Iron Chelation in the Treatment of Cooley's Anemia - ACS Symposium

15 Iron Chelation in the Treatment of Cooley's Anemia W. F R E N C H ANDERSON

Downloaded by CORNELL UNIV on June 13, 2017 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch015

Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20205

I will discuss iron chelation in the treatment of the human genetic disease beta-thalassemia, otherwise known as Cooley's anemia. Cooley's anemia, named after Dr. Thomas Cooley who first described the disease in detail in 1925 (1), is a lethal hereditary anemia in which the infants cannot make their own blood (2). They must be transfused every three to four weeks starting at six months of age for the rest of their lives. Failure to transfuse results in death from profound anemia. According to the World Health Organization, the group of diseases called the thalassemias, of which Cooley's anemia is the most severe, is the largest health problem in the world for single locus genetic diseases. The major problems these patients have, however, are caused not directly from their disease, but from the treatment. Every unit of transfused blood contains several hundred milligrams of iron. The body is not equipped to excrete such large amounts of iron and so it is deposited in the heart, liver, endocrine glands and other organs producing severe medical problems ultimately leading to death in early teenage. Iron chelation therapy was attempted by a few physicians back in the late 1960's, but in general such therapy was considered useless or even harmful (3). A major effort has been made over the past 5 years, however, to find means whereby the presently available iron chelator, Desferal , could be effectively administered to remove large quan tities of iron from iron-overloaded patients. This effort has proven successful, and Desferal is now used world-wide for iron chelation therapy (4). Life expectancy and quality of life of treated patients now appear greatly improved. From the beginning it was realized, however, that Desferal is not. an ideal drug (3). It can only be given by injection and these can sometimes be very painful, it becomes effective only when the body's iron load is already ten-times normal, and it is very expensive to produce. In 1973, under the sponsorship of the National Institute of Arthritis, Metabolism and Digestive Dis eases, a new program was instituted designed to develop new iron R

This chapter not subject to U.S. copyright. Published 1980 American Chemical Society Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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c h e l a t o r s f o r c l i n i c a l use (5). Several of the chemists here t o day have been a p a r t of that very s u c c e s s f u l program. Over 100 compounds have been t e s t e d and s e v e r a l are e i t h e r i n or being p r e pared f o r human c l i n i c a l t r i a l s (6). I am very pleased to have been asked to summarize t h i s f i e l d f o r you p r i o r to the major papers to f o l l o w . I w i l l review the disease Cooley's anemia, o u t l i n e the medical problems and complic a t i o n s , and then w i l l give you a b r i e f summary of the concepts behind i r o n c h e l a t o r development.


D e s c r i p t i o n of Cooley's Anemia P l a t e s 1A and B* a r e photographs of a p a t i e n t with Cooley's anemia. A t f i r s t glance, he appears p e r f e c t l y normal, but there are s e v e r a l c h a r a c t e r i s t i c s of h i s disease that are apparent. These p a t i e n t s cannot make u s e f u l blood because of t h e i r genetic block, but t h e i r bone marrows t r y to make blood and i n so doing become markedly o v e r a c t i v e . Therefore, c e r t a i n bones become gross l y enlarged because of the g r e a t l y expanded bone marrow i n s i d e them. The upper jaw enlarges, d i s p r o p o r t i o n a t e l y , producing prominent f r o n t teeth, a l a r g e space between the nose and upper l i p , and high prominent cheeks. The forehead s w e l l s or "bosses" out. Because of the l a r g e amount of t o t a l body metabolism going i n t o i n e f f e c t i v e blood production, body growth i s abnormal and g r e a t l y delayed. The arms and l e g s are s p i n d l y and the abdomen i s l a r g e and protuberant because of a g r e a t l y enlarged l i v e r . The spleen becomes so l a r g e e a r l y i n l i f e that i t f i l l s the whole abdomen and has to be removed. The x-ray shown i n F i g u r e 1A demonstrates the l a r g e amount of bone marrow i n the s k u l l . A normal s k u l l shows a s i n g l e t h i n l i n e of bony t i s s u e . As can be seen here, there i s a c o n s i d e r able q u a n t i t y of f u z z , and that f u z z represents the enormously enlarged bone marrow. T h i s c h a r a c t e r i s t i c i s c a l l e d " h a i r - o n end" because the x-ray looks l i k e h a i r standing up out of the skull. F i g u r e IB shows an x-ray of the hand. The i n s i d e s of the bones are dark on the x-ray, r e s u l t i n g from the h y p e r a c t i v e bone marrow which has hollowed out the normally s o l i d bones. This i s true of a l l the long bones: the f i n g e r s , arms, and l e g s . At times the bones become so t h i n that they f r a c t u r e e a s i l y . A blood smear of normal blood showing healthy r e d corpuscles i n P l a t e 2A*. In c o n t r a s t , P l a t e 2B* shows the blood smear of a p a t i e n t with Cooley's anemia. Note how pale the blood c e l l s are; they are of a l l d i f f e r e n t s i z e s and shapes, with many of them g r o s s l y abnormal i n shape. A scanning e l e c t r o n microscope p i c ture of these c e l l s i s shown i n F i g u r e 2. Most of these c e l l s have, not s u r p r i s i n g l y , a very short h a l f - l i f e . A photograph of Dr. Max Perutz's two angstrom-per-centimeter x-ray d i f f r a c t i o n model of hemoglobin i s shown i n P l a t e 3*. Hemoglobin, the blood pigment that c a r r i e s oxygen and makes the blood red, i s composed of four chains, two alpha g l o b i n chains * Color p l a t e s a r e located i n the Appendix.

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

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Figure 1.

Figure 2.

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X-rays of the skull and hand of a patient with Cooley's anemia

Scanning electron microscope photographs of red blood cells in Cooley's anemia (12)



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( i n black) and two beta g l o b i n chains ( i n w h i t e ) . The r e d d i s c represents heme which contains one molecule of i r o n . In Cooley's anemia, the bone marrow c e l l s cannot make the beta (or white) chains. Chromatograms of the g l o b i n chains synthesized by the immature red blood c e l l s o f p a t i e n t s a r e shown i n Figure 3 (7). The chains a r e separated on carboxymethyl-cellulose using a urea-phosphate b u f f e r . The top panel shows a chromatograph of normal human alpha and beta g l o b i n chains. There i s a roughly equal amount of alpha g l o b i n (on the r i g h t ) and beta g l o b i n (on the left). In c o n t r a s t , the middle panel shows the g l o b i n chains of a p a t i e n t with homozygous beta thalassemia ( i . e . , Cooley's anemia). There i s a normal amount of alpha g l o b i n but a g r o s s l y decreased amount of beta g l o b i n and a s l i g h t compensatory i n crease i n gamma (or f e t a l ) g l o b i n . The s m a l l number of beta and gamma g l o b i n chains complex with alpha g l o b i n chains to produce a l i m i t e d number of hemoglobin molecules. This produces hypochromic red blood c e l l s (RBC). What i s even worse i s that the e x t r a uncomplexed alpha g l o b i n chains, which are i n s o l u b l e , prec i p i t a t e w i t h i n the c e l l , cause membrane damage, and as a r e s u l t the c e l l s break down. The abnormality here i s the i n a b i l i t y of the s m a l l number of non-alpha g l o b i n chains to complex with a l l the alpha g l o b i n chains. The i r o n i n these broken down RBCs i s r e l e a s e d and i s deposited i n body organs. The bottom panel shows the g l o b i n chains from a newborn i n f a n t . The baby has f e t a l blood normally and i s only beginning to switch to a d u l t hemog l o b i n . Here, however, the number of beta plus gamma g l o b i n chains e x a c t l y equals the number of alpha g l o b i n chains so that no g l o b i n chain imbalance e x i s t s . This i n a b i l i t y to make the beta g l o b i n chains i n Cooley's anemia, which consequently means an i n a b i l i t y to make hemoglobin, together with the p r e c i p i t a t i o n of uncomplexed alpha g l o b i n chains causing a l l the r e d c e l l s made t o be l y s e d , accounts f o r the signs and symptoms seen i n the disease Cooley's anemia. Medical Complications from Iron Overload The only treatment f o r t h i s disease i s blood t r a n s f u s i o n , but with every u n i t of blood, as we s a i d before, s e v e r a l hundred mg of i r o n a r e deposited i n the body and t h i s d e p o s i t i o n of i r o n r e s u l t s i n severe symptoms and u l t i m a t e l y i n death. The body i s normally equipped to handle about one mg of i r o n a day i n the d i e t . What happens to the organs of p a t i e n t s with t h i s massive d e p o s i t i o n of excess i r o n ? P l a t e 4* i s a photograph of the heart from a p a t i e n t who d i e d from t r a n s f u s i o n a l hemosiderosis - i . e . , i r o n overload from t r a n s f u s i o n s ; i t shows l a r g e q u a n t i t i e s of i r o n . Note the dark brown or r u s t c o l o r a t i o n ; that i s i r o n . One cannot s l i c e an organ with t h i s much i r o n - one has to saw i t . I t i s hard to imagine how a heart l i k e t h i s could have kept f u n c t i o n i n g f o r as * Color p l a t e s are l o c a t e d i n the Appendix.



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long as i t d i d . This p a t i e n t was 23 years o l d when he d i e d . A s e c t i o n of c a r d i a c muscle from the heart shown i n P l a t e 4 * i s shown i n P l a t e 5 * . The i r o n d e p o s i t i o n i n each c e l l i s shown by the blue s t a i n . As can be seen, every c e l l has l a r g e q u a n t i t i e s of i r o n . P l a t e 6 * shows a photograph of the l i v e r from the same pat i e n t . The i r o n deposits can be seen as brown patches a l l over the surface. This l i v e r i s very heavy and, obviously, functioned very poorly. An i r o n s t a i n e d s e c t i o n of the p i t u i t a r y gland i s shown i n P l a t e 7*. I t i s no wonder that these p a t i e n t s grow slowly and have delayed and abnormal sexual development. A bar graph which summarizes the amount of i r o n i n each of the body t i s s u e s i n t h i s p a t i e n t i s shown i n Figure 4 . Most t i s s u e s had l a r g e amounts of hemosiderin and f e r r i t i n and many organs are g r o s s l y loaded. Most normal t i s s u e s have no d e t e c t able hemosiderin or f e r r i t i n . Now, what can be done? Obviously, the i d e a l s o l u t i o n would be a treatment which would allow the bone marrow to make normal amounts of alpha and beta g l o b i n chains, thereby c o r r e c t i n g the abnormality. U n t i l t h i s becomes p o s s i b l e , however, the next a l t e r n a t i v e i s to improve techniques f o r removing excess i r o n from the body t i s s u e s or even to chelate and excrete excess i r o n bef o r e i t i s ever deposited i n the body. Development of New

Iron

Chelators

Let me now spend a few minutes summarizing the general f i e l d of i r o n c h e l a t o r development. The best organic chemists f o r producing e f f e c t i v e i r o n c h e l a t o r s are the microbes (8). They have been doing i t f o r m i l l i o n s of years, and have developed extremely e f f e c t i v e molecules f o r c h e l a t i n g i r o n . Dr. Neilands, the expert on b a c t e r i a l i r o n c h e l a t o r s , w i l l be the next speaker. Microbes u t i l i z e p r i m a r i l y two types of chemical groups f o r c h e l a t i o n : hydroxamic acids [-N(OH)-CO] (Figure 5 ) and catechols which are two adjacent OH groups on a phenyl r i n g (Figure 6 ) . The simplest i r o n c h e l a t o r i n existence appears to be hadac i d i n (Figure 5 ) . This l i t t l e molecule has one hydroxamic a c i d group and, t h e r e f o r e , has two bonds a v a i l a b l e to chelate i r o n . I t i s not p a r t i c u l a r l y e f f e c t i v e s i n c e i t can only form two bonds, while i r o n u t i l i z e s s i x . At the other extreme i s ferrachrome, an a b s o l u t e l y b e a u t i f u l molecule, which i s l i k e a Venus F l y Trap with three hydroxamic a c i d groups - one i n the back of each of three arms. Once i r o n goes i n , i t i s h e l d i n place very t i g h t l y by s i x bonds. Ferrachrome i s one of the most e f f e c t i v e i r o n c h e l a t o r s i n nature. But n e i t h e r ferrachrome, which i s too complex, nor hadac i d i n , which i s too simple, are e f f e c t i v e i n c h e l a t i n g i r o n from the t i s s u e s of animals. The type of molecule that one wants i s one which i s f a i r l y simple and which has three hydroxamic acids * Color p l a t e s are located i n the Appendix.


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Normal (Folic Acid Deficiency)

Figure 3. Globin chain synthesis in the immature blood cells of an adult with no globin abnormality (top), a patient with Cooley's anemia (middle), and a newborn with no globin abnormality (bottom); for experimental details see Ref. 7

TUBE NUMBER

11,000 10,000 9000

•

Ferritin Iron

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Hemosiderin Iron

8000 7000 6000 5000 4000 3000 2000 1000

ν

Figure 4.

Iron content of body organs from the same patient


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Ο

II

OH

I

H — C — Ν — CH — C0 H 2

2


hadacidin

ferrichrome

HN 2

CONH

\

(CH )

(CH )

2 5

2 5

^N

CONH \ (CH )

(CH )

2 e

2

—C

I

\ (CH ) \ N—C

2 5

e

I

OH Jl Ο 1

OH

CH

II

OH Ο

desferrioxamine (Desferal ) R

Ο

Il

OH

*N — C —

I

HC — C — N 3

I

II

HO

Ο

CH

3

rhodotorulic acid

Figure 5.

Microbial iron chelators containing hydroxamic acids


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ψ;

C=0-H


I

c=o

f

\

O—C

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HCH

H—C—CH,—OH

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enterobectin (enterocheNn)

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Iron Chelation in the Treatment of Cooley's Anemia - ACS Symposium

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