25 r e g i o n of PTH. Although bPTH-(3—34) was a weak a n t a g o n i s t , [Nle » ,T y r ] b P T H - ( 3 — 3 4 ) N H 2 was a potent, s e l e c t i v e and competitive a n t a g o n i s t using r e n a l and c e r t a i n bone assays i n v i t r o (96-98). In vivo. however, [Nle » ,Tyr ]bPTH-(3--34)NH was n o t an a n t a g o n i s t . Instead, t h i s agent was a weak a g o n i s t d i s p l a y i n g a l l of the p r o p e r t i e s of PTH a t approximately 0.3-1% the potency of PTH (89,90,99). S i m i l a r l y , t h i s peptide was a p a r t i a l a g o n i s t i n the ROS 17/2.8 e e l 1 adenylate c y c l a s e assay as w e l l as the GppNHpa m p l i f i e d canine r e n a l adenylate c y c l a s e assay (28,43). In an attempt to develop an in. v i t r o and i n v i v o a n t a g o n i s t , the e f f e c t s o f f u r t h e r N-terminal t r u n c a t i o n s o f [ N l e » , T y r ] b P T H - ( 3 — 3 4 ) N H were s t u d i e d (96,98). The 7-34 analog was 10-100 f o l d weaker than the 3-34 analog but s t i l l showed s i g n i f i c a n t a f f i n i t y f o r the PTH r e c e p t o r . F o l l o w i n g replacement of methionines i n p o s i t i o n s 8 and 18, [ T y r ] b P T H - ( 7 — 3 4 ) N H was found t o be a potent PTH a n t a g o n i s t both i n v i t r o and j_n v i v o without p o s s e s s i n g p a r t i a l a g o n i s t p r o p e r t i e s (43.87.100). Using [ T y r ] b P T H - ( 7 — 3 4 ) N H a s the p r o t o t y p i c a l a n t a g o n i s t , the goal o f present s t u d i e s i s t o develop more potent and l o n g - a c t i n g i n v i v o PTH a n t a g o n i s t s . T h i s o b j e c t i v e i s being addressed using s e v e r a l approaches i n c l u d i n g 1) s u b s t i t u t i n g amino a c i d s t h a t may enhance r e s i s t a n c e t o p r o t e o l y t i c d e g r a d a t i o n , 2) understanding the conformational f e a t u r e s o f both a g o n i s t s and a n t a g o n i s t s t h a t are r e q u i r e d f o r b i o l o g i c a l a c t i v i t y s o t h a t r a t i o n a l changes can be made i n the a n t a g o n i s t sequence which i n c r e a s e PTH r e c e p t o r a f f i n i t y , and 3) the s y n t h e s i s of h y b r i d molecules of PTH and hHCF t o i d e n t i f y new d i r e c t i o n s f o r PTH a n t a g o n i s t d e s i g n . Once optimal s u b s t i t u t i o n s from each approach are i d e n t i f i e d , h y b r i d molecules c o n t a i n i n g combinations of s t r u c t u r a l f e a t u r e s can be s y n t h e s i z e d . In an e f f o r t t o p r o t e c t [ T y r ] b P T H - ( 7 — 3 4 ) N H against p r o t e o l y t i c degradation by aminopeptidases, the e f f e c t s o f 3 N-terminal s t r u c t u r a l m o d i f i c a t i o n s on a n t a g o n i s t potency were i n v e s t i g a t e d (84). F i r s t , the amino group of Phe was removed 8

18

34

2

1

8

1 8

34

8

18

34

2

8

2

34

2

34

2

34

2

7


1 8

3 4

256

PROBING BIOACTIVE MECHANISMS J4

( a - d e s a m i n o [ T y r 1 b P T H - n - - 3 4 ) N H ) . Second, Phe' was r e p l a c e d bv N-MePhe ([N-MePhe',Tyr ]bPTH-(7—34)NH ). T h i r d , by r e p l a c i n g M e t with N-MeMet an N-methylated peptide bond between r e s i d u e s 7-8 was formed ( [ N - M e M e t , T y r ] b P T H - ( 7 — 3 4 ) N H ) . Using r e n a l c o r t i c a l membrane b i n d i n g and adenylate c y c l a s e assays, these t h r e e s t r u c t u r a l m o d i f i c a t i o n s were found t o be t o l e r a t e d i n terms o f p r e s e r v a t i o n of b i o l o g i c a l a c t i v i t y i n v i t r o ( i . e . i n h i b i t i o n of PTH b i n d i n g and PTH-stimulated adenylate c y c l a s e a c t i v i t y ) , suggesting t h a t a charged N-terminal amino group i s not r e q u i r e d f o r a n t a g o n i s t a c t i v i t y . In o r d e r t o v a l i d a t e the enhanced s t a b i l i t y o f these analogs t o aminopeptidases, they w i l l be evaluated i_n v i v o . The second approach f o r developing potent peptide PTH a n t a g o n i s t s i s based on o b t a i n i n g an understanding of the conformational r e q u i r e ments f o r r e c e p t o r occupation and a c t i v a t i o n . Analogs which promote r e c e p t o r - f a v o r e d conformational f e a t u r e s can then be s y n t h e s i z e d . In the PTH r e g i o n of r e s i d u e s 12-15, f o r example, a 0-turn was p r e d i c t e d by the Chou-Fasman a l g o r i t h m (101). P o s i t i o n 1 2 - s u b s t i t u t e d analogs of P T H - ( 1 — 3 4 ) N H and P T H - ( 7 — 3 4 ) N H were, t h e r e f o r e , synthe s i z e d t o t e s t t h i s hypothesis (102.103). Replacement of G l y with amino a c i d s t h a t favored the formation o f an a - h e l i x r e s u l t e d i n the r e t e n t i o n of e i t h e r a g o n i s t or a n t a g o n i s t p r o p e r t i e s (Table I I ) . S u b s t i t u t i o n o f Pro ( a known a - h e l i x breaker) i n t h i s p o s i t i o n , however, caused a marked d i m i n u t i o n o f a c t i v i t y . Taken t o g e t h e r , these s t u d i e s f a v o r the presence of an a - h e l i x i n t h i s r e g i o n and suggested t h a t p o s i t i o n 12 would be a r e l e v a n t s i t e f o r f u r t h e r substitution studies. D-Trp was chosen as a p o s i t i o n 12 replacement i n [ T y r ] b P T H (7—34)NH and [ N l e » , T y r ] b P T H - ( 7 — 3 4 ) N H since this non-natural amino a c i d was shown t o i n c r e a s e analog p o t e n c i e s i n o t h e r p e p t i d e systems (104-107). [ D - T r p l , T y r ] b P T H - ( 7 — 3 4 ) N H and [ N l e . ' , D - T r p , T y r l b P T H - ( 7 — 3 4 ) N H were c o m p e t i t i v e i n h i b i t o r s of [monol *I-Tyr ,Nle »1 ]bPTH-(1—34)NH binding t o renal c o r t i c a l membranes and bone c e l l s in. v i t r o , with p o t e n c i e s 10-20-fold g r e a t e r than t h e i r n o n - p o s i t i o n 1 2 - s u b s t i t u t e d c o u n t e r p a r t s (Table I I I ) (108). These new analogs were a l s o more potent i n h i b i t o r s o f [Nle .T^7Tyr ]bPTH-(l--34)NH -stimulated adenylate c y c l a s e a c t i v i t y i n both r e n a l - and bone-based t i s s u e p r e p a r a t i o n s . In a d d i t i o n , [D-Trpl , T y r ] b P T H - ( 7 — 3 4 ) N H i n h i b i t e d b P T H - ( l — 8 4 ) - and hHCF(1 — 3 4 ) N H - s t i m u 1 a t e d a d e n y l a t e c y c l a s e a c t i v i t y . In c o n t r a s t t o [N1e »1 ,Tyr 4]- PTH-(3—34)NH (43), however, these agents d i d not possess p a r t i a l a g o n i s t p r o p e r t i e s in. v i t r o , suggesting t h a t they w i l l not be weak a g o n i s t s i n v i v o . The t h i r d approach towards PTH a n t a g o n i s t development i n v o l v e s using s t r u c t u r e - a c t i v i t y experiments o f nature t o i d e n t i f y new PTH antagonist leads. Based upon precedents from the PTH system, t r u n c a t i o n of hHCF t o h H C F - ( 7 — 3 4 ) N H was hypothesized t o generate a PTH a n t a g o n i s t . T h i s fragment i n h i b i t e d PTH b i n d i n g and PTHs t i m u l a t e d adenylate c y c l a s e a c t i v i t y using r e n a l membranes and bone c e l l s i n v i t r o (43). Although h H C F - ( 7 — 3 4 ) N H was more potent than [ T y r ] b PTH-(7 — 3 4 ) N H on bone c e l l s (but not renal membranes), the hHCF fragment d i s p l a y e d p a r t i a l a g o n i s t p r o p e r t i e s , suggesting t h a t i t may be a weak a g o n i s t in. v i v o , l i k e [ N l e » , T y r ] b P T H ( 3 — 3 4 ) N H (43). 2

7

34

8

2

8

8

34

2

2

2

1 2

34

8

18

34

2

2

2

34

2

8

8

12

34

2

2

34

8

8

2

8

34

2

2

34

2

2

8

8

3

D

2

2

2

3 4

2

8

18

2


34

17.



Table II. Biological Activities of PTH Analogs Substituted in Position 12

Analog

Biological Activity Binding K-j

Adenylate Cyclase K (Agonist) K^ (Antagonist) m

nfi

" 34

[Tyr ]hPTH-(l—34)NH

2

[Ty 34]bPTH~(l--34)NH r

2

0.7 ± 0.3

0.7 t 0.1

1.1

1,,1 t* 0.4

0.3

[Tyr ]hPTH-( 7—34 )NH

257 ± 36

842 ± 182

[Tyr34]bPTH-(7—34)NH

75.0 ± 8.3

835 i 65

144.0 ± 9.0

1550 ± 33

34

2

2

8

18

[Nle . ,Tyr34]bPTH-(7—34)NH

2

12

[Ala ,Tyr34]hPTH-(l— 34)NH

2

12

[D-Ala Tyr34 ]hPTH-( 1 —34) NH t

2

12

34

[Aib ,Tyr ]hPTH-(l—34)NH

2

12

[ Pro , Tyr34 ] hPTH-( 1 —34) NH 2

12

[D-Ala ,Tyr34]hPTH-(7—34)NH

2

12

[Pro , Tyr34 ]hPTH-( 7— 34) NH

2

2

[Aibl ,Tyr34]bPTH-(7—34)NH

1,.5 t 0.2

0.8 ± 0.1

1,.4

0.1

0.8 ± 0.1

0..6

0.2

587

2

2

[Alal ,Tyr34]hPTH-(7—34)NH

1.0 ± 0.04

2448 t 769

196

114 ± 32

413 ± 67

113 ± 4

612 ± 116

471 i 50

1400 ± 668 536

51.0 + 8.7

2

2

[B-Ala1 ,Tyr34]bPTH-(7—34)NH

304 ± 74

128 ± 21

2

12

[Sar ,Tyr34 ]bPTH-( 7—34 )NH

2506 + 732

503 ± 91.4

2

144

Table III. Relative potencies of PTH antagonists in kidney (bovine renal cortical membrane) and bone (R0S 17/2.8 cell) systems Analog

Bone Cells

Kidney Membranes

[x]bPTH(7-34)NH

Binding Kb

2

Adenylate Cyclase K,

Binding *b

Adenylate Cyclase Ki

nM

Tyr Nle

34

8,18 12

Jyr

34

0-Trp ,Tyr

80 ± 7

879 ± 68

767 ± 199

5620 ± 1670

145 ± 13

1631 ± 350

964 ± 170

1550 ± 361

69 ± 5

60 ± 20

211 ± 116

125 + 7

182 +32

69 + 17

34

7± 1 8

18

12

Nle ' D-Trp .Tyr

34

f

15+1

Values are the mean + S.E.M. of at least three experiments.


257

258


By p r e p a r i n g h y b r i d molecules c o n t a i n i n g v a r i o u s m o t i f s from PTH and hHCF, i t may be p o s s i b l e t o i d e n t i f y regions o f t h e hHCF molecule that a r e responsible f o r t h e partial agonist a c t i v i t y of hHCF-(7—34)NH2 as w e l l as determine new d i r e c t i o n s f o r a n t a g o n i s t d e s i g n . F o r example, s i n c e t h e r e i s l e s s sequence homology between PTH and HCF a f t e r t h e f i r s t 13 amino a c i d s , t h e 7-13 r e g i o n of each p e p t i d e could be coupled with t h e 14-34 r e g i o n o f t h e o t h e r ( e x : bPTH-(7—13) + h H C F - ( 1 4 — 3 4 ) N H o r hHCF-(7—13) + b P T H - ( 1 4 — 3 4 ) NH ). More s u b t l e h y b r i d molecules could help p i n p o i n t t h e r e l a t i v e importance and r o l e o f v a r i o u s amino a c i d s f o r PTH a n t a g o n i s t and p a r t i a l a g o n i s t a c t i v i t i e s . 2

2

Future prospects f o r non-peptide PTH a n t a g o n i s t s The u l t i m a t e goal o f PTH a n t a g o n i s t drug design i s t h e development o f o r a l l y e f f e c t i v e non-peptide ( o r pseudo-peptide) agents. Approaches towards accomplishing t h i s o b j e c t i v e i n c l u d e c o n v e r s i o n o f p e p t i d e agents t o non-peptide agents and d i r e c t i d e n t i f i c a t i o n o f n o n p e p t i d e agents from n a t u r a l products. Several avenues have been used f o r t h e c o n v e r s i o n o f p e p t i d e s t o non-peptides. One approach i n v o l v e s f i r s t i d e n t i f y i n g a minimal amino a c i d sequence r e q u i r e d f o r b i o l o g i c a l a c t i v i t y , then s y n t h e s i z i n g analogs p o s s e s s i n g a l t e r e d peptide bonds, thus d e c r e a s i n g s u s c e p t i b i l i t y t o p r o t e o l y s i s . Replacement o f s p e c i f i c p e p t i d e bonds with CH -NH i n t e t r a g a s t r i n , f o r example, r e s u l t e d i n t h e s y n t h e s i s o f a g a s t r i n a n t a g o n i s t from t h e parent a g o n i s t molecule (109). Another approach i n v o l v e s t h e use o f peptide s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s t o develop h y p o t h e t i c a l models f o r the mechanisms o f b i n d i n g o f an a n t a g o n i s t t o i t s t a r g e t ( r e c e p t o r o r enzyme), then s y n t h e s i z i n g small molecules t h a t i n t e r a c t with t h e a c t i v e s i t e o f t h e t a r g e t . F o r example, based upon an understanding of t h e i n t e r a c t i o n o f nonapeptides with a n g i o t e n s i n c o n v e r t i n g enzyme (ACE) and t h e s i m i l a r i t i e s between ACE and carboxypeptidase A, s m a l l , o r a l l y - a c t i v e and c l i n i c a l l y - e f f e c t i v e molecules were prepared t h a t s p e c i f i c a l l y i n h i b i t ACE a c t i v i t y (110-112). The i s o l a t i o n of pharmacological substances from n a t u r a l product e x t r a c t s provides a p o t e n t i a l l y u n l i m i t e d source o f new t h e r a p e u t i c e n t i t i e s . In recent y e a r s , t h i s approach has l e d t o t h e i d e n t i f i c a t i o n of many drugs i n c l u d i n g i o n channel i n h i b i t o r s (avermectins f o r t r e a t i n g p a r a s i t i c i n f e c t i o n s ) , enzyme i n h i b i t o r s (the HMGCoA reduc t a s e i n h i b i t o r , l o v a s t a t i n , f o r t r e a t i n g h y p e r l i p i d e m i a ) and p e p t i d e r e c e p t o r a n t a g o n i s t s ( a s p e r l i c i n as an i n h i b i t o r o f c h o l e c y s t o k i n i n ) . In attempts t o i d e n t i f y p e p t i d e hormone r e c e p t o r a n t a g o n i s t s , t h i s process i n v o l v e s s c r e e n i n g crude n a t u r a l product e x t r a c t s f o r recep t o r b i n d i n g i n h i b i t o r y a c t i v i t y , c o n f i r m i n g a c t i v i t y i n a secondary ( f u n c t i o n a l ) assay, determining r e c e p t o r s p e c i f i c i t y o f t h e e x t r a c t o r n a t u r a l product, and using a p p r o p r i a t e assays t o monitor i s o l a t i o n and p u r i f i c a t i o n e f f o r t s . F o r example, t h e i s o l a t i o n of t h e s e l e c t i v e p e r i p h e r a l c h o l e c y s t o k i n i n a n t a g o n i s t a s p e r l i c i n from A s p e r g i l l u s a l l i a c e u s (113). l e d t o t h e s y n t h e s i s o f benzodiazepines p o s s e s s i n g s e l e c t i v i t y and enhanced potency f o r e i t h e r t h e p e r i p h e r a l c h o l e c y s t o k i n i n r e c e p t o r (L-364,718, MK-329) (114.115) o r t h e g a s t r i n / c e n t r a l c h o l e c y s t o k i n i n r e c e p t o r (L-365,260) (116; L o t t i , V.J.; Chang, R.S.L. Eur. J . Pharmacol. 1989, i n p r e s s ) . 2


17.



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