BdtishJoumal ofHaematology, 1976, 33,477.

Chelation Studies with 2,3-Dihydroxybenzoic Acid in Patients with P-Thalassaemia Major

C. M. PETERSON, J. H. GRAZIANO,* R. W. GRADY, R. L. JONES, H. V. VLASSARA, V. C. CANALE,* D. R. Mmm* AND A. CERAMI

Rockefeller University and *Division of Pediatric Hematology-Oncology, The N e w York Hospital-Cornell Medical Center

(Received 5 January 1976; acceptedfor publication 28 lanuary 1976)

SUMMARY. a,3-Dihydroxybenzoic acid was evaluated as a potentially useful, orally effective iron-chelating drug by performing iron balance studies in patients with p-thalassaemia major. The administration of this substance at 25 mg/kg/d to five patients for 8 d caused an average increase in iron excretion of 4.5 mg/d. When the drug was administered at 2s mg/kg q.i.d. to eight patients for 21 d, iron excretion increased to 6.5 mg/d. Chelation was highly specific for iron with changes in magnesium and calcium excretion being insignificant. The drug was well tolerated with side effects limited to gastrointestinal complaints which ameliorated when the drug was taken with food. These studies provide a rationale for hrther evaluation of 2,3-dihydroxybenzoic acid in patients with iron overload.

Transfusion therapy on a continual basis is the only available treatment for the severe anaemia associated with the genetic disease, p-thalassaemia major. Since the body lacks an effective means of excreting iron, virtually all of the iron administered as transfused erythrocytes is retained and stored in various tissues as ferritin or haemosiderin. This accumulation of iron, particularly in the liver, heart and pancreas, leads to progressive fibrotic changes of these tissues resulting in organ failure and early death. Most patients with thalassaemia on a regular transfusion regimen die by the end of their second decade (Ellis et a!, 1954).

Since there is no means at present to treat the specific genetic defect of 8-thalassaemia major, therapeutic approaches have dealt with ways to remove excess iron. Most attempts to prevent iron accumulation have centred on the use of desferrioxamine (DF), an iron chelator produced by Streptomyces pilosus. Although DF is not widely accepted by physicians in the United States, recent reports from England suggest that chronic DF therapy retards the accumulation of iron, as evidenced by lower concentrations of both serum ferritin and liver iron in DF-treated patients compared with untreated patients who received a comparable number of transfusions (Barry et al, 1974). Nevertheless, several drawbacks are associated with the use of DF in the treatment of patients with 8-thalassaemia major. The drug must be given daily as an intramuscular injection. A number of adverse side effects have been described in humans, including allergic hypotensive reactions (Fairbanks et al, 1963 ; fIwang & Brown, 1964; Smith, 1964; Gevirtz et al, 1965). Accordingly, we have undertaken a

Correspondence: Dr Charles M. Peterson, Rockefeller University, New York, N.Y. 10021, U.S.A.

477

478 C. M, Peterson et a2

programme to design and evaluate new iron-chelating drugs with emphasis on those which might be orally effective.

Using the hypertransfused rat as an animal model of transfusion-induced iron overload, we identified 2,3-dihydroxybenzoic acid (2,pDHB) as an orally effective iron chelator (Graziano et a!, 1974). The toxicity of this compound is minimal in both animals and man (Graziano et al, 1974; Martini & Ponzio, 1952; Clarke et a!, 1958). In the present communica- tion we present our preliminary evaluation of 2,3-DHB in patients with /I-thalassaemia major. .

MATERIALS AND METHODS

Patients with P-thalassaemia major were referred from the clinics at New York Hospital or New York University Medical Center. Patients were hospitalized either at the Rockefeller University Hospital or at the Clinical Research Center of the Cornell Medical Center. Protocols were approved by the Food and Drugs Administration (FDA) and the appropriate committees of both hospitals. The first protocol consisted of a 21 d study period. The patients were hospitalized and placed on a low-iron diet (4-13 mg/d) which necessitated eating the same meals every other day of the duration of the study. The first 8 d were a baseline control period. The second 8 d were a drug dosage period with a single oral dose of 25 mg/kg of 2,3- dihydroxybenzoic acid. The last 5 d constituted a follow-up observation period. The second protocol involved a 29 d hospitalization period during which the patients were placed on similar low-iron diets. Once again, the first 8 d consisted of a baseline control period, while the next 21 d were a drug dosage period at 25 mg/kg p.0. q.i.d. During the course of both studies, 24 h collections of urine and stool were made. Estimations of both caloric and iron intake were made by the dietary departments of the respective institutions. Dietary iron was determined for each patient by homogenizing a representative diet and measuring the iron content of an aliquot by atomic absorption analysis. The degree of iron overload in the patients involved was estimated from their transfusion histories, and serum ferritin levels together with nitroprusside staining of a bone marrow aspirate. Patient acceptance of the drug regimen was evaluated by a subjective questionnaire in addition to verbal questioning. Physical examinations were performed before, during and after drug administration. Tem- perature, blood pressure, pulse and respiration rates were monitored at least twice a day during the study period. Photographs of the face and hands were obtained before and after drug administration. All women with childbearing potential had a negative urinary preg- nancy test prior to admission to the study. Informed witnessed consent was obtained prior to all studies from both the patient and his parents if the patient was under the age of majority.

2,3-Dihydroxybenzoic acid was synthesized at the Rockefeller University by standard procedures (Dallacker et a!, 1971). The purity of each preparation was monitored by mixed melting-point determination, thin-layer chromatography, infrared and NMR spectroscopy. The drug was packed into gelatin capsules by the pharmacies of the respective institutions. The iron content of the urine, stool and diet of each patient was determined by atomic absorption spectrophotometry. Similar determinations were made of copper, zinc, calcium, magnesium and potassium levels in the urine and stool (Graziano et al, 1974).

Erythrocytes were labelled for determination of faecal blood loss by standard methods (Gray & Sterling, 1950). A 50 ml aliquot of the patient’s erythrocytes was incubated with

~,3-Dikydroxybenzoic Acid and Tkalassaemia 479

50 pCi of 51chromium and reinfused into the patient after washing three times in isotonic saline. Blood samples were drawn at 30 min and every other day thereafter for determination of the specific activity of the blood. Faecal blood loss was determined by counting the 51Cr-radioactivity of an aliquot of homogenized stool. The levels of serum ferritin were determined by radioimniunoassay (Addison et al, 1972). Bone marrow aspirates were ob- tained from the left posterior superior iliac crest and smears were made immediately. One set of smears was stained with Wright’s stain counterstained with Giemsa and another set of slides was stained with nitroprusside counter-stained with safranin.

Laboratory evaluation of patients took place prior to admission to the study and weekly thereafter. This evaluation included urinalysis, complete blood count with haematocrit, haemoglobin, white cell count, differential, erythrocyte sedimentation rate, reticulocyte count, platelet count, T, resin uptake and T, by column, fasting blood sugar, serum glutamic oxalate transaminase, glutamic pyruvate transaminase, bilirubin (direct and indirect), alkaline phosphatase, lactic dehydrogenase (LDH), uric acid, calcium, phosphorus, magnesium, iron, total iron binding capacity, sodium, potassium, chloride, creatinine, BUN, prothroinbin time, visual acuity and electrocardiogram. These tests were performed by standard methods. Guaiac examinations were performed on all stool samples.

Statistical comparisons were made using the Student’s t test.

RESULTS 2,3-DHB (25 mglkgld)

Patient acceptance with subjective response. The drug was well accepted by the five patients and none wished to stop taking the compound. The questionnaire elicited complaints related to the gastrointestinal system following drug administration on an empty stomach. The complaints included intestinal gas (four patients), increased bowel movement (two patients), diarrhoea (two patients) and nausea (two patients). No complaints were elicited when the drug was administered with meals. All of the patients felt that their skin colour was lighter following drug administration.

Clinical and laboratory evaluation. There were no detrimental changes noted in the physical examinations, vital signs or laboratory evaluations of the patients. Of note was the fact that the patients consistently lost about z kg throughout the study period despite a diet of 35 cal/kg. It was felt that this weight loss reflected an increased metabolic demand due to bone marrow hyperplasia rather than a drug effect since the weight loss continued through the second control period, yet was regained on returning to the home environment. Subsequent studies have revealed that these patients require about 40 callkg to maintain weight.

The only significant change in blood chemistry was a decrease in the serum LDH (P =

0.03). Whether this decrease represents a true decrease in inflammation or haemolysis due to the drug or a general response to hospitalization remains to be determined. That the patients were severely iron overloaded at the time of the study is indicated by their serum ferritin levels. The mean serum ferritin in patients was 10 746 pg/l. while the normal serum ferritin is 60 &I.

Balance studies. Iron balance studies before and during the period of drug administration are summarized in Table I. Administration of the 2,3-DHB as a single oral morning dose of 25 mg/kg yielded an average net drug-induced iron excretion of 4.5 mg/d (2.40-6.87) over

e, 0

TA

BL

S I. M

ean 8

d iro

n ex

cret

ion

(mg/

d) in p

atie

nts w

ith &

thal

assa

emia

m

ajor

Mea

n po

st-

trans

jiusio

n Hb (s

ldl)

Uri

ne

Stoo

l D

ieta

ry i

ron

(mg/

d)

Cont

rol

DH

B*

Cont

roI

Dm

1/20

15450

2/19

9300

3/26

9900

4/24

7380

5/25

11700

10.1

9.1

9.7

11.5

10.1

7.91

1.38

2.13

7.73

11.19

8.82

3.94

5.41

10.42

13.52

8.41

3.12

4.62

9.33

12.31

13.02

2.52

3.71

8.26

13.93

8.32

1.44

2.47

12.80

14.17

Drug

-indu

ced

Net

iron

exc

retio

n iro

n ((

Uri

ne+ S

tool

) - die

t) ex

creti

on

cont

rol)

(mg/

kg/d

) (

DS

.r,

Cont

rol

DII

B

(mgl

kgld

) is kr

1.20

5-41

3.21

c 4.57

9 5.

55

10.12

4-05

8.52

4.47

- 2.24

4.63

6.87

a 5-91

8.13

2.40

%

Mea

n 23

10746

10.1

9.29

2.48

3.67

2.89

7.40

4.50

9.71

13.02

sEh4

I

I363

0.4

0.94

0.49

0.62

0.90

0.56

1-53

1.03

P 0.16

0.01

0.04

* 2,3-Dihy&oxybenzoic a

cid;

25

mgl

kgld

.

TABLE

11. M

ean

iron

exc

retio

n (m

g/d)

of

patie

nts

with

fith

alas

saem

ia m

ajor

Iron

exc

retio

n (m

glkg

ld)

Net

iron

exc

retio

n ((

Uri

ne+

Stoo

l) -

diet

) Dr

ug-in

duce

d Se

rum

M

ean

post

- U

rine

Stoo

l (m

glkg

ld)

iron

excr

etion

Pt

lAge

fe

nitin

tra

nsfu

sion

Die

tary

iro

n (D

HB

conf

rol)

St

ool b

lood

(p

gll.)

H

b (g

/dZ)

(m

g/d)

Co

ntro

l D

HB

* Co

ntro

l D

HB

Co

ntro

l D

HB

(m

g/kg

/d)

loss

(mlld

)

A12

4 73

80

10.1

13.0

2 2.52

1.69

8.

26

14.18

-2

.24

2.82

5.06

3.

01

B/I

9 93

00

9.1

8.82

3.94

4-

17

10.4

2 20.82

5.55

16

.18

10.6

3 0.

66

c/20

12000

8.5

5.17

1.

89

4.57

8.

32

19.6

1 5-

03

24.0

5 19

.02

2.71

3.87

4.

13

Dl1

9 El

12

8520

9.

2 7.

92

0.87

1.

38

7.92

8.

84

0.87

2.

30

1.43

F

/n

85

20

9.

1 6.

12

0.43

0.

73

13.8

4 15

.23

8.15

9.85

1.

70

G/n

11

040

9.0

4.40

2.

77

4.76

9.

12

12.20

7.48

12

.56

5.08

H

I14

8640

9.

2 6.

27

0.54

1.

71

12.8

6 16

.94

7.13

12

.38

5.25

Mea

n 16

.5

1070

7 9.

3 7.

25

1.71

2-

55

9.51

14

.58

3.96

10.50

6.54

2.

13

P 0.1

8 0.0

2

0.0

5

2025

0 10

.3

6.3 I

0.

71

1.39

5.

33

8.80

-0

.26

SEh4

1.6

1462

0.

209

0.96

0.

45

0.58

0.

98

1.59

I .

40

2.65

0.

74

Not

e: C

ontr

ol p

erio

d =

8 d

; dru

g pe

riod

= 2

1 d

. * ~

,3-D

ihyd

roxy

benz

oic a

cid;

25

mg/

kg q

.i.d.

N

?

P 3

SL

Y g 3 f;'

P,

n

482 C. M. Peterson et a1

an 8 d period. Most of this loss appeared in the stool rather than the urine in contrast to the pattern seen in the iron-overloaded rat (Graziano et al , 1974). No changes were seen in either urinary or faecal Cu, Zn, Ca or K. Although this group of patients appeared to excrete increased amounts of Mg (121 k 26 to 272k 26 mg/d) in response to the drug, the serum Mg levels remained constant. At the higher dosage regimen Mg excretion remained constant. We are unable to explain this discrepancy. The increase in faecal iron excretion was highly significant (P<o.oI) as was the increment in net iron excrction (P<o.o4). Thcre was no change in serum iron or iron binding capacity.

2,3-DHB (25 q / k g 2.i.d.) Following the initial trials with 2,3-DHB, approval was obtained from thc FDA and the

appropriate hospital committees to evaluate the compound over a 21 d period at 25 mg/kg q.i.d. in patients 7 years of age and older. Previous experiments in animals demonstrated that administration of the drug q.i.d. led to iron excretion greater than that seen when the total daily amount was administered as a single dose (Graziano et a/ , 1974). In addition the com- pound appeared to be excreted by humans within approximately 6 h thereby providing a rationale for such a dosage regimen (Peterson et a!, 1974).

Patient acceptance arid sirbjectiue response. The drug was generally well tolerated by the eight patients although gastrointestinal complaints were noted if the drug was not taken with food. Two of the eight patients experienced transient nausea when they first began taking the capsules. Three noted transient stomach pain, five noted an increase in eructation and four noted an increase in intestinal gas. No patient requested that the drug be stopped, and most felt that their skin colour was lighter. Attempts to document a change in skin colour by comparison of photographs was not satisfactory.

Clinical arid laboratory eualrratiort. No deleterious changes werc noted in the physical examina- tions, vital signs or laboratory evaluations. In this group of patients the serum alkaline phosphatase decreased significantly (P< 0.02) whereas the LDH did not.

Balance sttidies. Iron balance studies before and during treatment with 2,3-DHB are sum- marized in Table 11. The administration of 2,3-DHB at a dose of 25 nig/kg q.i.d. led to an average net iron excretion of 6.54 mg/kg/d (1.43-19.02).

Although stool guaiac examinations were consistently negative, a more quantitative estimation of faecal blood loss was determined by 5 1 Cr-labelling of erythrocytes in three of the older patients and found to be within normal limits (Table 11). It is of interest to note that two of the younger patients excreted substantial amounts of iron. This result is encouraging since it suggests that the drug may be of use in younger children. More studies are required to determine the minimum age and degree of iron overload at which 2,3-DHB is effective. In the small number of patients studied, iron excretion could not be correlated with serum ferritin levels. Ca and Mg analyses of stool and urine were also performed in five of these patients. There was no significant difference in the excretion of these metals between control and drug dosage periods.

DISCUSSION

The results presented here suggest that 2,pDHB may be of use in the chronic treatment of

z,j-Dih yhoxy benxoic Acid and Thalassaemia 483 transfusion-induced haemosiderosis. The patients who participated in these studies were transfused at the rate of approximately I nil of blood/kg/d corresponding to an average iron load of approximately 20 mg/d. Chelation therapy employing 2,3-DHB led to a net excretion of iron in all patients although thc response of individuals varied significantly (1.4-19.0 mg/d). The reasons for this variation remain unclear. Whereas desferrioxamine appears to be less effective in younger patients who have received fewer transfusions, the effectiveness of q - D H B does not seem to be related solely to either of these factors. Two of the youngest patients responded better than several of the adults. Furthermore, a comparison of the results of the two protocols reveals that some patients who received q - D H B at a dose of 25 mg/kg once a day excreted more iron than others who received the same dose four times a day. Such incongruous results might well be expected considering the small number of individuals examined.

Several other aspects of the study deserve comment. Of primary importance is the fact that the drug proved to be both orally effective and non-toxic with only mild side effects which were alleviated when the drug was taken with meals. Moreover, the specificity of 2,3-DHB for iron was demonstrated. Nevertheless, one of the major goals of the programme was not attained. It was not possible to put any of the patients into iron balance such that excretion matched input. Therefore the use of q - D H B could be expected to retard, but not prevent, the accumulation of iron in these patients. In this respect its action is similar to that of desferrioxamine. A recent report suggests that the use of the latter compound over a 6 year period caused a reduction in the rate of iron accumulation (Barry et al, 1974) and Letsky et al(1g74) showed that the serum ferritin concentration can be used as an index of body iron load in this type of patient. From the evidence to date, it would appear that no single iron chelator will be sufficient to cause a net zero iron balance in patients who are chronically transfused. On the other hand, combination therapy utilizing a number of iron chelators may produce the desired result. Experiments in iron-overloaded rats indicate that the simul- taneous administration of 2,pDHB and desferrioxamine leads to a net excretion of iron which is the sum of that excreted in response to either drug alone (Graziano et al, 1974). Thus, tissue iron may be located in a number of pools not all of which are available to a given chelator. Recent evidence for specific chelatable pools of iron has been found in both animal models and tissue culture systems although the mechanisms whereby pool size is regulated remain unknown (Hershko et al, 1973 ; White et al, 1976).

While we were evaluating the results of the first protocol an interesting hypothesis came to light. The five patients who participated in the study felt that their skin colour was some- what lighter after taking 2,3-DHB. Since melanin production is stimulated by free radicals and iron is known to catalyse the formation of such radicals, it occurred to us that q - D H B might be trapping free radicals generated in vivo thereby reducing melanin production. This hypothesis was substantiated in vitro by the demonstration that q - D H B can prevent lipid peroxidation in hydrogen peroxide-stressed erythrocytes (Graziano et a!, 1976). Although it was not possible to document skin lightening in vivo through sequential photographs, it is hoped that the ability of ZJ-DHB to trap free radicals may be of significant pharmacological importance. The pathological changes seen in the tissues of iron-overloaded patients are believed to result from free-radical damage induced by iron. Among such changes are the accumulation of lipofuscin and increased utilization of vitamin E, a known scavenger of

484 C. M. Peterson et a1

free radicals (Hynian et al, 1974). The Gee radical scavenging property of ZJ-DHB may prove useful in retarding tissue damage due to iron.

Another finding that emerged from these studies was the fact that during the control period most patients were excreting more iron than was contained in their diet. Previous studies utilizing radiolabelled iron suggested that thalassaemic patients absorb significant amounts of dietary iron (Bannerman, 1962). The degree to which the patients in our pro- gramme are transfused may account for the observed results. The stimulus for ‘hyper- absorption’ may be reduced in that bone macrow hyperplasia is somewhat reduced. Of perhaps more significance is the fact that the haem content of the low-iron diets which these patients consumed was somewhat less than that of a normal diet. Since haem iron is absorbed more efficiently from food than similar quantities of inorganic iron (Walsh et a!, 195s; Turnbull et al , 1962), minimizing this factor may have unmasked a potentially useful state of iron flux in the chronically transfused patient. Although further studies are needed to clarify the potential role of the diet in the management of these patients, it is suggested that a strict vegetarian diet may significantly reduce the rate of iron deposition.

In summary, these studies indicate that a multifactorial therapeutic programme may be needed to deal with the problem of iron overload in chronically transfused patients. Currently no single chelator can remove sufficient quantities of iron from the body so as to maintain normal iron levels in these patients. There is some evidence that a combination of chelators may be more satisfactory, yet additional studies are needed to determine the optimum dosage of such drug combinations as well as the appropriate age at which to begin chelation therapy. Within the next few years it may be possible to design a maintenance programme based on chelating drugs and dietary restrictions which has the capability of maintaining heavily transfused patients in optimum iron balance.

ACKNOWLEDGMENTS

We would like to thank Dr E. Amorosi for patient referral and are indebted for the assistance of L. Meckl, J. Figueiredo, D. J. Lewis, R.N., J. Leong, E. McCracken, A. de Bari, Dr N. Luban, Dr A. J. Markenson and Dr A. de Ciutiis.

The funds for this work were provided by the National Foundation-March of Dimes. The American Hellenic Educational Progressive Association, Cooley’s Anemia Volunteers, Inc., National Institutes of Health (Contract AM 2202), The Children’s Blood Foundation and National Institutes of Health Clinical Research Center Grant (RR 00102).

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