Inorganic Chemistry in Biology and Medicine - American Chemical

22 Computer-Predicted Drugs for Urolithiasis Therapy

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch022

M. RUBIN and R. GOHIL Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007 A. E. MARTELL and R. J. MOTEKAITIS Department of Chemistry, Texas A&M University, College Station, TX 77843 J. C. PENHOS and P. WEISS Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007

To attain the goal of developing therapeutic agents on a predictive basis challenges the state of knowledge of physiology, metabolism and the relation of chemical structure and biologic activity. The fact that new drug development still depends upon extensive - and expensive - screening programs for initial leads of biological activity is a marker of the long path ahead. However, in the case of alkaline earth urolithiasis, which makes up approximately eighty-five percent of renal stones, it seems possible to predict potentially useful therapeutic compounds and subject the predictions to experimetnal verification. With the known composition of stones, the quantitative stability data now available for metal ligand interactions, and the current knowledge of the composition of biologic compartments in health and disease, there is now sufficient information for computer calculation of metal speciation in certain biological fluids. Also, it appears that proposed regulatory mechanisms for calcium and magnesium metabolism correspond to i n v i v o responses, and enough pharmacolog i c a l data i s a t hand to allow a t e s t of t h i s approach to drug development. T h i s paper d e s c r i b e s the background of the c l i n i c a l problem, the r a t i o n a l e , methods, r e s u l t s , and conclusions drawn from the computer s i m u l a t i o n s t u d i e s , and t h e i r s u c c e s s f u l e x p e r i mental j i n v i v o v e r i f i c a t i o n demonstrating the p o s s i b i l i t y of d i s s o l v i n g calcium hydrogen phosphate (brushite) bladder and magnesium ammonium phosphate stones or i n h i b i t i n g t h e i r formation by treatment with an o r a l l y administered c h e l a t i n g agent. The Problem of U r o l i t h i a s i s In a nationwide study based on h o s p i t a l admission and d i s charge diagnosis (1) i t has been reported that f o r a d u l t p a t i e n t s an average of 9.47 persons per 10,000 p o p u l a t i o n were admitted with a diagnosis of u r i n a r y c a l c u l i . Evidence of g e o g r a p h i c a l v a r i a t i o n was obtained with r a t e s of 19.25 and 18.42 f o r South C a r o l i n a and Georgia at the highest s i d e of the s c a l e and 5.81 and 4.31 f o r Wyoming and M i s s o u r i at the low end. In general, 0-8412-05 88-4/ 80/47-140-3 81 $07.00/0 © 1980 American Chemical Society In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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the southeastern and northeastern area c e n t e r - s t a t e s around Massachusetts and Maine had the highest incidence with the mid and southwestern s t a t e s such as Wyoming, Arizona and Colorado at a lower l e v e l . Appropriate r e s e r v a t i o n s were expressed concerning the scope and adequacy of the sample but nonetheless the data f u l f i l l e d b a s i c requirements of s t a t i s t i c a l v a l i d i t y . "Stone b e l t s " where the incidence of r e n a l c a l c u l i i s unusually high have a l s o been w e l l documented i n other parts of the world. For the l a r g e m a j o r i t y of these p a t i e n t s a complex d i a g n o s t i c workup to exclude a d e f i n i t i v e e t i o l o g i c b a s i s f o r the formation of u r i n a r y c a l c u l i i s n e i t h e r f e a s i b l e m e d i c a l l y or on economic grounds. Thus, i n the present absence of a g e n e r a l l y u s e f u l medical treatment modality, s u r g i c a l i n t e r v e n t i o n i s r e q u i r e d when u r i n a r y obstruct i o n takes place. A l k a l i n e earth r e n a l l i t h i a s i s occurs when the ion s o l u b i l i t y product i s exceeded, a c r y s t a l n u c l e a t i o n s i t e i s present, and endogenous i n h i b i t o r s of c r y s t a l l i z a t i o n are decreased or absent ( 2 , · Mucoproteins (6) , c o l l a g e n (7), b r u s h i t e (8,9,10) , a p a t i t e (11), and monosodium urate (12), have been i m p l i c a t e d as p o s s i b l e n u c l e a t i o n s i t e s of a l k a l i n e earth stones (13). A high u r i n a r y concentration of magnesium (14) and pyrophosphate (15) on the other hand, have been c i t e d as p o s s i b l e i n h i b i t o r s of c r y s t a l l i z a t i o n and c r y s t a l growth. Urinary c i t r a t e , by v i r t u e of i t s c a p a b i l i t y to form unionized s o l u b l e calcium c h e l a t e s , may serve to decrease the ion c o n c e n t r a t i o n of calcium and thus a s s i s t i n prevention of calcium stone formation (16). Therapeutic attempts to i n h i b i t stone formation d e r i v e , as would be expected, from current understanding of the mechanisms r e s p o n s i b l e f o r t h e i r development (17). Numerous e f f o r t s have been made to b r i n g about a r e d u c t i o n i n the u r i n a r y concentration of p o t e n t i a l p r e c i p i t a t i n g i o n s . A l a r g e i n c r e a s e i n f l u i d intake r e s u l t s i n a general decrease i n the i o n a c t i v i t y of the consequent d i l u t e d u r i n e . This form of therapy i s d i f f i c u l t to s u s t a i n f o r a lengthy p e r i o d . Various approaches have been used to b r i n g about a r e d u c t i o n i n u r i n a r y calcium, by f a r the most frequent of stone forming c a t i o n s . O c c a s i o n a l l y the presence of h y p e r c a l c u r i a can be traced to an underlying pathophysiologic process such as hyperparathyroidism, malignancy, hyperthyroidism or s a r c o i d o s i s . E f f e c t i v e therapy of the disease process can sometimes m i t i g a t e the extension of the r e n a l l i t h i a s i s . Increased u r i n a r y e x c r e t i o n of calcium may a l s o occur as a d i r e c t consequence of i t s increased i n t e s t i n a l absorption or as a r e s u l t of a defect of i t s r e n a l absorption. I n t e s t i o n a l absorption can be modulated to some extent by d i e t a r y r e s t r i c t i o n but more e f f e c t i v e l y by o r a l a d m i n i s t r a t i o n of non-absorbable c e l l u l o s e phosphate. This compound i n h i b i t s calcium absorption i n the gut by complex formation (18). Correct i o n of a r e n a l tubular deficency i n calcium reabsorption has been achieved by a d m i n i s t r a t i o n of t h i a z i d e s (19). Although untoward s i d e e f f e c t s of p r o t r a c t e d t h i a z i d e therapy are w e l l e s t a b l i s h e d , t h e i r b e n e f i c i a l response f o r a s e l e c t e d group of p a t i e n t s appears unequivocal.

In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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Oxalate and phosphate are the anions a s s o c i a t e d with the a l k a l i n e earth c a l c u l i , calcium oxalate, calcium hydrogen phosphate and magnesium ammonium phosphate. Hyperoxaluria may r e f l e c t increased oxalate s y n t h e s i s or i n t e s t i n a l absorption. Although a procedure f o r decreasing oxalate synthesis i s p r e s e n t l y a v a i l a b l e , i t has a l s o been p o s s i b l e to modify the increased oxalate absorpt i o n o f t e n a s s o c i a t e d with v a r i o u s types of e n t e r i c malfunction. R e s t r i c t i o n of o r a l oxalate (20), a d m i n i s t r a t i o n of calcium c a r bonate (21), choiesterylamine (22) and aluminum hydroxide a n t i a c i d preparations (23) can reduce u r i n a r y oxalate output by reducing i t s g a s t r o i n t e s t i n a l absorption. Although a decrease i n u r i n a r y phosphate can be a t t a i n e d by inducing systemic phosphate d e p l e t i o n through decrease of i n t e s t i n a l phosphate absorption using o r a l aluminum hydroxide (24), the process i s a s s o c i a t e d with a s i g n i f i c a n t number of s i d e e f f e c t s (25). The view that n u c l e a t i n g s i t e s are an e s s e n t i a l i n d u c t i o n f a c t o r i n stone formation has l e d to a number of e f f o r t s to decrease t h e i r presence. Thus, o r a l c e l l u l o s e phosphate has been u t i l i z e d to decrease the b r u s h i t e (CaHP0^2H 0) a c t i v i t y i n u r i n e . A l l o p u r i n o l (26), administered apparently with some success, r e duces the h y p e r u r i c o s u r i a observed concomitantly with some oxalate stone formation. E f f o r t s have a l s o been made to d i m i n i s h c r y s t a l formation and growth by i n c r e a s i n g the u r i n a r y c o n c e n t r a t i o n of i n h i b i t o r s of these processes. The s i d e e f f e c t s of o r a l magnesium therapy limit the p o s s i b i l i t y of a t t a i n i n g the high concentrations i n u r i n e needed to provide e f f e c t i v e c r y s t a l formation and growth i n h i b i t i o n . Methylene blue, reputed to serve the same purpose, does not appear to be a u s e f u l agent (27). The p o s i t i v e e f f e c t s obtained by the a d m i n i s t r a t i o n of o r a l phosphate have been asc r i b e d to the r e s u l t i n g increase i n the u r i n a r y e x c r e t i o n of the c r y s t a l l i z a t i o n i n h i b i t o r , pyrophosphate (28). The most e f f e c t i v e agent of t h i s type developed to date i s ethane-l-hydroxy-1,1diphosphonate (29). This carbon analogue of pyrophosphate appears to f u n c t i o n by serving as a s i g n i f i c a n t i n h i b i t o r of phosphate and oxalate p r e c i p i t a t i o n as w e l l as slowing the aggregation of c a l cium oxalate c r y s t a l s . I t s use, however, r e s u l t s i n hyperphosphatemia by i n c r e a s i n g the tubular r e a b s o r p t i o n of phosphate i n c r e a s i n g bone r e s o r p t i o n and a f f e c t i n g the r e n a l metabolic formation of 1,25-dihydroxyvitamin D 3 (30-36). The formation of magnesium ammonium phosphate ( s t r u v i t e ) stones frequenly occurs i n an a l k a l i n e u r i n e r e s u l t i n g from u r i nary t r a c t i n f e c t i o n by urea c l e a v i n g gram negative organisms. Appropriate a n t i b i o t i c therapy, or the u t i l i z a t i o n of acetohydroxamic a c i d to i n h i b i t the a c t i v i t y of urease, coupled with management of phosphate e x c r e t i o n appear to be the treatment method of choice (37). The c a p a b i l i t y of e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d EDTA to s o l u b i l i z e otherwise i n s o l u b l e calcium p r e c i p i t a t e s l e d to i t s 2


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a p p l i c a t i o n i n the attempted d i s s o l u t i o n of a l k a l i n e e a r t h urol i t h i a s i s (38,39,40). There are a number of disadvantages of t h i s approach to therapy. EDTA i s reported to be poorly absorbed by mouth (41), i s p o t e n t i a l l y nephrotoxic (42), i s not s e l e c t i v e f o r calcium c h e l a t i o n i n the presence of magnesium, and as we and others have observed ( l o c . c i t . ) causes intense pain and hematuria when administered as a retrograde i r r i g a t i n g s o l u t i o n . Despite i n t e n s i v e e f f o r t s a t m o d i f i c a t i o n of the i r r i g a t i n g s o l u t i o n . Despite i n t e n s i v e e f f o r t s a t m o d i f i c a t i o n of the i r r i g a t i n g s o l u t i o n , we were unable to solve t h i s l a t t e r problem. Other attempts to develop l i t h o l y t i c s o l u t i o n s (43-51) have had only modest and i n c o n s i s t e n t success. There would be obvious advantages i n the use of a calcium c h e l a t i n g l i g a n d which could be given by mouth and end up i n the u r i n e i n a form which would combine with u r i n a r y calcium to reduce calcium i o n a c t i v i t y below the s o l u b i l i t y product l e v e l . This p o s s i b i l i t y was examined i n s t u d i e s with ethylene bis-(3-aminoethylether)-N,N,N ,N -tetraacetic a c i d , EGTA (52). This compound appears promising on the b a s i s of i t s low acute o r a l t o x i c i t y (3.96 ± 0.50 g/kg), l a c k of e f f e c t on serum calcium, absence of t o x i c i t y on o r a l feeding, and the evidence that 30-35% of the compound could be recovered i n the u r i n e a f t e r a s i n g l e o r a l dose. I t was observed, however, that when the compound was excreted i n t o the u r i n e i t c a r r i e d with i t an equivalent of calcium d e r i v e d from i t s combination with serum calcium. The net r e s u l t consequently was an i n c r e a s e i n the t o t a l u r i n e calcium e x c r e t i o n with no decrease i n the i o n i c calcium f r a c t i o n . f

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The Urinary Compartment. Recognition of the f a c t that the stone forming s a l t s i n u r i n e a r e u s u a l l y present i n s o l u t i o n a t a c o n c e n t r a t i o n exceeding t h e i r s o l u b i l i t y products l e d to s t u d i e s of the physico-chemical aspects of the f a c t o r s involved i n s o l i d phase formation (53-63). For calcium oxalate i t could be demons t r a t e d that pH v a r i a t i o n i n the p h y s i o l o g i c range was not s i g n i f i c a n t . On the other hand, the " s a l t i n g i n " e f f e c t of u r i n a r y e l e c t r o l y t e s and other c o n s t i t u e n t s were potent p o s i t i v e s o l u b i l i z a t i o n f a c t o r s . Sodium, potassium, magnesium, c h l o r i d e , b i carbonate, phosphate, s u l f a t e , c i t r a t e i o n , and urea increased the s u p e r s o l u b i l i t y of calcium oxalate i n u r i n e . These e a r l y pragmatic s t u d i e s were given a t h e o r e t i c a l b a s i s when computer programs provided a more f a c i l e s o l u t i o n of the numerous simultaneous equations governing the e q u i l i b r i a i n u r i n e . Using t h i s approach i t was p o s s i b l e to t e s t the p o t e n t i a l e f f e c t s of v a r i o u s present modes of therapy on the s o l u b i l i t y consequences f o r p r e c i p i t a t i n g s a l t s . Urine d i l u t i o n , as would be a n t i c i p a t e d a. p r i o r i , decreases p r e c i p i t a b i l i t y . A c i d i f i c a t i o n of u r i n e was l i k e w i s e c a l c u l a t e d to have a p o s i t i v e e f f e c t f o r calcium hydrogen phosphate, but i n the p h y s i o l o g i c range t h i s i s of l i t t l e consequence f o r the s o l u -



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b i l i t y of calcium oxalate. The l a t t e r r e s u l t agrees with e a r l i e r studies with r a t s . The computer c a l c u l a t i o n s a l s o suggested that a high u r i n a r y magnesium i o n concentration i n an a c i d i f i e d u r i n e would increase calcium oxalate s o l u b i l i t y , a f i n d i n g a l s o i n keeping with animal s t u d i e s . Therapeutic procedures, such as the a d m i n i s t r a t i o n of o r a l aluminum hydroxide gels to reduce u r i n a r y phosphate, were c a l c u l a t e d to be of i n c r e a s i n g p o t e n t i a l e f f e c t iveness with a u r i n a r y pH of 5 and above. However, the l a c k of p r e c i s e q u a n t i t a t i v e values f o r the s t a b i l i t y constants of u r i n a r y species placed some l i m i t a t i o n s on the a p p l i c a b i l i t y of t h i s approach to the problem of i o n i c e q u i l i b r i a i n u r i n e . Improvement i n the computer s i m u l a t i o n became p o s s i b l e with the development of an i t e r a t i v e c a l c u l a t i o n process, u t i l i z i n g b e t t e r values f o r the s t a b i l i t y constant of calcium oxalate. Good agreement was obtained between measured and c a l c u l a t e d s o l u b i l i t y and formation products of calcium oxalate i n the urine of normal i n d i v i d u a l s and stone formers. In continued s t u d i e s , a p p l i c a t i o n of t h i s physico-chemical background together with laboratory measurements of the concentration of u r i n a r y c o n s t i t u e n t s have f u r t h e r extended our understanding of the p o s s i b i l i t y of the formation and growth of a l k a l i n e earth r e n a l stones i n the c l i n i c al setting. The Plasma Compartment. Computer c a l c u l a t i o n s have a l s o been u t i l i z e d to provide data on the probable d i s t r i b u t i o n of metal ions among the many p o t e n t i a l binding l i g a n d s i n plasma (64-69). The low r e l a t i v e concentrations of the plasma metal ions enhances the p r o b a b i l i t y that the c a l c u l a t e d r e s u l t s approximate the s i t u a t i o n . Two major f a c t o r s p r e s e n t l y l i m i t the p o t e n t i a l a p p l i c a b i l i t y of such s i m u l a t i o n e f f o r t s . The f i r s t i s the e x i s i t n g u n c e r t a i n t y of the accuracy of some of the metal-ligand e q u i l i b r i a i n t h i s compartment. The second i s the modulating i n f l u e n c e of k i n e t i c p h y s i o l o g i c r e g u l a t o r y systems upon s h i f t s i n ion e q u i l i b r i a p r e d i c t e d by computer c a l c u l a t i o n based upon a s t a t i c compartment model. However, i t should be noted that the computer c a l c u l a t i o n s of i o n i z e d calcium i n plasma u l t r a f i l t r a t e are i n good agreement with measured values by spectrophotometry and potentiometry. Rationale of Study F i r s t c o n s i d e r a t i o n suggests that i t would be unreasonable to expect that potent a l k a l i n e earth binding ligands would be able to move through calcium and magnesium laden body compartments to a r r i v e i n the u r i n e i n a form capable of a d d i t i o n a l a l k a l i n e earth combination and s o l u b i l i z a t i o n . Nonetheless, a more searching examination of the problem i n d i c a t e s that meeting the physicochemical requirements may be p o s s i b l e w i t h i n the l i m i t s of change imposed by p h y s i o l o g i c t o l e r a n c e . The plasma compartment i s i l l u s t r a t i v e of t h i s s i t u a t i o n . Calcium bound to p r o t e i n repre-



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sents almost h a l f of the t o t a l calcium, low molecular weight complexes account f o r a small percentage, and the balance i s i o n i c calcium. The entry of a c h e l a t i n g l i g a n d such as EDTA upsets t h i s c a r e f u l l y regulated homeostatic b u f f e r with decrease i n i o n i c calcium as a f u n c t i o n of the r a p i d i t y and amount of EDTA i n t r o duced (70), and the formation of the p h y s i o l o g i c a l l y u n a v a i l a b l e calcium of the calcium c h e l a t e . The f a c t that plasma i s a calcium b u f f e r impies, however, that a calcium binding l i g a n d can be s e l e c t e d which w i l l provide calcium b u f f e r i n g a c t i v i t y i n the same range of i o n i c calcium as e x i s t s normally at the plasma pH of 7.4. Such c h e l a t i n g l i g a n d s would e x i s t i n plasma p a r t l y f r e e of c a l cium and p a r t l y as calcium c h e l a t e s . In c o n t r a s t to calcium p r o t e i n a t e , a l a r g e molecule, they would be f r e e l y f i l t r a b l e at the kidney glomerulus f o r passage i n t o the u r i n a r y t r a c t . Trans i t of the glomerular f i l t r a t e to the u r i n e i s by way of the nephron ( F i g . 1) which i s uniquely c o n t r o l l e d and s e l e c t i v e i n i t s c a p a b i l i t y to adjust the composition of the passing f l u i d . With the r e j e c t i o n of the high molecular weight calcium p r o t e i n a t e at the glomerulus and the r e a b s o r p t i o n of almost a l l of the i o n i c calcium i n the proximal and d i s t a l tubular r e g i o n s , the e q u i l i b r i a w i l l readjust to r e f l e c t the new i o n i c m i l i e u . I t could be a n t i c i p a t e d that the c h e l a t i n g l i g a n d would consequently have an enhanced c a p a c i t y f o r calcium binding i n the u r i n a r y compartment. A second f a c t o r may a l s o serve to increase i t s e f f e c t i v e n e s s i n s o l u b i l i z i n g u r i n a r y calcium. In the passage of f i l t e r e d plasma from the glomerulus to the u r i n a r y bladder sodium i o n i s reabsorbed with replacement by hydrogen and ammonium i o n s . To the extent that hydrogen ion a c i d i f i c a t i o n precedes and i s d i s s o c i a t e d from calcium i o n r e a b s o r p t i o n i n the nephron the r e s u l t could be the cleavage of f i l t e r e d calcium c h e l a t e i n an a c i d i f i e d environment, followed by r e a b s o r p t i o n of l i b e r a t e d calcium i o n . The r a t i o of calcium f r e e l i g a n d to calcium i o n i n u r i n e would increase to prov i d e a d d i t i o n a l calcium b u f f e r i n g c a p a c i t y . The g a s t r o i n t e s t i n a l compartment i s l e s s complicated. Calcium b i n d i n g l i g a n d s administered i n the f a s t i n g s t a t e would be present i n the absoprtive area p r i m a r i l y i n the unchelated form. The l o c a l pH and the presence of endogenous c a t i o n s and metal b i n d i n g l i g a n d s would determine the extent of calcium c h e l a t i o n . In the presence of food, however, one could a n t i c i p a t e that the food p o l y v a l e n t c a t i o n composition, the competitive metal binding l i g a n d s such as amino a c i d s and the l o c a l pH would e s t a b l i s h the r a t i o of c h e l a t e and metal f r e e l i g a n d . I t i s probable that i n f o r maximal e f f e c t i v e n e s s the p o t e n t i a l therapeutic agent should reach the plasma compartment i n metal f r e e form. Thus c o n s i d e r a t i o n must be given to the p o s s i b l e i n t e r a c t i o n of a l l the metal b i n d i n g l i g a n d s that would be present i n the i n t e s t i n a l absorption region.


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S e l e c t i o n of Test Compounds N i t r i l o t r i a c e t i c a c i d was s e l e c t e d as a t e s t compound to explore the a p p l i c a b i l i t y of the c o n s i d e r a t i o n s that have been enumerated. At the plasma pH of 7.4 i t b u f f e r s calcium i o n at concentrations between 10" and 10"^ M (71). This value i s c l o s e enough to the normal c o n c e n t r a t i o n of calcium i o n i n plasma, 1.2 χ 10~3 M, to suggest that an animal could accommodate e a s i l y to an a n t i c i p a t e d minor hypocalcemia. F o r t u i t o u s l y , because of i t s a p p l i c a t i o n i n detergents as a polyphosphate s u b s t i t u t e , an ex tensive l i t e r a t u r e has accumulated on i t s pharmacology and t o x i cology. I t i s known that the compound i s r e a d i l y absorbed from the g a s t r o i n t e s t i n a l t r a c t of r a t s and dogs, i s non-metabolized and i s almost q u a n t i t a t i v e l y excreted i n the u r i n e . I t s acute, subacute and c h r o n i c t o x i c i t y i s low, with a no e f f e c t l e v e l of 0.5 percent i n the d i e t of r a t s and dogs (72-75).


3

M a t e r i a l s and Methods N i t r i l o t r i a c e t i c a c i d was s u p p l i e d by the Hampshire Chemical Co., D i v i s i o n of W. R. Grace Co., Nashua, New Hampshire. For feeding s t u d i e s i t was n e u t r a l i z e d with 1 M sodium hydroxide to provide a 1% NTA s o l u t i o n . E t h y l n i t r i l o t r i a c e t a t e was obtained as a c l e a r colorless v i s c o u s o i l by r e f l u x i n g 30g of n i t r i l o t r i a c e t i c a c i d i n 1 l i t e r of absolute a l c o h o l i n the presence of 3g of toluenesulphonic a c i d f o r f o r t y - e i g h t hours. The a l c o h o l was removed by d i s t i l l a t i o n from a water bath i n vacuo, the r e s i d u e mixed with 100 ml of Q.5 percent sodium bicarbonate s o l u t i o n and extracted with one l i t e r of benzene. The benzene s o l u t i o n was extracted twice more with bicarbonate s o l u t i o n , washed with water and concentrated from a water bath i n vacuo. The c l e a r c o l o r l e s s r e s i d u a l o i l was d i s s o l v e d by shaking with an aqueous s o l u t i o n of 1.5 M c i t r i c a c i d and the volume adjusted by a d d i t i o n of d i s t i l l e d water to provide a f i n a l s o l u t i o n of 1% NTA e t h y l e s t e r as c i t r i c acid salt. Animal Studies Mixed b r u s h i t e / s t r u v i t e bladder stones i n r a t s were produced according to the method of Vermeulen et a l . (76). Pieces of z i n c sheet, 20-30 mg., were s u r g i c a l l y implanted i n the bladders of 40 Sprague-Dawley male r a t s . Three c o n t r o l groups of ten animals were maintained on an ad l i b d i e t of Purina r a t chow f o r eight weeks. The f o u r t h group of ten animals was given the same d i e t to which a n e u t r a l sodium NTA s o l u t i o n had been added to provide 5000 ppm (0.5%) of NTA. At the end of the study period one group of c o n t r o l animals and the group of NTA t r e a t e d animals were s a c r i f i c e d and examined f o r gross evidence of u r o l i t h i a s i s . One of the two remaining groups was continued as a c o n t r o l on the r e g u l a r d i e t while the other was now fed the treatment


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proximal

Figure 1.

Diagram of the blood supply and functional anatomy of the kidney nephron


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d i e t c o n t a i n i n g 0.5% NTA. Both groups were s a c r i f i c e d a f t e r an a d d i t i o n a l period of four weeks and examined f o r the presence of r e n a l stones. Studies with e t h y l n i t r i l o t r i a c e t a t e were conducted i n the same manner. For the t h e r a p e u t i c t r i a l i n a dog the n e u t r a l NTA s o l u t i o n was added to the r e g u l a r d i e t to provide an NTA concen t r a t i o n of 0.5%. Feeding was continued f o r a p e r i o d of two weeks. The animal was a four-year o l d female S c o t t i s h t e r r i e r . X-ray examination was done before and a f t e r the treatment p e r i o d .


Computer Studies The program u t i l i z e d f o r t h i s study provides the c a p a b i l i t y of e s t a b l i s h i n g s o l u t i o n and s o l i d phase e q u i l i b r i a f o r a m u l t i component system of up to t h i r t y f i v e components and 500 s o l u t i o n and s o l i d s p e c i e s . Using e q u i l i b r i u m constant data f o r metals, i n o r g a n i c anions, and organic l i g a n d s (77) the i t e r a t i v e c a l c u l a t i o n provides f o r the determination of metal s p e c i a t i o n as a f u n c t i o n of pH and component c o n c e n t r a t i o n . Table I and I I pro v i d e c a l c u l a t e d c o n s t i t u e n t concentrations f o r the Purina r a t chow used i n the animal feeding s t u d i e s f o r the b a s i c u r i n e composition. Results of Computer Studies The Plasma Compartment. A computer c a l c u l a t i o n of the s p e c i a t i o n of plasma calcium and other ions i n the presence of the c h e l a t i n g agent n i t r i l o t r i a c e t i c a c i d (NTA) was performed on a t y p i c a l plasma composition i n v o l v i n g the l i g a n d s l i s t e d i n Table III. The appropriate s t a b i l i t y constants have been s e l e c t e d by r e s t r i c t i n g c o n s i d e r a t i o n to those that undergo s i g n i f i c a n t i n t e r a c t i o n s with Ca2 , Mg^ , Cu^ , and Z n ^ ions i n s o l u t i o n . Since n e a r l y a l l amino a c i d s (the monoamino monocarboxylic a c i d s ) have s i m i l a r a f f i n i t i e s f o r metal i o n s , an "average" s t a b i l i t y constant and t o t a l c o n c e n t r a t i o n were employed. This procedure e l i m i n a t e d the need f o r c o n s i d e r a t i o n of a l a r g e number of unknown mixedl i g a n d s t a b i l i t y constants f o r ternary "mixed-ligand" complexes. Data i n the l i t e r a t u r e have been adjusted where p o s s i b l e (78), to 1.6 M i o n i c s t r e n g t h and 37°C. T y p i c a l data f o r plasma metal i o n s p e c i a t i o n i n the presence of 1 χ 10"^ M NTA i s provided i n Table IV. I t may be noted that although the c o n c e n t r a t i o n of i o n i c calcium has decreased only somewhat (from a normal range of about 49-52% to a l e v e l of 45%) there has been a r e d i s t r i b u t i o n of the non-ionic calcium from the plasma p r o t e i n s to NTA. I t can be a n t i c i p a t e d f o r other c h e l a t e s that the extent of t h i s r e d i s t r i b u t i o n w i l l be dependent upon the s t a b i l i t y constants of the calcium chelates at phasma pH. Data f o r the e f f e c t of up to 1.5 M NTA on the i o n i c calcium l e v e l of plasma i s provided i n F i g u r e 2. I f the plasma compartment were s t a t i c then up to 1.5 M NTA could be present before i o n i c clacium l e v e l s were reduced to a nonphysiological level. In f a c t , as we have reported (79-82), the +

+

+

+


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TABLE I


INTESTINAL CONCENTRATION OF FOOD COMPONENTS Calcium

3

X

Phosphorus

3

X

Potassium

2

X

Magnesium

8

X

Sodium

1.3

X

Iron

6.4

X

Zinc

8

X

Manganese

3

X

Copper

1.7

X

Cobalt

1

X

Amino A c i d s

1.3

-1 M 10 ^ -1 M 10 ^ -1 M 10 -2 M 10 -1 M 10 -3 M 10 -4 M 10 -3 M 10 -4 M 10 , -5 M 10 Ν

C a l c u l a t e d from the analyzed content of Purina r a t chow i n a wt/wt d i l u t i o n with i n t e s t i n a l fluid.

TABLE I I SIMULATED URINE COMPOSITION Phosphate Sulfate

3.35

5

X X

ίο"

2

M

ίο"

2

M M

Citrate

2.3

X

ίο"

2

Chloride

1.12

X

icf

1

M

Bicarbonate

1.6

X

ίο"

2

M

Sodium

9.7

ίο"

2

M

ίο"

2

M

ίο"

2

M

X

l(f

3

M

X

ίο"

2

M

Potassium

3.7

Calcium

3.2

Magnesium Ammonia

7.7

3

X X X

Urea and t r a c e concentrations of other c o n s t i t u e n t s have been omitted.


MEDICINE

In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980. (78).

2.5; Mg

S t a b i l i t y constants from Smith R. M.; M a r t e l l , A. E.

T o t a l m i l l i m o l a r c o n c e n t r a t i o n s of metal i o n s : Ca

0.9; Zn

0.05; Cu

9.4 6.7 10. 2

2.3

2.2

0.4

Albumin (SH)

5.9

(16.8), 5.7, 4.3, 2.9

2.2

2.2

1.5

Globulin (no SH)

4.7

3.7

10.1, 1.9

3. 7

5. 9

3.2

3.2

0.11

Citrate

1.8

2. 5

0.9

1.6

1.0

0.21

Proline 1.1

9. 7 5.1,

8. 6, 15.,9

1.6

1.0

0.07

Histidine

2.67

6.3, 11. 7

9.8, 17..5

1.6

1.0

2.51

Alanine (etc.)

Lactate

9.0, 2.3

4.5,

7.8, 14.,3

1.8

1.3

1.13

HPO^~

8.8, 5.8,

11.6, 5.7

2.4

3. 3

0.8

+

0.8

H

28.80

8. 5

9.8 6.1

5

-

2+

6. 1

Zn

-

2+

2.7

r,

Cu

2.4

2+

_

M*

Ca

3

(Μ χ 10 )

T o t a l Ligand Concentration

HC0~

CO^~

Complexing Ligand

METAL COMPLEXES IN BLOOD PLASMA (t = 37°, μ = 0.16 M, pH = 7.4)

TABLE I I I


0.001.

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TABLE IV


DISTRIBUTION OF METAL IONS IN BLOOD PLASMA ([NTA] Ligand

= 1.0 χ 10 Per

Ca

2 +

3

M)

Cent Metal Bound M

2 + S

Cu

r, 2H Zn

CO3-

1.9

1.3

-

-

HCO3

7.6

10.2

-

-

HPoJ"

-

4.0

-

-

ALA et a l .

-

-

9.0

-

Histidine

-

-

25.2

-

Proline

-

-

-

-

Lactate

1.5

1.3

-

-

Citrate

2.1

2.9

-

-

Globulin

8.2

11.0

-

-

-

-

-

-

99.6

Albumin Uncomplexed

45.1

60.5

NTA

32.5

8.7

64.2

% Distribution of NTA

8.13

7.8

-

5.0

C a l c u l a t e d from Table I I I according to Morel, F.; Morgan, J . (77).


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presence of calcium c h e l a t i n g l i g a n d s evokes the u s u a l homeostatic r e g u l a t o r y responses which r e s t o r e the i o n i c calcium concentra tion. In chelates given by mouth and absorbed from the g a s t r o i n t e s t i n a l t r a c t the f i n a l determinant of complexation w i l l be the balance between the k i n e t i c s of l i g a n d absorption, plasma calcium homeostasis, u r i n a r y e x c r e t i o n of the calcium c h e l a t e and p o s s i b l e r e n a l d i s s o c i a t i o n of the compound with r e a b s o r p t i o n of calcium and e x c r e t i o n of the c h e l a t i n g l i g a n d . For NTA i t appears that even at low plasma NTA concentrations some of the compound w i l l be a v a i l a b l e f o r e x c r e t i o n i n t o the urine i n calcium f r e e form. The Urinary Compartment. The computer program permits a wide-ranging e x p l o r a t i o n of the p o t e n t i a l e f f e c t of calcium b i n d ing ligands on the species d i s t r i b u t i o n as a f u n c t i o n of changes i n the c o n c e n t r a t i o n of any c o n s t i t u e n t and changes i n the pH of the medium. A number of these p o s s i b i l i t y have been explored f o r the presence of NTA i n the u r i n a r y compartment. V a r i a t i o n s i n u r i n e pH i n the range of 4.8 to 8.0 at v a r i o u s concentrations of ammonia, oxalate, phosphate, s u l f a t e , c i t r a t e , sodium, potassium, c h l o r i d e , calcium, magnesium, and carbonate provide i n s i g h t i n t o the p o s s i b l e consequences f o r a l k a l i n e earth s a l t p r e c i p i t a t i o n i n the presence of NTA. F i g u r e 3 demonstrates the sharp depend ence upon pH, calcium and oxalate concentrations f o r concentra t i o n s of NTA that e f f e c t i v e l y i n h i b i t p r e c i p i t a t i o n when the t o t a l u r i n a r y calcium c o n c e n t r a t i o n i s 1 χ 10"" M and the oxalate con c e n t r a t i o n i s between 1 χ 10"^ and 6 χ 10"^ M. An i n c r e a s i n g l y high c o n c e n t r a t i o n of u r i n a r y NTA i s r e q u i r e d as the pH i s de creased i n the moderately a c i d i c r e g i o n . The e f f e c t of v a r i a t i o n s i n the calcium i o n d i s t r i b u t i o n i n u r i n e as a f u n c t i o n of v a r i a t i o n s i n NTA c o n c e n t r a t i o n has a l s o been c a l c u l a t e d f o r lower concentrations of NTA equivalent to those o b t a i n a b l e i n feeding s t u d i e s . Figure 4 provides data i n terms of the change i n the f r a c t i o n of i o n i c calcium that would be present i n s o l u t i o n s of v a r y i n g pH from 5.0 to 8.0 with NTA con c e n t r a t i o n s of 3 χ 10""3 and 6 χ 10~3 M. C l e a r l y , i n these ranges of u r i n a r y NTA c o n c e n t r a t i o n , a major s h i f t i n the i o n i c concen t r a t i o n of calcium i o n can occur. This takes p l a c e with a corresponding change i n the f r a c t i o n of f r e e NTA l i g a n d a v a i l a b l e f o r a d d i t i o n a l calcium c h e l a t i o n . At u r i n e concentrations of 1 χ Ι Ο " M f o r calcium, 6 χ 10~3 M f o r c i t r a t e , and 6 χ 10~ M f o r NTA, and a t pH 7.65 e s s e n t i a l l y a l l the calcium i n u r i n e i s i n the form of a non-ionized chelated s p e c i e s . Approximately 65% of the t o t a l NTA w i l l be present as the unchelated calcium f r e e l i g a n d . Related c a l c u l a t i o n s f o r the s o l u b i l i z a t i o n of magnesium ammonium phosphate are presented i n Figures 5, 6, and 7 at the s t a t e d concentrations of magnesium phosphate and ammonium i o n s . The high u r i n a r y concentrations of NTA r e q u i r e d to i n h i b i t s o l i d phase formation at the a l k a l i n e pH a s s o c i a t e d with the c l i n i c a l circumstances f o r t h i s type of stone formation renders the p o t e n t i a l a p p l i c a b i l i t y of NTA of u n c e r t a i n value. 3

3

3


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N T A / C A L C I U M IN PLASMA

Figure 3. Computer-calculated concentration of NTA required to inhibit the precipitation of calcium oxalate at 1 X 10~ M Ca and varied concentrations of oxalate in a simulated urine (Table III) as a function of urine pH 3


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ΙΟΟι-

Figure 4. Computer-calculated percentage of NTA unchelated and Ca as Ca ion in simulated urine at two NTA and citrate concentrations as a function of urine pH

Figure 5. Computer-calculated concentration of NTA required to inhibit the precipitation of MgNH, PO,, in a simulated urine as a function of Mg concentration and urine pH f


395

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396

Figure 7. Computer-calculated concentration of NTA required to inhibit MgNH, POt, precipitation in a simulated urine as a function of ammonia concentration and urine pH

r


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The s i t u a t i o n appears to be q u i t e d i f f e r e n t i n the case of calcium hydrogen phosphate (brushite) stones. At a pH above 6.5 t h i s species i s converted, F i g u r e 8, to that of the s o l u b l e c a l cium NTA. The important i n f l u e n c e of the pH on the s o l u b i l i z i n g r o l e of NTA i s c l e a r l y evident i n the data p l o t t e d i n Figure 9. As would be a n t i c i p a t e d , an increased u r i n e pH markedly reduces the c o n c e n t r a t i o n of the l i g a n d r e q u i r e d to form calcium NTA. The data suggest that NTA could e f f e c t i v e l y i n h i b i t the formation or p o t e n t i a l l y cause the s o l u b i l i z a t i o n of calcium hydrogen phosphate stones i n the u r i n a r y t r a c t . The G a s t r o i n t e s t i n a l Compartment. In the pH range of 6.0 to 8.0 which may occur i n the a b s o r p t i v e area of the g a s t r o i n t e s t i n a l t r a c t the amount of f r e e or unchelated NTA present upon i n g e s t i o n of the usual r a t d i e t i s given i n Figure 10. Thus, when NTA i s added to the d i e t at 1.8 χ 10"" 2 M approximately 45 to 80 percent of the compound w i l l not be chelated to a p o l y v a l e n t c a t i o n . Once again, the sharp pH dependence of c h e l a t i o n i s evident. The f r a c t i o n of unchelated NTA i s d i r e c t l y r e l a t e d to the hydrogen i o n c o n c e n t r a t i o n . S i g n i f i c a n t l y a high percentage of NTA i n a d i e t containing 5000 ppm NTA w i l l be a v a i l a b l e f o r a b s o r p t i o n i n un complexed form even i n the presence of the competitive calcium i n t e r a c t i o n of the d i e t a r y c o n s t i t u e n t s . I m p l i c a t i o n s of the Computer Studies f o r U r o l i t h i a s i s The r e s u l t s of the computer c a l c u l a t i o n s a p p l i e d to simulated g a s t r o i n t e s t i n a l , plasma and u r i n e compartments suggest that the i n c l u s i o n of a non-toxic c o n c e n t r a t i o n of 5000 ppm NTA i n the d i e t of r a t s would: a) provide a s i g n i f i c a n t c o n c e n t r a t i o n of the calcium binding l i g a n d a v a i l a b l e f o r absorption i n i t s uncomplexed form; b) r e s u l t i n plasma concentrations of NTA which would maintain plasma calcium i o n i n the p h y s i o l o g i c range while a l l o w i n g f o r a s i g n i f i c a n t f r a c t i o n of the compound to be excreted i n t o the u r i n e f r e e of bound calcium; c) convert e s s e n t i a l l y a l l of the u r i n e calcium to the s o l u b l e calcium NTA c h e l a t e . Maintenance of the (calcium) Chelated form would be d i f f i c u l t f o r NTA i n the presence of i n c r e a s i n g concentrations of oxalate at a u r i n e pH below 6.0, and f o r i n c r e a s i n g concentrations of magnesium, ammonium, and phos phate ions with i n c r e a s i n g pH. However, NTA appears to have the p o t e n t i a l f o r i n h i b i t i n g the formation and inducing the s o l u b i l i z a t i o n of calcium hydrogen phosphate i n u r i n e . Animal Studies Rat Studies. The computer c a l c u l a t i o n s were tested by induc ing the experimental formation of b r u s h i t e / s t r u v i t e bladder stones in rats. The i n h i b i t i o n of stone formation by the feeding of 5000


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398

Figure 9. Computer-calculated percentage of the total Ca in a simulated urine present as solid-phase calcium hydrogen phosphate as a function of NTA concentration and urine pH



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ppm of NTA i n the d i e t of t r e a t e d animals compared to a group of untreated c o n t r o l s i s i l l u s t r a t e d i n P l a t e 9*. In a f i r s t study eight of ten animals i n the untreated c o n t r o l group had s i n g l e large stones or medium-sized stones with m u l t i p l e small stones. The two remaining c o n t r o l animals had i n c i p i e n t stone formation as manifested by c r y s t a l l i n e e n c r u s t a t i o n on the z i n c implant. In the treatment group seven of the ten animals were f r e e of bladder stones. In two animals, a l s o without evidence of stone formation, we could not f i n d the implanted z i n c sheet. In the remaining animal there was a small bladder stone. The study of i n h i b i t i o n of stone formation was repeated. One animal i n the c o n t r o l group died during the four week period and was not autopsied. The nine c o n t r o l animals at the end of the study had a l a r g e stone or m u l t i p l e stones. Eight of the ten treated animals had no evidence of stones. In three of these animals the implanted zinc p e l l e t was not r e t r i e v e d . The two r e maining animals i n the t r e a t e d group y i e l d e d a p e l l e t with a minor amount of z i n c e n c r u s t a t i o n . A t h i r d study was undertaken to determine whether the a d d i t i o n of NTA and i t s e t h y l e s t e r to the animal d i e t could cause the d i s s o l u t i o n of e x i s t i n g c a l c u l i . Forty animals were implanted with z i n c p e l l e t s . They were maintained on Purina r a t chow f o r four weeks. At that time, ten animals were s a c r i f i c e d and examined f o r bladder stones. M u l t i p l e stones were observed i n 9/10 animals i n t h i s c o n t r o l group. The remaining t h i r t y animals were d i v i d e d i n t o one c o n t r o l group of ten and two treatment groups of ten animals. One treatment group was given 0.5% of NTA i n i t s diet. The second treatment group was given 0.5%, as NTA, of the NTA e t h y l c i t r a t e . A l l animals were s a c r i f i c e d at the end of four weeks. A l l of the c o n t r o l animals had developed bladder stones. Eight of the ten NTA treated animals were g r o s s l y f r e e of stones. Two animals had small to moderate s i z e d stones. The NTA ester c i t r a t e treated group showed some evidence of t o x i c i t y as i n d i c ated by dehydration and weight l o s s . S i x animals were f r e e of bladder stones. Four had medium s i z e d stones. Dog Study. A female four-year o l d S c o t t i s h t e r r i e r with c l i n i c a l and X-ray evidence of phospaturic bladder stone format i o n , F i g u r e 11a, was treated f o r two weeks by the i n c o r p o r a t i o n of 0.5% of NTA i n the d i e t i n the form of the n e u t r a l 1% s o l u t i o n . The X-ray at the end of the treatment p e r i o d , F i g u r e l i b , showed no evidence of the presence of c a l c u l i . The animal was symptom free. Discussion The experimental v e r i f i c a t i o n of the computer generated pred i c a t i o n s provides some assurance of the soundness of the underl y i n g assumptions. I t i s consequently of i n t e r e s t to explore p o s s i b l e l i m i t a t i o n s of t h i s approach. * C o l o r plates are located i n the Appendix.


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ppm 2000

100

5000

15,000

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PH 90

-0 6.0

-

-o6.5

-o7.0


7.5 8.0

30 0

2

3 4 5 6 C o n e . N T A x l O " M in Diet

7

8

9

2

Figure 10. Computer-calculated percentage of unchelated Γ free") NTA present in the simulated intestinal contents during digestion as a function of NTA concen tration and intestinal pH

Figure 11. X-rays of female Scottish terrier (4 years) with clinical evidence of brushite bladder calculi. Treatment consisted of the incorporation of 0.5% Na NTA wt/wt, in the diet for a period of two weeks. 2



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The problem of the t r a n s p o r t of metal-free l i g a n d s from g a s t r o i n t e s t i n a l t r a c t to plasma compartment i s not a s i g n i f i c a n t one. A d m i n i s t r a t i o n of the t h e r a p e u t i c agent to the f a s t i n g p a t i e n t , i n c o r p o r a t i o n a t d i e t a r y l e v e l s s u f f i c i e n t to provide some metal f r e e c h e l a t i n g agent f o r a b s o r p t i o n , encapsulation to bypass metabolic conversion and ensure absorption from the i n t e s t i n e , and s t r u c t u r e m o d i f i c a t i o n to provide f o r post a b s o r p t i v e metabolic conversion to the a c t i v e agent, a l l o f f e r promising avenues. A more d i f f i c u l t problem concerns the nature of the r e l a t i o n s between the p o t e n t i a l t h e r a p e u t i c agent and the plasma compartment c o n s t i t u e n t s and homeostasis. A c h e l a t i n g agent potent enough to cause the s o l u t i o n of s o l i d phase calcium oxalate i n the u r i n a r y t r a c t would f i r s t combine with i o n i c and p r o t e i n bound calcium of the plasma. This f o l l o w s s i n c e the s t a b i l i t y constant of calcium p r o t e i n a t e at a plasma pH of 7.4 i s s e v e r a l orders of magnitude lower than the s t a b i l i t y constant of calcium o x a l a t e . As we have shown, the r e s u l t i n g hypocalcemia w i l l be a f u n c t i o n of the r a t e of entry of the agent i n t o the plasma compartment and the homeos t a t i c r e g u l a t i o n o f i o n i c calcium (79-82). In any event, even i f the hypocalcemia were to be maintained with p h y s i o l o g i c a l l y t o l e r able l e v e l s , the t h e r a p e u t i c agent would have been calciumsaturated before i t entered the u r i n a r y compartment. Whether i t could s t i l l be of p o t e n t i a l u t i l i t y f o r the s o l u t i o n of u r i n a r y calcium oxalate c a l c u l i may depend upon some p r e s e n t l y unresolved questions concerning the mechanisms of calcium metabolism i n the nephron. There i s , however, an a l t e r n a t i v e approach to the problem. By the s e l e c t i o n of potent calcium b i n d i n g l i g a n d s with b a s i c donor groups, advantage could be taken of the pH d i f f e r e n t i a l between the r e g u l a t e d 7.4 of plasma and the p o t e n t i a l of i n c r e a s i n g u r i n e pH to a maximum of 8.0. Increased calcium b i n d i n g c a p a b i l i t y would ensue i n the passage of such a compound from plasma to u r i n e . P o t e n t i a l l i m i a t i o n s are l e s s severe f o r e f f o r t s that would be d i r e c t e d toward i n h i b i t i n g the formation of r e c u r r e n t urinary c a l c u l i . In t h i s case the need f o r reducing u r i n a r y calcium i o n a c t i v i t y can be met by a p p l i c a t i o n of a l e s s potent calicum binding c h e l a t i n g agent i n combination with d i e t a r y r e s t r i c t i o n s of calcium, phosphate and o x a l a t e . The complexity of calcium metabolism i n the nephron coupled as i t apparently i s with that of sodium, hydrogen, bicarbonate and ammonium i o n s , o f f e r s some unusual o p p o r t u n i t i e s f o r c o n t r o l of the i n t e r a c t i o n of the c h e l a t i n g l i g a n d with calcium and magnesium i o n s . The glomerular f i l t r a t e as i t enters the proximal tubule i s e s s e n t i a l l y p r o t e i n f r e e , i s o h y d r i c and isoosmotic to the plasma. The calcium c o n c e n t r a t i o n of the f l u i d compared to plasma i s decreased by approximately h a l f due to the n o n - f i l t r a t i o n of the plasma calcium p r o t e i n a t e . In i t s f u r t h e r passage along the proximal tubule the f l u i d l o s e s sodium and calcium i o n by t h e i r r e a b s o r p t i o n across the c e l l s l i n i n g the area. One can a n t i c i p a t e



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that some e q u i l i b r i u m readjustment could occur i n the i n t e r a c t i o n of calcium and c h e l a t i n g l i g a n d . Depending on the r e l a t i v e conc e n t r a t i o n s of the s p e c i e s , the r e s u l t would be an increased r a t i o of c a l c i u m - f r e e l i g a n d to calcium c h e l a t e . This could increase the p o t e n t i a l f o r decreasing calcium i o n a c t i v i t y . Following water r e a b s o r p t i o n i n the loop of the nephron, a f u r t h e r set of i o n i c readjustments takes place i n the d i s t a l tubular p o r t i o n of the nephron. For purposes of present c o n s i d e r a t i o n s , the most important changes are those i n the concentrations of hydrogen, ammonium, sodium, bicarbonate, and calcium i o n s . The d i s t a l tubular r e a b s o r p t i o n of sodium ions i s a s s o c i a t e d with concomitant s e c r e t i o n of hydrogen and ammonium i o n s . The net r e s u l t i s an a c i d i f i c a t i o n of the r e s u l t i n g u r i n e . Such an e f f e c t , which i s v a r i a b l y c o n t r o l l a b l e according to systemic homeostatic needs by drugs such as the d i u r e t i c carbonic anhydrase i n h i b i t o r s and through d i e t a r y m o d i f i c a t i o n , could r e s u l t i n some pH dependent d i s s o c i a t i o n of the calcium c h e l a t e i n t r a n s i t . I f , as seems p o s s i b l e , a f u r t h e r c o n t r o l l e d reabsorption of l i b e r a t e d calcium i o n were to occur, there would again be an enhanced calcium-free l i g a n d calcium c h e l a t e r a t i o with f u r t h e r increase i n the calcium sequestering c a p a b i l i t y of the t h e r a p e u t i c agent. That these p o s s i b i l i t i e s have p o t e n t i a l r e a l i t y may be i n f e r r e d from some published s t u d i e s . In a r e p o r t on the long term i n f l u e n c e of NTA on mineral metabolism i t was pointed out that the only observed change was an i n c r e a s e i n u r i n a r y z i n c e x c r e t i o n (75). There was no change i n calcium balance. The c o n c l u s i o n can be drawn from t h i s work that the glomerular f i l t r a t i o n of plasma calcium NTA was followed by d i s s o c i a t i o n of the calcium NTA and r e a b s o r p t i o n of the calcium independently of the e s s e n t i a l l y q u a n t i t a t i v e u r i n a r y e x c r e t i o n of the l i g a n d . The extensive use of e t h y l e n e d i a m i n e t e t r a a c e t i c a i c d , EDTA, and i t s calcium c h e l a t e i n medicine has provided much information on the metabolism of the compounds i n humans. Calcium EDTA i s the treatment of choice i n lead poisoning (83-86). Spencer et a l . demonstrated that a d m i n i s t r a t i o n of i n t r a venous EDTA was followed by a v a r i a b l e and incomplete u r i n a r y e x c r e t i o n of calcium i n r e l a t i o n to the molar equivalence of the administered l i g a n d (87, 88). We subsequently reported that t h i s e f f e c t was c h a r a c t e r i s t i c of experimetnal hypocalcemia and could be overcome by a d m i n i s t r a t i o n of carbonic anhydrase i n h i b i t o r s (79). The c o n c l u s i o n may be drawn from these s t u d i e s that r e n a l d i s s o c i a t i o n and calcium r e a b s o r p t i o n can occur even with a potent calcium binding c h e l a t i n g agent such as EDTA. I t i s unc l e a r , however, that t h i s process could be c o n t r o l l e d f o r the purpose of d i s s o l v i n g r e n a l c a l c u l i . Therapeutic t r i a l of the compound by retrograde i r r i g a t i o n of the u r i n a r y t r a c t has been u n s u c c e s s f u l i n our hands, as w e l l as i n the other t r i a l s , due to l o c a l i r r i t a t i o n by these i r r i g a t i n g s o l u t i o n s .


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Abstract A method has been developed to predict the potential u t i l i t y of orally administered chelating ligands to inhibit the formation of alkaline earth urinary c a l c u l i or dissolve those present in animals. A computer program provides the calculated speciation of calcium and magnesium in simulated intestinal plasma and urinary compartments upon the addition of the ligand. Predicted levels of metal ion interaction with the added chelating agent provide suggested dosage levels and other parameters for biologic al testing. The addition of 0.5% of disodium n i t r i l o t r i a c e t a t e (Na2NTA) to the diet of rats inhibited the formation of experi mental mixed brushite (CaHPO4) struvite (MgNH4PO4) bladder c a l c u l i formed by the implantation of zinc pellets. Established c a l c u l i were dissolved by this treatment. A brushite calculus in a dog was dissolved by Na NTA treatment as established by X-ray and c l i n i c a l findings. 2

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