......... 4.1.4 Mobilization of microfilariae by diethylcarbamazine

Page

. . .

3.2 Anti-inflammatory effect

. . .

3.2.1 Anaphylaxis

. . .

3.2.1.1 In calves

. . .

3.2.1.2 In rats and monkeys

. . .

3.2.1.3 Cutaneous anaphylaxis

. . .

3.2.2 Rat peritonea1 cells

. . .

3.2.3 Experimental eosinophilia

. . .

3.2.4 Pros taglandins

. . .

3.2.5 Effect on skin tests

. . .

3.2.6 Sumaery of anti-inflanrmatory action

. . .

3.3 Bronchial asthma

. . .

4. Antifilarial activity

. . .

4.1 Actiononmicrofilariae.

. . .

4.1.1 In vitro

4.1.2 In vivo

. . . . . .

4.1.3 Mode of action on microfilariae

. . .

4.1.3.1 Experimental evidence

. . .

4.1.3.2 Theoretical conclusions

. . .

4.1.4 Mobilization of microfilariae by diethylcarbamazine

. . .

4.1.4.1 W. bancrofti

. . .

4.1.4.2 0. volvulus

. . .

4.1.5 Other actions of diethylcarbamazine upon microfilariae 4.2 A c t i o n o n a d u l t w o r m s . , . . .

4.2.1 In vitro

. . .

4.2.2 In vivo

. . . . . .

4.2.3 Action of piperazine upon Ascaris

. . .

4.3 Action on forms in the insect vector

. . .

4.4 Action on infective larvae and immature worms

. . .

4.5 Development of drug resistance

. . .

4.6 Action on other worms

. . .

5. Toxicity

. . .

5.1 Animals.

5.2 Man

-

uninfected

. . .

5.3 Man

-

infected with filariae

. . . . . .

5.3.1 O . v o l v u l u s . . .

. . .

5.3.1.1 Clinical

5.3.1.2 Histology of diethylcarbamazine reaction in

onchocerciasis

. . .

5.3.2 Other filariae

. . .

5.3.3 Local reactions

. . . . . .

5.4 Deaths reported as due to diethylcarbamazine treatment

5.4.1 Discussion of evidence

. . .

5.4.2 Encephalitis

. . .

6. Clinical use

. . . . . .

6.1 Individual patients

6.1.1 Patients with W. bancrofti

. . . . . . . . . . . . . . . . . . . . . .

³⁰

6.1.2 Patients with B. malayi and L. loa

. . . . . . . . . . . . . ^{. . .} . ^.

³⁰

6.1.3 Patients with 0. volvulus

. . . . . . . . . . . . . . . . . . . . . . .

^{3 0}

6.1.3.1 Treatment of light infections

. . . . . . . . . . . . . . . .

³⁰

6.1.3.2 Treatment for ocular onchocerciasis

. . . . . . . . . . . . .

³¹

6.1.3.3 Local application for ocular onchocerciasis

. . . . . . . . .

³¹

6.2 Mass therapy

. . . . . . . . ^{. . . . . . . . . . . .} . ^{. . . . . . . . . . . .}

^{3 2}

6.2.1 Bancroftian and malayan filariasis

. . . . . . . . . . . . . . . . . ^.

³²

6.2.1.1 Mass treatment with diethylcarbamazine

. . . . . . . . . . .

³²

6.2.1.2 Review of nmss therapy

. . . . . . . . . . . . . . . . . . .

³³

6.2.2 Diethylcarbamazine in cooking salt

. . . . . . . . . . . . . . . .

³⁴

6.2.3 Mass therapy of other filarial infections

. . . ^.. . . . . . . . . . . .

³⁵

6 . 3 Treatment of tropical eosinophilia

. . . . . . . . . . . . . . . . . . . . ^{. .}

³⁵

7. Recommendations for research on diethylcarbamazine

. . . . . . . . . . . . . . . . .

³⁵

R I ~ ~ u u I ~ . . .

. . .

³⁷

References quoted in text

. ^. . . . . . . . . . . . . . . . . . . . . . . ^. . . . ^.

³⁹

References consulted but not quoted in text

. . . . . . . . . . . ^{. . . . . . . . . . . .}

⁵¹

Recommendations for research on diethylcarbamazine

1. Mode of action of DEC upon microfilariae 2. Mansonella ozzardi

3. Skin test for onchocerciasis with DEC 4. Radioactive diethylcarbamazine

5. Action of DEC on adult worms

6. Therapeutic so-called allergic reactions to DEC in patients with onchocerca or other filarial infections

7. Local application of DEC to the eyes 8. Methods of mass administration 9. Toxicity of arsenical compounds

INTRODUCTION

Difficulties encountered from toxic reactions during treatment of onchocerciasis patients with diethylcarbamazine and suramin

-

the only two drugs at present considered suitable for

treatment

-

necessitated a thorough search of the literature in an attempt to throw light on the pharmacodynamics and toxicity of these drugs, both of which are used experimentally in other fields of pharmacology, biochemistry, etc.

A literature search was done on Medline and with the assistance of Professor Lammler (suramin). It was stimulated as a result of recommendations of a Scientific Advisory Panel Working Group on the Chemotherapy of Onchocerciasis, Drug Efficacy and Toxicity (Geneva, 28-29 November 19741, and of the Scientific and Technical Advisory Committee of the Onchocer- ciasis Control Programme in the Volta River Basin (OCP) at its second and third sessions

(Geneva, 3-5 June 1975 and 3-4 March 1976), and was financed by OCP (UNDP Project No. R A F / ~ ~ / 004).

The literature revealed by the search was studied and retrieved by Dr F. Hawking in two papers, one dealing with diethylcarbamazine and the other with suramin. This led to recom- mendations for future investigations; all possible sources of information in the literature to date have now been tapped.

1.1 Chemical and physical propertiks Name

l-die thylcarbamyl-4-me thyl-piperazine Formula

Synonyms

Hetrazan, Banocide, Notezine, Caricide, Carbilazine, Supatonin, 3799 R.P.

Diethylcarbamazine was first issued as the chloride, but is now produced as the dihydrogen citrate which contains only half its weight as base. In reporting doses it is important to indicate whether the doses refer to a specific salt or to the base; unless otherwise stated, it can generally be assumed that the dose refers to the citrate. Diethylcarbamazine is a white powder, freely soluble in water with a slightly unpleasant sweetish taste. It is stable under all ordinary laboratory conditions and is fully stable to autoclaving, when mixed with diet.

1.2 Relation of structure to activity

The piperazine ring is of fundamental importance as was shown during the original investi- gations of Hewitt et al. (1948) although piperazine itself has no antifilarial activity.

Piperazine is, however, active against Ascaris and other round worms in the intestine, and its ring structure may have specific action on parasitic n e w o d e s in general. Various compounds with slightly different rings, e.g. N, N-diethyl-4-methyl-l, 4-diazacycloheptane-l-carbamaxide hydrochloride (JGS-110), have some antifilarial activity, but diethylcarbamazine remains the most effective known antifilarial agent (Reinertson & Thompson, 1955). In the piperazine series, a carbethoxy radical in position 1 of the ring, with various substitutions in position 4 , produced high antimicrofilarial activity; but as the alkyl chain was increased the

toxicity became greater and the activity less. If the alkyl group is greater than -C4Hg, activity is lost. The alkyl group in position 4 is not particularly significant, however, since high activity continues after it is removed. The only compounds lacking the carbethoxy group, which show marked activity against microfilariae, contain an ethyl, diisopropyl,

dimethyl, or diethyl carbamyl group in position 1. A compound with the formula:

is also active (patra et al., 1969).

Apparently for the above compounds to be effective there must be two aliphatic amines connected by a saturated carbon framework; one amine must be basic and the other must be modified by some type of carbonyl function. The spatial separation of these amines in diethylcarbamazine is optimal; compounds with wider separation are less active.

A modification of the diethylcarbamazine structure has been described by Saxena et al.

(1970) and studied further by Thompson et al. (1973) and by Sturm et al. (1974). It is named

"compound 11" for convenience, and will be described in more detail belcu. In this compound, the terminal carbon of one ethyl group is connected to the piperazine ring at the 2 carbon position as follows:

The essential structure is similar to that of diethylcarbamazine but the change makes the molecular structure rigid and locks the urea moiety into a single rotational conformation. In contrast, the diethylcarbamyl group of diethylcarbamazine can rotate on all its single bonds.

The new compound is approximately planar with the carbon-oxygen bond of the carbamyl and all the nitrogens lying in the same plane. There are 2.8~" between N1 and Nq of the piperazine ring and 3 ~ " between N4 and N2. Since the compound is as active or perhaps more active than diethyl- l carbamazine, this configuration is apparently optimum.

In addition to this modification, various linkages across the piperazine ring by -CH2

-

CH2

-

have been investigated by S t u m et al. (1974). Linkage between positions 3 and 5 on the piperazine ring does not diminish activity. Other linkages between positions 1 and 6 and 2 and 6 diminish activity moderately.

1.3 Analogues of diethylcarbamazine 1.3.1 Compound I1 (Centperazine)

- This campound was first reported by Saxena et al. (1970); a brief description as well as its chemical formula has been given above. Its action is almost identical to that of diethyl- carbamazine. When tested by Saxena et al. (1970) and by Thompson et al. (1973) in cotton rats and jirds infected with Litomosoides, the antifilarial rksponse was not closely propottional to the dose, a situation which made accurate conparison difficult. Saxena et al. claimed that it was five times more active than diethylcarbamazine and Thompson et al. agreed that it was as active or slightly more active than diethylcarbamazine upon the microfilariae. Like diethyl- carbamazine it had no action on the adult worms of Litomosoides. The acute toxicity in mice

was slightly less than diethylcarbamazine. This compound should be investigated further and tested in man against Wuchereria bancrofti and against Onchocerca volvulus. Although similar in behaviour to diethylcarbamazine in cotton rats and jirds, it is possible that in man it might present some advantages of greater activity on adult worms. It had no action against

Hymenolepis nana or Nippostrongylus or against Chandlerella hawkingi in crows. Like diethyl- carbamazine it showed anti-inflammatory action in rats and inhibited passive cutaneous

anaphylaxis (Saxena et al., 1970).

1.3.2 Hoechst 33258

2-fi

-

(4-hydroxylphenol)

-

⁶

-

benzimidazolyfl

-

^6- (1-methyl-4 piperazyl) benzimidazole

-

tri HC1.

H HOE 33258

This was described by Raether & LHmmler (1971). It is fluorescent. The hydrochloride is water soluble, but the diphosphate is sparingly soluble and is thus only slowly absorbed after

oral and parenteral administration. In cotton rats infected with Litomosoides its action resembled that of diethylcarbamazine but was slower, more complete and more prolonged. The phosphate, given as 8 mg/kg i.p. on five consecutive days, caused all the microfilariae to disappear with 14-67 days; only very few had returned after 111-297 days. In half of the rats the adult female worms were dead but, in the other half, females were alive and producing microfilariae.

The maximum tolerated dose of the hydrochloride for mice was 250 mg/kg i.p. or 625 mg/kg

S.C. The prolonged action is probably due to a depot effect since the compound remains in the tissues for a long period of time. When administered to mice, the nuclei of the liver and kidney still showed fluorescence after 37 days (Llmler & Schutze, 1969). The compound binds to DNA, but whether such binding has a positive or negative value seems to depend upon which nuclei of the host or of the parasite are preferentially involved.

It has been tested by Duke (personal communication) on chimpanzees infected with human 0. volvulus. One animal was given 5 mg/kg i.m. on five consecutive days. Four weeks later

the skin had been completely cleared of microfilariae, but 10 days after this the chimpanzee was found dead. There were large sterile abscesses at the sites of injection. Adult worms were alive and contained both live and moribund microfilariae. WO other chimpanzees were

treated with an improved pharmaceutical preparation, 4 ling/kg i.m. twice weekly for two to four weeks. This preparation did not cause abscesses. One lightly infected animal was completely cleared of microfilariae for over 10 months. In the other case, the microfilariae were reduced to near zero but after the sixth dose the animal became very weak, relapsed into coma and died.

Most of the adult worms were dead, but a few were still alive although the microfilariae in them were deformed and mostly immobile. These worms were probably moribund. It appears that the compound kills the microfilariae of 0. volvulus, and many of the adult worms, but it may be dangerously toxic. Unlessthe danger of toxicity canbe removed, further trials seem inadvisable.

A further modification of Compound E has been introduced by Friedheim (1974) in which two molecules of Hoechst 33258 are combined with a molecule of an arsenical compound (~151) closely

related to melarsoprol. When this was given intramuscularly to dogs infected with Dirofilaria immitis (dose, 3-41.5 mg/kg one to four doses at two- to four-day intervals), it killed the

m i c r o f i l a r i a e and t h e a d u l t w o r n . This combination was t e s t e d a g a i n s t 0 . v o l v u l u s by Duke ( p e r s o n a l c-unication). One i n f e c t e d chimpanzee was g i v e n f o u r i n t r a m u s c u l a r d o s e s of 6 mg/

k g a t t h r i e - t o fout-day i n t e r v a l s and a n o t h e r was g i v e n 3 mg/kg weekly. Both a n i m a l s showed a n o r e x i a , l a s s i t u d e and l o s s of weight. I n b o t h animal8 t h e m i c r o f i l a r i a e and t h e macro- f i l a r i a e appeared t o have been k i l l e d . One h e a v i l y i n f e c t e d man was t h e n g i v e n f o u r weekly d o s e s of 1 mg/kg. The t r e a t m e n t was w e l l t o l e r a t e d ; b u t t h e m i c r o f i l a r i a l count was l i t t l e a l t e r e d and t h r e e weeks a f t e r t h e l a s t dose a n e x c i s e d nodule showed h e a l t h y a d u l t worms. S i x weeks a f t e r t h e l a s t d o s e , t h e p a t i e n t developed a l a r g e v i t r e o u s haemorrhage i n h i s r i g h t e y e producing b l i n d n e s s i n t h a t eye. A f t e r some months, t h e haemorrhage c l e a r e d and a d e g r e e of u s e f u l v i s i o n was r e s t o r e d . Another p a t i e n t r e c e i v e d f o u r weekly d o s e s of 2 mg/kg and a f t e r t h e l a s t i n j e c t i o n s u f f e r e d a n o r e x i a , l a s s i t u d e and p a i n i n t h e knee j o i n t s . H i s m i c r o f i l a r i a l count f e l l d r a m a t i c a l l y t o 3% of i t s i n i t i a l v a l u e d u r i n g t h e f o u r weeks, and a n e x c i s e d nodule showed no l i v i n g worms. Approximately s i x weeks l a t e r , however, he d i e d suddenly of what

appeared t o be a d i a b e t i c coma. I n view of t h e s e two mishaps, f u r t h e r t r i a l s were d i s c o n t i n u e d . 1 . 3 . 3 Hoechs t 28637a

Cyclohexane c a r b o x y l i c acid-M-methyl p i p e r a z i n e c i t r a t e . I t i s c l o s e l y s i m i l a r i n s t r u c t u r e and a c t i v i t y t o d i e t h y l c a r b a m a z i n e when t e s t e d on L. c a r i n i i i n Mastomys n a t a l e n s i s , b u t i t s t o x i c i t y i s l w e r ( U m n l e r e t a l . , 1971).

1.3.4 Hoechst 29691a

Tetrahydropyrane c a r b o x y l i c acid-N-methylpiperazide c i t r a t e . This i s a n o t h e r compound c l o s e l y r e l a t e d t o d i e t h y l c a r b a m a z i n e , I t i s s l i g h t l y more a c t i v e t h a n d i e t h y l c a r b a m a z i n e a g a i n s t t h e m i c r o f i l a r i a e of L. c a r i n i i i n M. n a t a l e n s i s , and s l i g h t l y l e s s t o x i c (Mmmler e t a l . , 1971).

1.3.5 Hoechst 37598

This belongs t o t h e b i s benzimidazole s e r i e s and has a l o n g - a c t i n g r e p o s i t o r y e f f e c t . I t was given by Duke ( p e r s o n a l colmmunication) t o a chimpanzee i n f e c t e d w i t h 0. v o l v u l u s , a t a dose of 6 mg/kg i n t r a m u s c u l a r l y d a i l y f o r f i v e days. There was temporary d i m i n u t i o n of micro- f i l a r i a e b u t they b u i l t up a g a i n i n two months. The animal was then t r e a t e d w i t h 10 mg/kg twice weekly f o r s e v e n d o s e s b u t t h e r e waa no change i n t h e m i c r o f i l a r i a l count d u r i n g t h e n e x t 1 2 weeks. A f t e r 24 weeks of t r e a t m e n t , however, t h e r e were two l a r g e a b s c e s s e s a t t h e s i t e s of i n j e c t i o n .

1.4 Chemical e s t i m a t i o n o f d i e t h y l c a r b a m a z i n e

E s t i m a t i o n i n blood and u r i n e can be made by a c o l o r i m e t r i c method d e s c r i b e d by Lubran (1950). I n t h i s method t h e body f l u i d i s made a l k a l i n e and e x t r a c t e d w i t h e t h y l e n e d i c h l o r i d e . The a d d i t i o n of one d r o p of carbon d i s u l f i d e t o u r i n e heJps remove primary and secondary amines which o t h e r w i s e g i v e a h i g h e r b l a n k value. The e t h y l e n e d i c h l o r i d e i s t h e n shaken w i t h an aqueous s o l u t i o n of bromthymol b l u e , s o t h a t a c o l o u r e d s a l t of d i e t h y l c a r b a m a z i n e i s formed which p a s s e s i n t o t h e s o l v e n t , w h i l e t h e dye i t s e l f remains i n t h e aqueous phase. Plasma o r serum should

not

be d e p r o t e i n i z e d b e f o r e e x t r a c t i o n . Amounts can be measured a s low a s t h e normal b l a n k v a l u e which i s about 1 mcg/ml f o r serum, and 3-10 mcg/ml f o r u r i n e . A s i m i l a r

t e c h n i q u e h a s b e e n d e s c r i b e d by Hujimaki ( 1 9 5 8 ) . A m o d i f i c a t i o n u s i n g bromphenol b l u e h a s been d e s c r i b e d b y Rao & Subrahmanyam ( 1 9 7 0 ) . A n o t h e r method o f e s t i m a t i o n by p i c r i c a c i d h a s b e e n r e p o r t e d b y Ramachandran ( 1 9 7 3 ) . Methods f o r p h a r m a c e u t i c a l e s t i m a t i o n d e p e n d i n g upon r e i n e c - k a t e s h a v e b e e n d e s c r i b e d by Sheng e t a l . ( 1 9 6 3 ) and u s i n g b r o m o c r e s o l g r e e n b y V a d o d a r i a e t a l . ( 1 9 6 8 ) . When d i e t h y l c a r b a m a z i n e h a s b e e n a d d e d t o t h e food o f a n i m a l s , i t s c o n c e n t r a t i o n c a n be m e a s u r e d by c o l o r i m e t r y , o r by g a s - l i q u i d c h r o m a t o g r a p h y b u t n o t by p o l a r o g r a p h y ( A l l e n &

Beckman, 1 9 6 4 ) .

D i e t h y l c a r b a m a z i n e h a s a l s o b e e n e s t i m a t e d w i t h r a d i o a c t i v e t e c h n i q u e s by Bangham ( 1 9 5 5 a , b ) , u s i n g s a m p l e s l a b e l l e d w i t h c14 i n t h e p i p e r a z i n e r i n g o r i n t h e m e t h y l g r o u p , and by F a u l k n e r & S m i t h ( 1 9 7 2 ) .

2 . ABSORPTION, EXCRETION, DISTRIBUTION AND METABOLISM

2 . 1 A b s o r p t i o n , e x c r e t i o n and d i s t r i b u t i o n

When d i e t h y l c a r b a m a z i n e i s a d m i n i s t e r e d o r a l l y t o e i t h e r a n i m a l s o r man, i t i s r a p i d l y a b s o r b e d from t h e a l i m e n t a r y c a n a l . One d o s e o f 10 mg b a s e p e r kg p r o d u c e s a peak b l o o d

l e v e l o f 4-5 mcg/ml i n t h r e e h o u r s , t h e t i m e when t o x i c symptoms a r e most p r o m i n e n t . The l e v e l t h e n g r a d u a l l y f a l l s t o z e r o w i t h i n a b o u t 48 h o u r s . Most o f t h e e x c r e t i o n i n t h e u r i n e o c c u r s i n t h e f i r s t 24 h o u r s , d u r i n g w h i c h t i m e 10-26% o f t h e d o s e may be r e c o v e r e d a s d i e t h y l c a r b a m a z i n e . When 3 mg b a s e p e r kg i s t a k e n t w i c e d a i l y f o r f o u r - a n d - a - h a l f d a y s , t h e b l o o d c o n c e n t r a t i o n o f d i e t h y l c a r b a m a z i n e r e a c h e s 4-5 mcg/ml by t h e t h i r d d a y and t h e n f a l l s e v e n t h o u g h t h e d o s a g e i s c o n t i n u e d . D i e t h y l c a r b a m a z i n e a p p a r e n t l y p e n e t r a t e s ' r e a d i l y i n t o h y d r o c e l e f l u i d , a n d p r e s u m a b l y i n t o o t h e r body f l u i d s ( ~ a w k i n g , 1 9 5 0 ; L u b r a n , 1950).

The d i s t r i b u t i o n o f t r i t i u m - l a b e l l e d d i e t h y l c a f b a m a z i n e i n m i c e , a f t e r i n t r a p e r i t o n e a l i n j e c t i o n , h a s been s t u d i e d by a u t o r a d i o g r a p h y (Sakuma e t a l . , 1967). The compound was r a p i d l y d i s t r i b u t e d , and 20 m i n u t e s a f t e r i n j e c t i o n t h e r a d i o a c t i v i t y i n t h e l i v e r , k i d n e y , a d r e n a l g l a n d , m u s c l e and g a s t r o i n t e s t i n a l t r a c t r e a c h e d i t s h i g h e s t d e n s i t y ; by s i x h o u r s i t had d i m i n i s h e d g r e a t l y . I t a c c u m u l a t e d i n t h e b r a i n a t 20 m i n u t e s and d i m i n i s h e d a f t e r o n e h o u r . S p e c i a l l y h i g h a c c u m u l a t i o n s o c c u r r e d a f t e r o n e t o t h r e e h o u r s i n t h e s a l i v a r y g l a n d , a d r e n a l m e d u l l a , p u t u i t a r y g l a n d and lymph nodes. The compound a c c u m u l a t e d i n and was e x c r e t e d from t h e k i d n e y , from t h e g l a n d u l a r p o r t i o n o f t h e stomach w a l l , and from t h e l i v e r i n t o t h e b i l e .

The r e l a t i o n s b e t w e e n d o s a g e , b l o o d l e v e l s and m i c r o f i l a r i c i d a l e f f e c t h a v e been s t u d i e d i n p a t i e n t s i n f e c t e d w i t h W. b a n c r o f t i by H u j i m a k i (1958). T r e a t m e n t was g i v e n by him f o r 10-14 d a y s . W i t h d a i l y o r a l d o s e s o f 0.3-0.6 mg/kg c i t r a t e g i v e n i n t h r e e a d m i n i s t r a t i o n s , t h e m o r n i n g b l o o d l e v e l was l e s s t h a n 0.65 mcg/ml and t h e r e was n o t h e r a p e u t i c e f f e c t . W i t h d a i l y d o s e s o f 1 . 5 mg/kg, t h e b l o o d l e v e l was 0 . 5 - 1 . 1 mcg/ml i n t h e morning and o v e r

1 . 0 mcg/ml l a t e r i n t h e d a y , and m i c r o f i l a r i a e w e r e e v e n t u a l l y a l l e x t e r m i n a t e d . A f t e r a s i n g l e d a i l y d o s e , e . g . 6 mg/kg, t h e r e was a p e a k b l o o d l e v e l o f 3.0 mcg/ml, which f e l l r a p i d l y w i t h i n s e v e r a l h o u r s . On t h e o t h e r h a n d , i f t h e same amount was d i v i d e d i n t o t h r e e o r s i x d o s e s , t h e b l o o d l e v e l r e m a i n e d s t e a d y t h r o u g h o u t t h e day. The minimum e f f e c t i v e

c o n c e n t r a t i o n i n t h e b l o o d seemed t o b e 0.8-1.0 mcg/ml. F o r t h e t r e a t m e n t o f b a n c r o f t i a n f i l a r i a s i s , H u j i m a k i recommended a d a i l y d o s e o f 6 mg/kg d i v i d e d i n t o s i x a d m i n i s t r a t i o n s , f o r 1 4 d a y s . T h i s s h o u l d g i v e a b l o o d l e v e l o f 1 . 8 - 2 . 6 w i t h a n a v e r a g e o f 2 . 1 mcg/ml i n t h e m o r n i n g and p r o b a b l y a h i g h e r l e v e l d u r i n g t h e day.

2 . 2 M e t a b o l i s m

A more s p e c i f i c a n a l y s i s o f t h e m e t a b o l i s m and d i s t r i b u t i o n o f DEC i n r a t s and monkeys was made by Bangham ( 1 9 5 5 a , b ) , who worked w i t h t h e d r u g l a b e l l e d w i t h

c l 4

i n t h e p i p e r a z i n e r i n g . An o r a l d o s e i s r a p i d l y a b s o r b e d , and a b o u t 7 0 % . o f t h e p i p e r a z i n e m e t a b o l i t e s a r e e x c r e t e d i n t h e u r i n e w i t h i n 24 h o u r s . When a n i n t r a v e n o u s d o s e o f 2.25 mg/kg i s

a d m i n i s t e r e d , 95% i s e x c r e t e d w i t h i n 30 h o u r s ; 10-20% o f t h i s amount a p p e a r i n g a s unchanged d r u g . T h i s v a r i e s s l i g h t l y , however, d e p e n d i n g on t h e d o s e l e v e l g i v e n . M e t a b o l i s m i s v e r y r a p i d a n d t h e d r u g i s e x c r e t e d i n f o u r d i f f e r e n t f o r m s , i n a l l o f w h i c h t h e p i p e r a z i n e r i n g r e m a i n s i n t a c t . D i e t h y l c a r b a m y l p i p e r a z i n e a c c o u n t s f o r 5-15%, m e t h y l p i p e r a z i n e f o r 2-5%,

W H O / O N C H O / ~ ~ . 142 page 1 0

and p i p e r a z i n e f o r 1-6% o f t h e 95% e x c r e t e d . The f o u r t h form was a n u n s t a b l e b a s i c compound which was n o t i d e n t i f i e d by Bangham. T h i s m e t a b o l i t e c o u l d be d e m o n s t r a t e d i n t h e b l o o d w i t h i n two m i n u t e s o f an i n t r a v e n o u s i n j e c t i o n .

The metabolism h a s been f u r t h e r i n v e s t i g a t e d i n r a t s by F a u l k n e r & Smith (1972) u s i n g d i e t h y l c a r b a m a z i n e l a b e l l e d w i t h

14c

i n t h e 3.5 p o s i t i o n s o f t h e p i p e r a z i n e r i n g . They found t h a t t h e main m e t a b o l i t e , n o t i d e n t i f i e d by Bangham, was diethycarbamazine-N-oxide ( s e e Fig. 1 ) . A second m a j o r m e t a b o l i t e was l - e t h y l carbamyl-4-methyl p i p e r a z i n e a c c o u n t i n g f o r 23% of t h e compound e x c r e t e d i n t h e u r i n e , w h i l e unchanged d r u g a c c o u n t e d f o r 15% o f t h e u r i n a r y

r a d i o a c t i v i t y . Thus when 20 mg/kg were g i v e n by mouth t o r a t s , t h e r a d i o a c t i v i t y was e x c r e t e d i n t h e u r i n e a s f o l l o w s :

Unchanged d r u g 10-20% of d o s e Methyl p i p e r a z i n e 2-5%

P i p e r a z i n e 1- 6%

D i e t h y l c a r b a m y l p i p e r a z i n e

-

i . e . m e t h y l group s p l i t o f f 5-10%

l - e t h y l carbamyl-4-methyl p i p e r a z i n e

-

i . e . one e t h y l group s p l i t o f f 23%

Diethylcarbamazine-N-oxide 50%

( S e e Fig. 1 . )

Although metabolism i s r a p i d and e x t e n s i v e , no m e t a b o l i t e t h u s f a r i s o l a t e d a p p e a r s t o h a v e an e f f e c t s u f f i c i e n t l y profound t o a c c o u n t f o r t h e e x t r a o r d i n a r y s p e e d o f a c t i o n o b s e r v e d on t h e c i r c u l a t i n g m i c r o f i l a r i a e . A c c o r d i n g l y , t h i s a c t i o n i s p r o b a b l y due t o

d i e t h y l c a r b a m a z i n e i t s e l f . S t u d i e s o f t h e d i s t r i b u t i o n of t h e r a d i o a c t i v e p r e p a r a t i o n , show t h a t d i e t h y l c a r b a m a z i n e soon e q u i l i b r a t e s w i t h a l l o r g a n s , blood c e l l s and t i s s u e s ( e x c e p t f a t ) , and t h a t n e i t h e r t h e m i c r o f i l a r i a e n o r t h e a d u l t worms of D. i m i t i s c o n c e n t r a t e i t t o any pronounced e x t e n t a s compared w i t h t h e s u r r o u n d i n g t i s s u e s o r f l u i d s . N e a r l y a l l i s e x c r e t e d i n t h e u r i n e ; t h e f a e c e s c o n t a i n o n l y s m a l l amounts of p i p e r a z i n e m e t a b o l i t e s . 2.3 L o c a l a b s o r p t i o n i n t o e y e

When d i e t h y l c a r b a m a z i n e s o l u t i o n s a r e a p p l i e d l o c a l l y t o t h e e y e s o f r a b b i t s , t h e compound e n t e r s t h e aqueous humour i n h i g h c o n c e n t r a t i o n ; no t o x i c e f f e c t was d e t e r m i n e d a f t e r an e i g h t week t r i a l ( L a z a r e t a l . , 1968). The m a t t e r w i l l b e d i s c u s s e d f u r t h e r i n s e c t i o n 6.1.3. A g e n e r a l a c c o u n t of t h e p e n e t r a t i o n o f many t y p e s of d r u g s i n t o t h e e y e a f t e r l o c a l a p p l i c a t i o n i s g i v e n by Benson (1974).

3. PHARMACOLOGY

3 . 1 G e n e r a l

When examined by t h e t e c h n i q u e s of c l a s s i c a l pharmacology, d i e t h y l c a r b a m a z i n e i s o n l y s l i g h t l y a c t i v e . R e a c t i o n s v a r y somewhat w i t h d i f f e r e n t s p e c i e s a n d t h e y c a n n o t b e assumed t o b e n e c e s s a r i l y t h e same i n man.

3.1.1 Smooth m u s c l e

On r a b b i t i n t e s t i n e , 1 0 pg/ml p r o d u c e s r e l a x a t i o n . On t h e i s o l a t e d u t e r u s of r a b b i t and r a t t h e r e was no a c t i o n a t c o n c e n t r a t i o n s o f 1/100 000 (10 )Ig/ml), b u t w i t h v i r g i n g u i n e a - p i g u t e r u s t h i s c o n c e n t r a t i o n c a u s e d weak c o n t r a c t i o n s ( ~ a r n e d e t a l . , 1948). Subbu & Biswas

(1971) r e p o r t e d t h a t 20 pg/ml c a u s e d c o n t r a c t i o n o f t h e u t e r u s i n t h e g u i n e a - p i g and r a t and concluded t h a t d i e t h y l c a r b a m a z i n e might c a u s e a b o r t i o n i n women. T h i s h a s , however, n o t been r e p o r t e d c l i n i c a l l y i n s p i t e o f w i d e s p r e a d u s e of d i e t h y l c a r b a m a z i n e t h r o u g h o u t t h e world. The q u e s t i o n was e x t e n s i v e l y i n v e s t i g a t e d by F r a s e r (1972) who found t h a t l a r g e d o s e s (100-200 mg/kg) g i v e n by mouth d a i l y t o p r e g n a n t ra'ts and r a b b i t s had no a b o r t i f a c i e n t a c t i o n and no h a r m f u l e f f e c t on t h e f o e t u s e s . S a r e e n e t a l . (19611, w h i l e t e s t i n g many s u b s t a n c e s a s a n t i f e r t i l i t y a g e n t s , found t h a t d i e t h y l c a r b a m a z i n e g i v e n t o b r e e d i n g mice a t 200 mg/kg d a i l y f o r 10 d a y s , produced 50% i n f e r t i l i t y ; many o t h e r compounds, however, were more a c t i v e .

WHO/ONCHO/~~. 14 2 page 11

3.1.2 ~ l o o d p r e s s u r e

When i n j e c t e d i n t r a v e n o u s l y i n t o a n a e s t h e t i z e d dogs. 2.5- 10 mg/kg o f d i e t h y l c a r b a m a z i n e c a u s e s a r a p i d r i s e o f blood p r e s s u r e , followed a f t e r about one minute by a moderate f a l l . Both t h e s e r e a c t i o n s a r e blocked by hexamethonium, a s u b s t a n c e which b l o c k s a c t i o n on g a n g l i a . The a c t i o n o f d i e t h y l c a r b a m a z i n e i s s i m i l a r t o t h a t o f n i c o t i n e . A p p a r e n t l y d i e t h y l c a r b a m a z i n e s t i m u l a t e s sympathetic g a n g l i a c a u s i n g v a s o c o n s t r i c t i o n , c a u s e s t h e a d r e n a l medulla t o r e l e a s e a d r e n a l i n e , and s t i m u l a t e s t h e r e s p i r a t o r y chemoreceptors i n t h e a o r t i c and c a r o t i d b o d i e s ( F o r b e s , 1972). These e f f e c t s a r e o f no c l i n i c a l s i g n i f i c a n c e . I n r a t s and c a l v e s , t h e i n t r a v e n o u s i n j e c t i o n of 20 mg/kg of d i e t h y l c a r b a m a z i n e c a u s e s changes i n t h e a r t e r i a l blood p r e s s u r e s i m i l a r t o t h o s e produced by t h e i n j e c t i o n of h i s t a m i n e . I n r a t s , d i e t h y l ~ a ~ b a m a z i n e c a u s e s t h e r e l e a s e o f h i s t a m i n e from t h e l u n g s , probably from t h e mast c e l l s ( D e l i n e e t a l . ,

1973).

3.1.3 R e s p i r a t i o n

I n t r a v e n o u s i n j e c t i o n of d i e t h y l c a r b a m a z i n e i n t o a n a e s t h e t i z e d dogs produces d e e p e r and more r a p i d r e s p i r a t i o n . This may be due t o d i r e c t a c t i o n on t h e r e s p i r a t o r y c e n t r e and a l s o

t o a c t i o n on t h e chemoreceptors j u s t mentioned (Harned e t a l . , 1948; F o r b e s , 1972).

3.1.4 C e n t r a l nervous system

P a r e n t e r a l i n j e c t i o n of 25 mg/kg i n c a t s and dogs o f t e n c a u s e s v o m i t i n g , a p p a r e n t l y by s t i m u l a t i o n of t h e vomiting c e n t r e . O r a l a d m i n i s t r a t i o n t o dogs of 50 mg/kg a l s o c a u s e s v o m i t i n g , which i s most l i k e l y due t o d i r e c t a c t i o n on t h e stomach. L a r g e r doses may c a u s e s l e e p i n e s s (Harned e t a l . , 1948). Toxic d o s e s , e.g. 560 mg/kg a d m i n i s t e r e d o r a l l y t o mice, c a u s e c o n v u l s i o n s . This c o n v u l s i v e a c t i o n i s probably due t o s t i m u l a t i o n of t h e c e r e b r a l c o r t e x s i n c e t h e same c o n v u l s i o n s have been s e e n i n c a s e s of p i p e r a z i n e c i t r a t e p o i s o n i n g i n c h i l d r e n (Savage, 1967 ; M i l l e r 6 C a r p e n t e r , 1967 ; M a r t i n d a l e , 1967). These c h i l d r e n

s u f f e r e d , from headache, i n c o o r d i n a t i o n , myoclonic j e r k s and coma, but r e c o v e r e d w i t h i n 24 h o u r s . The dose r e q u i r e d f o r producing a n e f f e c t i s v e r y l a r g e (1.5 g d a i l y f o r two t o t h r e e days i n s m a l l c h i l d r e n ) ; such c o n v u l s i o n s have never been seen w i t h normal d o s e s of d i e t h y l c a r b a m a z i n e . N e v e r t h e l e s s , t h e headache, s l e e p i n e s s and v o m i t i n g , which may o c c u r a f t e r t h e a d m i n i s t r a t i o n of c l i n i c a l doses of d i e t h y l c a r b a m a z i n e , a r e p r o b a b l y due t o t h e a c t i o n of t h e compound on t h e c e n t r a l nervous system and on t h e stomach.

3.2 Anti-inflammatory e f f e c t

Diethylcarbamazine has an a n t i - i n f l a n m a t o r y o r b l o c k i n g a c t i o n i n many a n a p h y l a c t i c r e a c t i o n s . These r e a c t i o n s a r e v e r y complex and d u r i n g t h e l a s t 10 y e a r s t h e y have been s t u d i e d by many workers i n a g r e a t v a r i e t y o f animals and organs. The u s u a l p r o c e d u r e has been t o provoke some s y s t e m i c o r l o c a l i z e d a n a p h y l a c t i c r e a c t i o n and t o s e e whether t h e

r e a c t i o n i s i n h i b i t e d by a long s e r i e s of b l o c k i n g a g e n t s , of which d i e t h y l c a r b a m a z i n e i s one.

Thus d i e t h y l c a r b a m a z i n e h a s been used a s an i m p e r f e c t l y understood t o o l f o r e l u c i d a t i n g

r e a c t i o n s which a r e a l s o n o t y e t understood. Furthermore, t h e r e s u l t s o b t a i n e d by one worker i n one s e t of experimental c o n d i t i o n s do n o t a g r e e w i t h t h o s e o f o t h e r workers i n o t h e r

c o n d i t i o n s and r e s u l t s w i t h one s p e c i e s of animal o f t e n d i f f e r from t h o s e w i t h a n o t h e r s p e c i e s . Consequently, i t i s d i f f i c u l t t o g i v e a c o h e r e n t account of t h e s e a c t i o n s of d i e t h y l c a r b a m a z i n e o r t o r e l a t e them t o a l o g i c a l system o f u n d e r l y i n g p h y s i o l o g i c a l e v e n t s .

During a n a p h y l a x i s and o t h e r r e a c t i o n s i n s e n s i t i z e d t i s s u e s , a n t i b o d i e s (mainly IgE) a d h e r e t o s i t e s on t i s s u e mast c e l l s , b a s o p h i l l e u c o c y t e s and s i m i l a r c e l l s and t h u s s e n s i t i z e them t o t h e a n t i g e n . When an a n t i g e n comes i n t o c o n t a c t and r e a c t s w i t h them, t h e s e c e l l s r e l e a s e v a r i o u s pharmacological a g e n t s c a l l e d m e d i a t o r s , e.g. h i s t a m i n e , s e r o t o n i n , slow

r e a c t i n g s u b s t a n c e o f a n a p h y l a x i s (sRS-A), p r o s t a g l a n d i n b , k i n i n s and o t h e r s y e t unknown. The r e l e a s e of t h e s e m e d i a t o r s from t h e c e l l s r e q u i r e s enzymes, c a l c i u m i o n s , and p r o b a b l y a l s o t h e breakdown of c y c l i c adenosine monophosphate. When t h e m e d i a t o r s have been r e l e a s e d , they s t i m u l a t e smooth muscles ( a r t e r i o l e s o r b r o n c h i o l e s ) t o c o n t r a c t , c a p i l l a r i e s t o d i l a t e and t o become more permeable, and l e u c o c y t e s of v a r i o u s k i n d s t o congregate. Thus t h e phenomena o f a n a p h y l a x i s and/or o f l o c a l i z e d inflammation a r e produced. These a n a p h y l a c t i c r e a c t i o n s may

WHO/ONCHO/~~. 142 page 12

be partially inhibited by a great variety of blocking compounds which can act in two ways.

They may directly antagonize the pharmacological reaction of histamine or serotonin or one of the other active substances, perhaps by blocking the receptor sites on the muscle or other cells sensitive to the substances. This is called direct neutralization of histamine etc.

Alternatively, they may act on the cells, e.g. mast cells, which release histamine or other substances, inhibiting one or more of the several stages which lead to such release, so that one or more of the mediators are not liberated.

It is believed that diethylcarbamazine usually does not neutralize histamine or serotonin directly, but that its main action is to block the release of SRS-A, an unsaturated hydroxy acid with a low molecular weight of around 400, and with biological activity at one nanogram or less. Sometimes, however, the action of diethylcarbamazine seems to be non-specific and in some circumstances it may antagonize the effects of histamine and serotonin directly

(Burka & Eyre, 1974a). Furthermore, diethylcarbamazine must normally be supplied in extremely high concentrations to produce its effects. Thus during in vitro experiments, concentrations of 0.4 or 4.0 mg/ml have been employed (Burka & Eyre, 1974b) and for in vivo experiments on anaphylaxis in calves, intravenous doses of 20 mg/kg have been supplemented by constant infusion to maintain the blood level. These concentrations are much higher than those of other inhibitory compounds and much higher than would be obtained by therapeutic doses in man.

Other important inhibitory compounds with which diethylcarbamazine is compared are as follows.

(a) Cromoglycate (FPL 670, INTAL) is a complex molecule.

It is believed to inhibit the release of histamine from cells, especially mast cells, started by reagin type antibodies (Cox, 1967). Possibly it stabilizes the membranes of the mast cells.

(b) Meclofenamate (Winder et al.

,

1965) is an "anti-inf lanrmatory agent"

CooH C1 CH?

C 1

which inhibits the action of SRS-A and antigenic bronchospasm of guinea-pigs. It also inhibits prostaglandin synthesis.

(c) Methysergide is an antagonist for serotonin (5-hydroxytryptamine, 5 HT).

(d) Isoproterenol (~soprenaline)

CHOH. CH2 NH CH (CH3)

is a sympathomimetic amine which acts almost exclusively,on beta receptors. It inhibits the in vitro allergic response in which antigen acts on sensitized leucocytes to cause a release of histamine (Lichtenstein & De Bernardo, 1971).

These compounds have no structural similarity to diethylcarbamazine.

The evidence for the above account is derived from numerous experiments by many workers;

the results and the interpretations are therefore sometimes controversial. As described

WHO/ONCHO/~ _8.142 page 1 3

above, t h e u s u a l p r o c e d u r e i s t o provoke some k i n d of a n t i g e n - a n t i b o d y r e a c t i o n which may be g e n e r a l i z e d o r l o c a l , i n v i v o o r i n v i t r o . An a t t e m p t i s t h e n made t o a n a l y s e t h i s by s t u d y i n g t h e i n h i b i t o r y e f f e c t of t h e v a r i o u s b l o c k i n g a g e n t s mentioned above. S i n c e t h e r e a c t i o n s a r e m a n i f o l d , h i g h l y complex, and s t i l l i m p e r f e c t l y u n d e r s t o o d , i t i s i m p o s s i b l e a t t h i s s t a g e t o p r e s e n t a c l e a r , c o n s i s t e n t p i c t u r e ; t h e main p i e c e s of e x p e r i m e n t a l evidence a r e , however, e x p l a i n e d below.

3.2.1 Anaphylaxis

3.2.1.1 I n c a l v e s

Calves a r e s e n s i t i z e d by i n j e c t i o n s of h o r s e serum a f t e r which a n a p h y l a c t i c shock i s produced by i n t r a v e n o u s i n j e c t i o n of a d d i t i o n a l h o r s e serum. I n c a l v e s t h e main r e a c t i o n

t a k e s p l a c e i n t h e l u n g s , where both t h e a r t e r i o l e s and t h e b r o n c h i o l e s a r e s t i m u l a t e d t o c o n t r a c t . A s describe'd above, t h e combination of a n t i g e n w i t h a n t i b o d y s t i m u l a t e s t h e r e l e a s e of h i s t a m i n e , s e r o t o n i n and SRS-A (Eyre, 1971). Eyre e t a l . (1973) s t u d i e d t h e a g e n t s which, when a d m i n i s t e r e d p r e v i o u s l y , i n h i b i t e d t h i s a n a p h y l a c t i c shock i n t h e i n t a c t animal. They found t h a t t h e most e f f e c t i v e i n h i b i t i o n was given by meclofenamate a t 1.3 mg/kg, o r by a combination of cromoglycate and d i e t h y l c a r b a m a z i n e a t 2 0 mg/kg. T h i s combination i n h i b i t e d almost a l l t h e symptoms of a n a p h y l a x i s such a s c o n t r a c t i o n of b r o n c h i o l e s and pulmonary a r t e r i o l e s , haemoconcentration, and hyperkalaemia, but d i d n o t i n h i b i t t h e leucopenia.

Diethylcarbamazine a l o n e produced 50% i n h i b i t i o n of t h e changes i n s y s t e m i c a r t e r i a l blood p r e s s u r e , b u t i t d i d n o t i n h i b i t changes i n pulmonary a r t e r i a l blood p r e s s u r e o r i n r e s p i r a t i o n

(Burka & Eyre, 1974a). Cromoglycate a l o n e was i n a c t i v e . I t i s concluded t h a t i n c a l v e s , a n a p h y l a c t i c shock i s due mainly t o t h e r e l e a s e of SRS-A, and t h a t d i e t h y l c a r b a m a z i n e blocks t h e r e l e a s e of SRS-A o r a n t a g o n i z e s i t s a c t i o n (Wells e t a l . , 1973; Eyre e t a l . , 1973;

Wray & Tomlinson, 1974; Burka & Eyre, 1974a). It might a l s o block t h e r e l e a s e of h i s t a m i n e , s e r o t o n i n o r p r o s t a g l a n d i n s .

I f s t r i p s of pulmonary v e i n from s e n s i t i z e d c a l v e s a r e s t u d i e d i n v i t r o , they can be seen t o c o n t r a c t when brought i n t o c o n t a c t w i t h a n t i g e n s . This r e a c t i o n i s i n h i b i t e d by a n t i - h i s t a m i n e s and by m e t h y s e r g i d e ( a n t i s e r o t o n i n ) . It i s a l s o 50% i n h i b i t e d by d i e t h y l c a r b a m a z i n e o r by cromoglycate and i s t o t a l l y i n h i b i t e d by t h e two combined (Eyre, 1971). It can be

concluded from t h i s t h a t t h e r e a c t i o n of s e n s i t i z e d c a l f pulmonary v e i n i s a complex one of h i s t a m i n e , s e r o t o n i n , SRS-A, p r o s t a g l a n d i n s and p o s s i b l y o t h e r a g e n t s .

3.. 2.1.2 I n r a t s and monkeys

I n r a t s which were f i r s t s e n s i t i z e d by c h i c k ovalbumin, and t h e n shocked w i t h albumin a d m i n i s t e r e d i n t r a v e n o u s l y , d i e t h y l c a r b a m a z i n e i n doses of up t o 40 mg/kg d i d n o t modify t h e c a r d i o v a s c u l a r c o l l a p s e caused by t h e shock. One can t h e r e f o r e conclude t h a t i n r a t s a s opposed t o c a l v e s t h e shock i s

not

due t o t h e l i b e r a t i o n of SRS-A (Lecomte & Salmon, 1972).

When s e r o t o n i n o r h i s t a m i n e was p e r f u s e d through r a t l u n g s , spasmogens (e.g. p r o s t a g l a n d i n s , SRS e t c . ) were r e l e a s e d , b u t t h i s r e l e a s e was blocked by d i e t h y l c a r b a m a z i n e a t 1 mg/ml o r by indomethacin a t 10 pg/ml. There i s a marked d i f f e r e n c e i n lungs among d i f f e r e n t s p e c i e s of animals (Bakhle & Smith, 1972).

I f s e n s i t i z e d monkey lung i s exposed t o a n t i g e n i n v i t r o , d i e t h y l c a r b a m a z i n e i n h i b i t s t h e r e l e a s e of SRS-A and a l s o of h i s t a m i n e ( I s h i z a k a e t a l . , 1971). I n t h i s r e a c t i o n i t i s s y n e r g i c w i t h i s o p r o t e r e n o l .

3.2.1.3 Cutaneous a n a p h y l a x i s

Many s t u d i e s have been made on cutaneous a n a p h y l a q i s i n r a t s and o t h e r animals. The r e s u l t s v a r y depending on t h e t e c h n i q u e by which t h e r e a c t i o n i s provoked and a l s o on t h e s p e c i e s of animal used. A c t i v e cutaneous a n a p h y l a x i s i s produced by immunizing t h e r a t w i t h a n t i g e n (albumin) and l a t e r i n j e c t i n g t h e same a n t i g e n i n t r a d e r m a l l y . P a s s i v e cutaneous a n a p h y l a x i s i s produced by i n t r a d e r m a l i n j e c t i o n of a n t i s e r u m from an immunized animal and by i n j e c t i n g t h e a n t i g e n i n t r a v e n o u s l y 48 hours o r more afterward. Both of t h e s e r e a c t i o n s a r e

WHO/ONCHO/~~. 142 page 14

inhibited by diethylcarbamazine and by antihistamines, but not by colchicine (Harada et al., 1971). Passive cutaneous anaphylaxis in calves was inhibited by diethylcarbamazine at 20 mg/kg; this inhibition was increased by cromoglycate (Eyre, 1971; Wells & Eyre, 1972).

Harada et al. (1971) found that diethylcarbamazine at 250 mg/kg i.p., also inhibited the reactions to intradermal injections of histamine or serotonin in rats, i.e. in this experimental model, diethylcarbamazine neutralized histamine and serotonin directly.

Pelczarska (1974), however, did not find this effect. In mice, passive cutaneous anaphylaxis was not prevented by diethylcarbamazine or cromoglycate, but specific antagonists for serotonin or histamine did prevent it (Casey & Tokuda, 1973).

3.2.2 Rat peritonea1 cells

When peritoneal cells from sensitized rats are stimulated in vitro by antigen, SRS-A is released from the polymorphs; this release is inhibited by diethylcarbamazine. By contrast, histamine is released from mast cells and the release caused by antigen is

not

inhibited by diethylcarbamazine (Orange et al., 1968). On the other hand, if the basophil cells are stimulated by concanavalin A, so that they release histamine, this histamine release

2

inhibited by diethylcarbamazine (Siraganian & Siraganian, 1974). As a further complication, if the rat peritoneal cells are stimulated by corticotrophin or compound 48/80 to release histamine from the mast cell granules, this release is inhibited by diethylcarbamazine citrate, which is used by most experimenters, but not by diethylcarbamazine HCL. Thus, some of the observed effects may be due to citrate rather than to diethylcarbamazine base (R~egg & Jaques, 1974).

3.2.3 Experimental eosinophilia

Repeated injection of Trichinella antigen into the footpads of guinea-pigs provokes

eosinophilia in the corresponding lymph nodes. This effect is inhibited by diethylcarbamazine, provided it is administered 5-180 minutes before the injection of antigen (Thevathasan & Litt, 1971). Probably this is again due to the inhibition of the release of SRS-A. Diethylcarba- mazine does not modify the eosinophilia occurring during anaphylaxis in rats or in guinea-pigs or in rats by repeated injections of histamine or 48/80. It does, however, prevent the rise of eosinophils which takes place between three and seven days after feeding guinea-pigs with ova or larvae of Ascaris. Perhaps this action may be due to the removal of the parasites

(Sanyal, 1961; Sanyal & Sinha, 1962). Diethylcarbamazine is effective in the treatment of tropical eosinophilia; this is discussed in section 6.3.

3.2.4 Prostaglandins

Other substances which are formed and released locally by enzymes during inflammation include prostaglandins, which are highly active unsaturated fatty acids with a 20 carbon atom structure and many different pharmacological reactions which vary depending on the particular

4

compound (Leopold, 1974). Their stimulatory or relaxant actions on ox pulmonary vein strips in vitro may be antagonized by diethylcarbamazine in high concentrations (0.4 and 4 mg/ml) and also, more powerfully, by phloretin or by SC-19220. This action of diethylcarbamazine seems to be non-specific (Burka & Eyre, 1974b).

3.2.5 Effect on skin tests

If patients were given diethylcarbamazine at doses of 1.5 g/day for five days and then skin tests were performed by intradermal injection of antigen from infected larvae of W. bancrofti, the resulting reaction was much diminished or even suppressed (Katiyar et al.,

1974). This is probably another example of the anti-inflammatory reaction described above.

3.2.6 Summary of anti-inflammatory action

The liberation of pharmacologically active agents such as histamine and SRS-A, during inflammation and during antigen-antibody reactions is still poorly understood, and it differs in different experimental models and in different species of animals. Diethylcarbamazine is only one of a dozen compounds which interfere in these series of reactions, and there is no clear chemical similarity among them. It seems clear that diethylcarbamazine has a general tendency to inhibit the release of SRS-A, and sometimes other agents, which takes place

WHO/ONCHO/~~. 142 page l5

following antigen-antibody reaction or in inflamnation; this may explain the palliative action in bronchial asthma. Unfortunately, the exact details differ greatly in different

experimental circumstances. Usually the concentration required in vitro of up to l mg/ml, is much higher than that of other inhibitors. It is not clear whether this anti-infla~nnatory

reaction is always due to the same basic mechanism, or whether multiple mechanisms are involved. Similarly, it is not clear whether this action is due to the same structural configuration as the antifilarial action, or whether piperazine might have a somewhat similar action; or even whether the action sometimes depends on the citrate part of the molecule

(RUegg & Jaques, 1974).

The chief clinical effect of diethylcarbamazine in onchocerciasis is to cause inflanmatory reactions in the skin, presumably due to release of microfilarial antigens in sensitized

tissues; this contrasts sharply with the anti-inflamnatory actions reported above. As is described below in section 4.1, the action of diethylcarbamazine upon microfilariae seems to be to cause them to release or to expose antigens inside a sensitized host. The relation between this action and the anti-inflamnatory action reported above is not clear. Perhaps the two . reactions are unrelated.

3.3 Bronchial asthma

Bronchial asthma is a complicated anaphylactic-like response in which some antigen-antibody reaction takes place releasing active substances, including SRS-A, which then provoke the

characteristic symptoms. Probably there are multiple causes and reactions (Orange, 1973).

In 1965, Salazar Ma1l& reported that diethylcarbamazine at daily doses of 10 mg/kg was effective in the treatment of asthma. This was confirmed by many workers (e.g. Srinivas 6 Antani, 1971; Sly, 1974; Thiruvengadam et al., 1974). Relief of subjective symptoms was usually greater than objective change in vital measurements. On the other hand, Benner &

Lowell (1970), and Koivikko & Rantanen (1971) found no significant improvement in their

patients. Good reports tend to come from geographical areas where human parasites are comnon;

this may explain the differences reported (Koivikko, 1973).

4. ANTIFILARIAL ACTIVITY

The action of diethylcarbamazine on microfilariae and on adult worms must be described separately. Moreover, the effectiveness differs according to the different species of worms involved.

4.1 Action on microfilariae 4.1.1 Invitro

In contrast to its powerful action in vivo, diethylcarbamazine has no action in vitro upon either microfilariae or adult worms, and they can survive in relatively high concentrations of it for several days. Furthermore, serum from animals treated with diethylcarbamazine is not microfilaricidal in vitro, i.e. there is no conversion into an active metabolite (Hawking et al.,

1950; Kobayashi et al., 1969).

Cavier et al. (1971) have stated that diethylcarbamazine at a concentration of 1/1000 at 2 8 " ~ immobilized the microfilariae of Dipetalonema viteae in 24 hours. The pH was not controlled, however, and in any case, diethylcarbamazine does not act upon the microfilariae of D. viteae in vivo. Gonzalez Barranco et al. (1962) reported that diethylcarbamazine at concentrations ranging from 1/100 000 to 1/1000 at room temperature killed 82% of the micro- filariae of Onchocerca in one day. This work probably has little relevance to what happens in the body, however, since 50% of the controls were killed, pH was not controlled, and there was no correlation between the concentration of the drug and the percentage killed.

Natarajan et al. (1973b) state that the microfilariae of Bru ia ser enti from the slow loris, are immobilized after 12 hours by concentrations of 1t-601/ 28 or 37'~. In

their experiments, the pH was stabilized at 7.2 and non-specific substances of similar structure viz. sodium benzoate, and methyl-piperazine HC1 had no effect. All the same, the concentra-

-

tions which they used are far higher than those reached in vivo, and the entire course of the reaction is so different in vivo that the weak in vitro action is probably not significant.

WHO/ONCHO/~~. 142 page 16

Hewitt et al. (1947) studied microfilariae in the frog filaria Folyella dolichoptera placed in solutions of diethylcarbamazine HC1 at concentrations of from 1/100 to 1/10 000 which may have been acidic. These microfilariae are very large and have' a narrow' whiplike

anterior end and a thicker posterior one. Immediately upon contact with the drug, the anterior end contracted into a tight coil and violent jerky movements occurred. Then the microfilariae straightened out and became motionless within 5-15 minutes. This behaviour probably has nothing to do with the way diethylcarbamazine kills microfilariae in vivo, but it may be a manifestation of its action upon acetylcholine and cholinesterases in the microf ilariae.

4.1.2 Invivo

When diethylcarbamazine is administered to infected animals or humans by any of the usual routes, microfilariae rapidly disappear from the circulation. Single intraperitoneal doses of 50 mg of diethylcarbamazine citrate per kg given to cotton rats, usually cause a marked diminution in the microfilarial count which passes off after four to eight days. Daily doses as low as 1-5 mg/kg given for six days, exert a recognizable action. Large doses of up to

1100 mg/kg in nine hours, do not remove all microfilariae from the blood. After these treat- ments, numerous living microfilariae can be found post mortem in the pleural cavities of cotton rats infested with L. carinii (Hawking et al., 1950).

If diethylcarbamazine at 60 mg/kg is injected intravenously for rapid absorption into cotton rats, 80% of the microfilariae disappear in one minute, which is an astonishingly rapid disappearance; the same occurs with W. bancrofti in man. By contrast, however, a few micro- filariae often persist for days in cotton rats or in man, even if large doses of diethylcarba- mazine have been given (Hawking et al., 1950). In cats infected with B. malayi and B. pahangi and treated with doses of diethylcarbamazine which kill all the adult worms (as shown by

autopsy), some microfilariae still persist in the blood (Edeson & Laing, 1959). The reason for this persistence has not been elucidated. It may be that persistent microfilariae are biologically different from the others, either new born or very old, or that there is some reservoir outside the circulation from which the microfilariae continually enter the blood.

The latter is a probable explanation, since diethylcarbamazine does

not

affect microfilariae found outside the circulation, e.g. those of L. carinii in the pleural cavity of a cotton rat, those of W. bancrofti in a hydrocele, or those of 0. volvulus in a fibrous nodule (Hawking, 1950, 1952). Another plausible exp'lanation is that the microfilariae which persist are in a different immunological state from the others. As regards the sensitivity of the different species, diethylcarbamazine is effective against most species of microfilariae including those of 0. volvulus and Dipetalonema streptocerca when they are in the skin.

There is surprisingly little information about the microfilariae of Manzonella ozzardi.

Mazzotti (1948) reported that diethylcarbamazine had no effect against these microfilariae in Mexico. Montestruc et al. (1950) treated five patients in Martinique with 400 mg of

diethylcarbamazine daily for 10 days and stated that the microfilariae disappeared from the blood in a few days. Botero et al. (1965) refer to one patient who was treated previously by Restrepo et al. (1962) without any effect on the microfilariae. Dr F. Biagi in a personal co~unication states that Mazzotti made extensive trials in Yucatan and found that 10 mg/kg of diethylcarbamazine per day was ineffective, but that 30 mg/kg per day for 15 days easily achieved total cure. This treatment was used successfully as mass chemotherapy in heavily infected areas of Yucatan, but unfortunately Mazzotti died before writing a final report on the subject. See also Biagi (1973).

Diethylcarbamazine is less effective, or even inactive, against the microfilariae of Dipetalonema perstans (Hawking, 19501, of D. viteae of jirds, and of Edesonfilaria malayensis from Thailand. It has little action upon the microfilariae of D. repens (Singh, 1962); two dogs were treated with 3.8 and 5.6 mg/kg, respectively, daily for five days, but there was no reduction of the microfilarial count; the microfilariae also developed normally in mosquitos which were allowed to feed during this treatment. The compound acts on the microfilariae of Icosiella neglecta of frogs (Minning & Ding, 19511, and upon those of Setaria equina in horses (Lapeyra & Zabala, 1970) and of S. cervi transplanted into rats or dogs (Singhal et al., 1972a, b).

WHO/ONCH~/~~. 142 page 17

4.1.3 Mode of action on microfilariae 4.1.3.1 Experimental evidence

When a search is made for the microfilariae of L. carinii which disappear from the circulation of cotton rats after treatment with diethylcarbamazine, they are often found in the liver, and to a lesser extent in the spleen and bone marrow. Within an hour after administration of the drug, phagocytes congregate around trapped microfilariae, and within 18 hours most of them have been destroyed (Hawking et al., 1950). This trapping of micro- filariae has been studied in the living liver by Taylor (19601, and by Schardein et al. (1968) with the electron microscope. Before the abinistration of diethylcarbamazine, the micro-

filariae could be seen freely circulating through the liver capillaries. Within five minutes of intravenous injection of diethylcarbamazine, most of the microfilariae had become adherent to the walls of the capillaries, usually by their tails. They remained like this, wriggling, for 4-60 minutes. Sometimes a leucocyte became attached to the tail of a stationary micro- filariae; occasionally the microfilariae became so numerous that they blocked the capiblary.

In the electron microscope studies, before the administration of diethylcarbamazine, the liver contained few microfilariae; those present were normal and enclosed in a sheath. Twenty minutes after the drug had been administered, there were many microfilariae in the liver.

Some were free in the sinusoids; an attachment by the tail would not, however, be seen under the electron microscope. They appeared similar to untreated microfilariae except that no sheath was visible. The microfilariae were surrounded by a clear space. There were occasional microfilariae inside hepatocytes which otherwise showed no localized cellular reaction or destruction. The microfilariae were partitioned from the cytoplasm by a clear space. Four hours after the administration of diethylcarbamazine, the microfilariae were less numerous in the liver, and many of those in the sinusoids were undergoing lysis. The adjacent Kupffer cells and hepatocytes contained many lysosomes and there were foci of inflammatory cells, mostly polymorphs, around the microfilariae which were undergoing lysis.

The collection and destruction of the microfilariae in the liver is probably a function of the reticuloendothelial system, which is concentrated in the liver of rodents more than in other organs. It seems to depend upon the large, fixed macrophage of the tissues.

Destruction of the microfilariae of Loa loa has also been shown to occur in the liver of man (.Woodruff, 1951). In a drill infected with L. loa which was treated with diethylcarbama-

zine and killed six hours later, many microfilariae were found to be undergoing destruction by the reticuloendothelial cells of the liver, but few or none were being destroyed in the spleen (Duke, 1960), although in the spontaneous immune reaction of the drill against L. loa, most of the microfilariae are destroyed in special nodules in the spleen, and none are

destroyed in the liver. The part played by the bone marrow has not been properly investigated.

Presumably, the microfilariae of

W.

bancrofti are destroyed in the same way as those of L. loa and Litomosoides carinii.

According to Kobayashi et al. (1969), who studied L. carinii in cotton rats, diethyl- carbamazine is not effective in removing microfilariae from the blood unless antibodies are present. Thus, if microfilariae or adult worms are transplanted into a clean host which is then treated with diethylcarbamazine, the microfilaricidal action of the drug is or nearly so absent. If microfilariae are soaked in diethylcarbamazine at 1500 yg/ml in vitro, and then transfused into a clean host, they are not destroyed. When cotton rats containing inoculated microfilariae were passively immunized by injecting infected cotton rat serum, diethylcarbamazine had an immediate but transient effect in reducing the number of micro- filariae in the blood. Kobayashi's conclusion is disputed by Zahner et al. (1977), but the tables given by Kobayashi et al. (1969) seem convincing.

4.1.3.2 Theoretical conclusions

Since the action of diethylcarbamazine depends on a specific chemical configuration, it would seem that there must be an attachment to the microfilaria. No such fixation of the

compound on microfilariae has yet been demonstrated, but it would be desirable to reinvestigate the subject with a radioactive, labelled compound. On the basis of experimental evidence it appears that, after fixation, diethylcarbamazine modifies the microfilariae in the following two ways, which may be independent of each other.

WHO/ONCHO/~~. 142 page 18

1. Effect on neuro-muscular system. Microfilariae are in a constant state of

muscular activity (wriggling); waves of contraction pass down them from head to tail producing forward movement, and less frequently from tail to head producing backward movement. By this means, microfilariae can move towards more favourable locations, or back away from unfavourable ones. Most important in the case of W. bancrofti, Loa loa and other periodic microfilariae in the blood, these reverse movements enable the microfilariae to hold themselves in the small arterioles of the lungs (Hawking, 1967). Microfilariae contain acetylcholine

ellan an by,

1955) and cholinesterase (Bueding, 1952) which doubtless play a part in their neuro-muscular activity. Diethylcarbamazine potentiates the action of acetylcholine in causing the

contraction of nerve-muscle preparations of Ascaris, such potentiation being shown in dilutions as high as those of eserine, i.e. about 50 n m w a t 2 7 " ~ (Natarajan et al., 1973a). Such interference with the acetylcholine mechanism might well disturb the normal waves of contraction in microfilariae perhaps blocking the power to reverse. Clinically this would explain why:

(a) The liberation of microfilariae of W. bancrofti from the lungs into the blood during the daytime is caused by provocative doses of diethylcarbamazine (see below). (b) One of the first reactions of the microfilariae of Onchocerca to the compound is that many of them pass from the dermis to the epidermis which is an unfavourable environment in which they are not normally found. (c) Similarly, the number of Onchocerca microfilariae in the urine, blood and sputum is increased. (d) After administration of diethylcarbamazine, some micro- filariae of Litomosoides have penetrated into the hepatocytes of the liver, where they are never normally found (Schardein et al., 1968). Admittedly no obvious effect of diethyl- carbamazine upon the mobility of microfilariae in vitro has yet been reported, but the usual technique of inspection under a coverslip would not reveal the more subtle changes of reversal of waves of contraction. This should be studied by cinematographic technique with the micro- filariae compressed on an agar pad (Hawking & Clark, 1967).

This action of diethylcarbamazine in deranging the muscular activity of microfilariae .ight depend upon the piperazine ring part of the molecule rather than upon the microfilaricidal

tructure (see section 4.2.3). It would be interesting to investigate whether piperazine alone can liberate microfilariae into the blood stream. In any case this deranging action, although interesting, may not contribute much to the ultimate destruction of the microfilariae (see also 4.1.4).

Apparently organophosphorus compounds, particularly haloxon, have a microfilaricidal action very similar to that of diethylcarbamazine, i.e. when tested on L. carinii in Mastomys they cause a rapid reduction in the number of microfilariae in the blood, which does not last more than three days. Reduction by 86% takes one hour after an oral dose of haloxon at 100 mg/kg, or 10 minutes af ter an oral dose of diethylcarbamazine at 500 mg/kg (Lllmmler &

GrUner, 1975). Organophosphorus compounds are well known to inhibit cholinesterases of worms and of mammals, but such inhibition has not been demonstrated for diethylcarbamazine (except as discussed above. It would be interesting to investigate this similarity of action further with particular reference to the following questions.

(a) Does haloxon cause microfilariae of

L.

carinii to be accumulated and destroyed in the liver, as diethylcarbamazine does?

(b) Does the microfilaricidal action of haloxon require the presence of antibodies in the host?

(c) Does haloxon or diethylcarbamazine alter the waves of contraction/relaxation which pass up and down microfilariae? This should be examined by cinematography on an agar pad as mentioned previously.

2. Effect on the surface layers of microfilariae. As described above, after diethylcarbamazine is given in vivo, microfilariae are seized by phagocytes and destroyed, but the finer mechanism of this reaction is not yet completely understood. Within five minutes of injecting diethylcarbamazine, microfilariae of L. carinii begin to adhere by their tails to the walls of liver sinusoids (Taylor, 1960). According to electron microscopy, the micro- filariae of L. carinii had lost their sheath in less than 20 minutes (Schardein et al., 1968).

In the case of the microfilariae of Onchocerca which do not have a sheath but which have a five layer cuticle, Rougemont et al. (1974) reported that when examined after 12 hours, the cuticle