Studies on the Mode of Action of Psoralen Derivatives

Studies on the Mode of Action of Psoralen Derivatives

STUDIES ON THE MODE OF ACTION OF PSORALEN DERIVATIVES I. THEIR EFFECT ON COPPER AND GLUTATHIONE LEVELS IN BLOOD AND LIVER* ANWAR EL-MOFTY, M.B., C.H...

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STUDIES ON THE MODE OF ACTION OF PSORALEN DERIVATIVES I. THEIR EFFECT ON COPPER AND GLUTATHIONE LEVELS IN BLOOD AND LIVER*

ANWAR EL-MOFTY, M.B., C.H.B., M.D., ABDEL MONEM EL-MOFTY, M.B., C.H.B., D.V.D. (lioNs.), M.D., HASSAN ABDELAL, M.B., C.H.B., D.V.D. AND M. F. S. EL-HAWARY, B.Sc. (lioNs.) CAIRo, Pn.D. Psoralens were first introduced into medical use glutathione). They found that treatment with as a treatment for vitiligo 1948 as ammoidin psoralen compounds caused definite rise in serum

(8-methoxypsoralens) and ammidin (5-isoamyl- copper levels of both leukodermic patients and ene oxypsoralen) which were separated from the normal controls. It was therefore thought of interest to explore the extract of fruits of an Egyptian herb Ammi &tajus Linn (1). The efficiency of this group of in detail the action of psoralens on copper metabodrugs promoting melanogenesis has been es- lism and glutathione levels in rats in an attempt tablished by many workers in this field (2—7).

From clinical observations A.M.El-Mofty (8)

(in press) reported that the supplementation of copper as copper sulfate—given orally—to the treatment of vitiligo with psoralen compounds

to determine the mode of action of the psoralens on pigmentation. Analysis of blood and liver for their copper and glutathione contents in experimental animals receiving the psoralen derivates was done and compared with control groups. We

showed great improvement in repigmentation tried to compare the effect of pure crystalline and raised the percentage of improvement and psoralen, ammoidin (8-methoxy psoralen) and complete cure to 47%. ammidin (5-isoamylene oxypsoralen) to study It is well established that under normal con- the effect of each drug separately since the drug ditions the process of melanin formation goes used in treatment was usually a mixture of on steadily as a result of the enzymatic activity psoralen compounds. of tyrosinase, a copper metalo enzyme. In 1951 EXPERIMENTAL Lerner et al. (9) reported that copper essential In this work albino rats weighing from 200 gm. for tyrosinase activity in mammalian tissues inwere used. Ammoidin (8-methoxypsoralen) and cluding human epidermis, and more recently in 1958 Breathnach (10) found that the addition of dilute copper sulphate solution to the tyrosinase solution leads to a speeding up of the action in

the melanocytes of both freckles and pale epi-

dermis. On the other hand it was found that

ammidin (5-isoamylene oxypsoralen) were kindly supplied by Memphis Chemical Company as pure chemical materials. Doses of 800 mgm./Kg. body weight of either drug were used in the brief experiments whereas doses of 300 mgm./Kg. body weight were used in

many organic sulfur compounds such as cysteine the longer experiments. The doses were given and glutathione are active inhibitors of enzymatic orally mixed with food as an emulsion, forced-fed melanin formation in vivoas well as in vitro (11, 9). with a polythene tube attached to a syringe. The

It is thus evident that all factors leading to disturbance in melanin formation could be traced

to an upset of a final balance between copper tyrosinase and the inhibiting-SH group.

A study was carried out by A.M.El-Mofty, Fawzy, El-Hawary (12) to investigate the biochemical effects of psoralen therapy on the two important factors controlling tyrosinase activity, namely, the concentration and availability of copper and—SH group (represented mainly as * From the Departments of Medicine and Dermatology, Cairo University, Cairo, Egypt, and

experimental period for the short experiments was 24—48 hours and for the longer experiments it was 16 days.

The life span of albino rat is assumed to be 3 years. This may be considered as the equivalant of 90 years of human life (19). Sixteen days in

the life span of albino rats can be considered equal to 16 months of human life.

At the end of the experimental period the animals were sacrificed by decapitation. The blood

was collected from the neck; and the animal was immediately dissected and tissue (liver) needed for experimentation was quickly removed,

the Serum and Vaccine Institute, Biochemistry washed in physiological saline, blotted against filter paper to remove any extra fluid, then weighed

Research Unit, Agouza, Cairo. Received for publication September 19, 1958.

and kept in the deep freeze until it was analyzed. 645

646

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TABLE I Tissue copper and glutathione contents in normal rats Results are expressed as mg. glutathione/100

ammoidin in doses of 800 mg/Kg. body weight causes a dramatic increase in blood copper, a

ml and as microgram Cu/l00 ml in blood and

microgram %.

/100 gm. fresh weight in case of tissues.

decrease in the liver copper content from a normal

Meao Value

Tissue 2

4

3

normal mean value of 131 to mean value of 325

This was

associated with a marked

mean value of 113 to 37 mierogram/100 gm.

Rat No. 1

These figures show that administration of

6

5

SE.

fresh tissue weight.

The results are graphically represented in Fig. 1.

Blood

140

122

138

126

133

127

131

Cu Liver

131

104

110

115

121

109

115

Cu Blood

Regarding the effect of brief experimentation with ammoidin on tissue glutathione, the figures

show a marked decrease in liver glutathione content from a normal mean value of 94 to 48

21.2 22.2 24.6 24.3 23.5 26.8 23.8

G.S.H. Liver 109 G.S.H.

mg./100 gm. tissue fresh weight;whereas in blood,

79.6 83.7 96

98.3 87.4 94

the decrease was not great (only from a normal mean value of 23.8 to a mean value of 20.4 mg. glutathione/100 ml. blood). Yet in some rats (No. 1, 2, 6) the decrease in the levels of blood gluta-

The method for estimation of blood glutathione, worked out by Thompson and Watson in 1952 (13), was adopted in this work. Tissue glutathione was estimated by the method of El-Hawary (14).

The method of Gubler, G. J. et at. (15) for the

estimation of serum copper was followed.

thione is significant.

The results obtained from blood and liver copper and glutathione content in rats receiving

doses of 800 mgm. ammidin/Kg. body weight in the short experiments are given in Table III.

These figures show that the administration of

ammidin in the previously mentioned doses has

For the estimation of tissue copper content the no appreciable effect on copper and glutathione same procedure was applied using tissues homocontent of blood and liver. genates prepared as follows: It was thought that the results obtained in About one gm. of the liver was homogenized the short experiment may not represent the and made up to a volume of 4.5 ml. Three ml. of this homogenate were transfered to a conical cen- changes occurring in treating the patients with trifuge tube (capacity 15 ml.). To it was added 3 theraputie doses of psoralen compounds. For ml. of 2N HC1. The contents were mixed well and this reason we carried out some chronic experileft to stand for 10 minutes. Then 3 ml. of 20% ments using sublethal doses of ammoidin, 300 trichioro acetic acid solution were added, the con- mgm./Kg. body weight daily for 16 days. The tents again mixed well and left to stand for 10 results of these prolonged experiments are repreminutes. The tubes were then centrifuged for 30 sented in Table J1/ minutes at 300 r.p.m. Two and a half microgram of the supernatant-fluid were used for the deterTABLE II mination of copper content following the same Liver Blood procedure of Cubler et at. (15). Rat No. RESULTS

The results obtained for the different rats are

1

G.S.H.

Cu

G.S.H.

15.2



82

35.4

46

Cu

2

19.8

294 245

Table I shows the values obtained for tissue

3

20.2

403

53.2

37.4

copper and glutathione contents in rats receiving no drugs.

4

26.4

217

37.1

36.2

5

20.2

392

48.6

74.7

The results obtained for blood and tissue

6

16.8

395

64.8

66.2

glutathione and copper content in rats receiving doses of 800 mg. ammoidin/Kg. body weight in the short experiments are given in Table II.

7

24.0 20.4



53.5



325

48.8

37.1

noted in the following tables.

Mean S .E.

647

MODE OF ACTION OF PSORALEN DERIVATIVES Liver Copper

360

Blood Copper

340 325

320

After Ammoidin

300 280 260 240

220 200 180 160

140 120

Normal

100

80

60 40 20

Fio. 1. Effect of Ammoidin on copper levels. Rat TABLE IV

TABLE III

Liver

Blood

The Effect of Ajumidin on

No. of Rats No. of Rats

G.S.H.

1

2 3

4 5 6 Mean S.E.

Liver

Blood

18.8 21.2 21.2 20.3 18.4 20.5 19.7

Cu

— 112

112 108 115 113 112

G.S.E.

72.4 68.2 74.6 73.4 71.2 74.6 72.4

Cu

110 121 101 103

116 115

G.S.H.

Cu

G.S.H.

1

26

433

107

2 3 4

29.2 24.3 30.8 25.2

315 344 275 357 325

120 120

78

115

86 59

106

57 62

341

100

84.5

5 6

Mean S.E.

29.6 27.4



94

Cu

111

is again a huge rise in blood coppr from a normal

From the data given in Table IV it is clear mean value of 131 to a mean value of 341 microthat prolonged doses of ammoidin (8-methoxy- gram%, and this was associated with a decrease psoralen) have almost the same effect as the in the level of liver copper from normal value of doses of the shorter ammoidin treatment. There 115 to 84.5 mg./100 gm. tissue fresh weight. The

648

360

THE TOURNAL OF INVEBTIGATIVE DERMATOLOGY Blood Copper

Liver Copper

341

340

After Ammoidin

320 300

280 260 240 220 200 180 160 140 131

120

Normal

Normal

100

80 60 40 20 0 FIG. 2

latter decrease is less dramatic than the decrease amylene oxypsoralen) is completely inert and occurring in the short ammoidin course of treat- has no effect at all on copper or glutathione levels in blood or liver. ment. On the other hand, the effect of prolonged With regard to the effect on —811 group, it doses of ammoidin on blood and tissue glutathi- appears that the blood changes are not striking. one is less than the corresponding effect in If the changes are compared to changes in blood the shorter course dosage. copper one is tempted to relate the therapeutic The effect of ammoidin in prolonged doses on value to its effect on blood copper rather than its tissue and blood copper and glutathione is effect on —Sil group. graphically represented in Fig. 2. The explanation of hepatic depletion of —511 group under the effect of psoralens is not clear. DISCUSSION

We are tempted to suggest that the cause is The findings show beyond any reasonable disruption of glutathione rather than its mobi-

doubt that ammoidin (8-methoxy psoralen) is a strong hyper-cupremic agent and that it leads lization from liver since no striking elevation of to elevation of blood copper through mobilization —SU level in the blood was obtained. The doses used in both experiments are far above maximum of liver stores of copper. This was demonstrated in both the short and therapeutic effective doses. This was intended to the prolonged experiments. Ammidin (5-iso- accentuate the changes for purposes of demon-

RODE OF ACTION OF PSORALEN DERIVATIVES

649

stration. The theraputic doses in man (12) lead by Memphis Chemical Co., Cairo, Egypt to whom to definite rise in blood copper levels. The mechanism by which psoralens mobilize

copper from liver to blood was in urgent need of investigation not only to elucidate the mode of action, but also to exclude the possibility that copper is mobilized as a result of a direct toxic effect on the liver by the drug. The results are going to be reported in the following paper. But

it suffices here to report that the drug loses completely its mobilizing effect on copper (from

liver to blood) in hypophysectomized animals. This excludes completely a direct toxic effect on the liver, causing cellular disruption and mobilization and escape of copper to blood. Keeping in mind the importance of copper and copper tyrosinase in melanogenesis, the elevation

of blood copper under ammoidin might throw light on its mode of action and its therapeutic

we are grateful.

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Linn, Pharmacognostical study of Isolation

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SUMMARY AND CONCLUSION

1. Ammoidin (8-methoxy psoralen) causes marked rise of blood copper levels and drop of copper contents of liver in rats in both short and prolonged experiments.

2. Ammidin (5-isoamylene

oxypsoralen)

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Under publication.

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