To aid development of more efTective second generation analogues of m-AMSA,

possible quantitative molecular structure-biological activity relationships (QSAR)

have been investigated employing multiple regression analysis and a modification of

the model pioneered by Hansch (1971). As DNA is the purported site-of-action of

m-AMSA and its analogues (Waring , 1976), direct measures of agent-DNA

association constants have been employed in regression equations rather than

attempting more indirect modell ing of drug-s ite interactions by use of extrathermodynamic parameters (Han sch, 1971).

The most prominent route of in vivo degradation of m-AMSA is a nonenzyrriically mediated react ion with thiols (Scheme I) (Wilson, Cain & Baguley,

1977). To examine if ease ofthiolytic clea vage influences ana!ogu e potency, the halflives ofthe agents, in the presence of excess 2-mercaptoethanol , have been measured

and employed as input in regression ana lyses.

AMINOACRIDINE STR UCTURE - ANTITUMOUR CORRELATES 437

Methods

Details ofthe synthesis a nd characterisation ofthe agents con sidered (Table I) have

been published (Denny, AtwelI & Cain, 1979; and references quoted therein).

CH 30DNHS02lCH 21nCH)

NH ,

'<:::: 2

R

Figure 1 Scheme 1: Generic fonnula for m-AMSA analogues examined and the nature ofthe

thiolytic cleavagereaction.

Ll210 Screening

The requirements for reproducibility in Ll 210 assays have been detailed and the

methods employed for maintenance of an d th e perform an ce of tests with the Ll2l0

leukaemia ha ve been described in fulI (Cai n & AtwelI, 1976). Briefl y, an imals were

implanted with lOs Ll210 celIs i.p. on day O. Drug treatment was i.p., once daily, on

days 1-5 and animal deaths were monitored at twelve hour intervaIs. A dose-response

profile ofantileukaemic act ivit y, measured as perc entage increase in mean lifespan of

tre ated an imals (T ), in relation to that of contro ls (C) [100(T-C)/C] , was accumulated

employing doses separated by 0.09 log do se intervals. In determination of the LDIO

the doses employed were separated by 0.05 log do se intervals and animals observed

for a 50 da y period. The LD IO was derived by line ar correlation of per cent probit

mortalities and the logarithms of the correspondi ng drug doses employed , as before

(Denny & Ca in , 197 8). Signifi cant percentage life extensions in leu kaemia Ll 210

assays, obtained at and below the LD IO dose, were linearly correlated with the

logarithms of the corresponding doses employed . The do se in moles/kilogram body

weight providing a 40 % increase in lifespan (D40) was derived from th is regression

line .

Table 2 Square correlation matrix forthe independent variables employed

Rd Rm logK logT lf2

Rd I 0.64 0.01 0.03

Rm I 0.06 0.07

logK I 0.28

logTlf2 I

Table 3 Stepwisedevelopment of multivariable equations for log(I/D40)

Intercept

-1.94

-2.94

(}~K

1.20

1.28

Rd

1.71

r

0.62

0.78

s

0.52

0.42

Fl.(x/

36.6(60)

32.3(59)

a FI.(x) is the F statistic for introduction of each single variable, with the degreesof freedom(x)

in parentheses.

Table 1 Physicochemical and biological prope rties of m-AMSA analogues .j::. \,;.)

00

Form ula J log (JID40)d

Entry No. n

R Rma LogKb T'/,(min)" Obs. Calc. Diff.

2 0

H 0.18 5.57 13.2 5.29 4.94 4).35

3 I H 0.38 5.57 13.4 5.27 4.74 4).53

4 2 H 0.53 5.48 13.4 4.82 4.51 4).31

5 3

H 0.62 5.66 13.6 4.49 4.33 4).16

6 4

H 0.68 5.43 13.5 4.48 4.20 4).28 :"I

7 5

H 0.75 5.46 13.9 4.51 4.03 4).48 !)

> 8 6 H 0.80 5.59 13.7 4.23 3.90 4).33 7-

9 7

H 0.83 5.56 14.0 4.12 3.81 4).31

10 0 3- NHCOCHJ 0.07 6.07 23.6 5.98 5.62 4).36 :;

I1 1 3- NHCOCHJ 0.30 6.07 23.9 5.69 5.48 4).21 I:l:l

12 2 3- NHCOCHJ 0.48 6.05 23.5 5.37 5.24 4).13

>Cl

13 3 3-NHCOCHJ 0.56 6.09 23.8 4.93 5.09 0.16 c

14 4 3-NHCOCHJ 0.62 6.04 24.1 4.63 4.97 0.34 r[Tl

15 5 3-NHCOCHJ 0.70 6.07 23.9 4.57 4.79 0.32

<

16 6 3- NHCOCHJ 0.75 6.11 23.7 4.58 4.67 0.09 $E

17 0 2-NHl -0.15 5.95 233 5.87 5.44 -0.43

>

18 0 2-CHJ 0.40 5.35 32.9 3.69 4.68 0.99 CI tT1

19 0 2-CH(CHJ)l 0.66 5.39 49.7 3.58 4.01 0.43 z

20 0 2-F 0.32 5.28 10.3 4.73 4.44 4).29 z-<

21 0 2-1 0.36 5.70 6.32 3.67 4.93 1.26 Ro

22 0 3-N= -0.14 5.09 2.01 4.33 4.34 0.01 Cl

23 0 3-NHl 0.06 6.21 1284 5.52 5.80 0.28 c24 0 3-NHCOOCHJ 0.25 6.37 35.1 5.48 5.91 0.43

>-l

25 0 3-NHCHJ 0.17 6.17 1956 6.38 5.71 4).67

26 0 3-NOl 0.10 5.65 1.9 5.38 5.08 4).30 tT1

r

27 0 3-CHJ 0.44 5.95 32.6 5.52 5.14 4).38 r28 0 3-CHlCH J 0.56 5.66 29 4.77 4.57 4).20

29 0 3-CH(CHJ)l 0.68 5.46 28.3 3.40 4.06 0.66

30 0 3-üCHJ 0.29 5.83 51.3 5.66 5.18 -0.48

31 0 3-F 0.31 5.54 6.34 5.07 4.79 4).28

32 0 3-C1 0.32 6.06 4.6 5.42 5.44 0.02

33 0 3-Br 0.34 6.29 4.4 5.36 5.71 0.35

34 0 3-1 0.41 6.35 4.5 5.56 5.70 0.14

35 0 3-e

N 0.06 5.65 1.83 4.26 5.09 0.83

36 0 3-eONH2 "'{).4 1 5.66 3.57 4.83 4.82 0.01

37 0 3-eF1 0.54 5.24 2.26 4.17 4.07 ...{).IO

38 0 4-N= ...{).07 5.27 1.12 4.4 1 4.60 0.19

39 0 4-OCH1 0.19 5.94 13.4 5.34 5.40 0.06

40 0 4-OCH2CH1 0.43 5.77 13.1 4.80 4.93 0. 13

4 1 0 4-OCH CH 2 2OH 0.10 5.74 14.8 5.4 1 5.19 ...{).22 ;l>

42 0 4-OCH2CH(OH)CH OH 2 ...{).09 5.90 11.4 5.60 5.40 ...{).20 3::

43 0 4-OCH2CONHCH1 ...{).02 5.80 10.8 5.51 5.28 "'{).23

Z

44 0 4-O(CH2hCONCH2 ...{).15 5.74 10.9 5.26 5.17 ...{).09 0;l>

45 0 4-eON

H2 ...{).27 5.47 2.52 4.65 4.74 0.09

n

;:c

46 0 4-e

ONHC

H1 0.06 5.54 3.01 5.23 4.94 "'{).29 Cl

47 0 4-eON(CH1h 0.09 5.04 3.29 4.74 3.42 -1.32

Z

48 0 4-eONcC.

HgO' ...{).18 4.96 3.55 3.91 4. 15 0.24 rntIl

49 0 4-eONH(C H2hCH1 0.47 5.40 2.89 4.38 4.39 0.01 """

50 0 4-eONH(CH2hOH ...{).16 5.42 3.29 5.11 4.75 0.36 ;:c

c

51 0 4-eONCH(CH2)lOH ...{).15 5.40 3.37 4.84 4.73 0.11

n

""" 52 0 4-eONHCH2CHOHCHl 0.09 5.34 2.94 5.53 4.68 ...{).85 c

53 0 4-e

ONHC

H2CHOHCH2OH ...{).36 5.26 2.84 4.52 4.37 ...{).15 ;:crn

54 0 4-eONHCH2CON H2 ...{).50 5.39 3.9 1 4.65 4.72 0.07 I;l> 55 0 4-eHl 0.25 6.03 11.10 5.74 5.47 ...{).27 z

56 0 4-{CH2)CONH2 ...{).17 5.65 8.44 5.56 5.04 "'{).52 =1""" 57 0 4-e6Hs 0.45 5.60 3.20 3.92 4.68 0.76 c

58 0 4-F 0.21 5.65 4.28 5.10 5.02 ...{).08 3::

59 0 4-e1 0.23 5.76 2.42 4.43 5.14 -+D.71 0

c

60 0 4-Br 0.25 5.57 2.36 4.50 4.88 0.38 ;:c

61 0 4-eN ...{).03 5.01 3.78 4.08 4.27 0. 19

n

0

62 0 4-N0 2 0.09 5.20 2.81 3.90 4.50 0.60 ;:c;:c

rn

r

;l>

a Rm value, relative measure ofagent lipophilic - hydrop hilic balance, see text. rn""" b The logarithm ofthe agent- DNA association constant (K) for poly d(A-T) measured by the ethidium displacement techniq ue. tIl

c Half-life ofreac tion oft he agent in the presence of2-mercaptoethanol , see text.

d D.o is the dose in mol kg-I providing 40% increase in mean lifespa n in Ll210 leukaemia assays in mice under standard conditions. Observed (Obs.)

are the values measu red. (Calc.), those calculated from the regression equation derived. The differences between Obs. and Calc . figures are provided

in the last column.

e NcC.HsO is an abbreviation for th e morpholide. v.> '00

440 B. F. CA IN, B. C. BAG ULEY. W. A. DE NNY & G . J. ATWELL

Agent ph ysiochemical properties

Rm values [= log (I/Rr- I)] from partition chromatography have been shown to be of

equivalent utility, in multiple regression analyses, to measured log P values obtained

with the N-octanol-water system (Hanseh, 1971). Rm values were determined in a

reversed phase, thin layer chro matographic system employing Merck Fm cellu lose

DC cards (Denny & Cain, 1978). For aseries of sta nda rd compounds, Rm values

measured in this system and log P (N-octanol- water) values were related by the

equation: log P = 2.00 (± 0.I 5) Rm + 0.51 (±0.1O)

n= 21, r=0.99 , s = 0.2 1, FI.19=678

(Denny, At well & Cain , 1979). In th is equation, n is the number of data points; r is

the correlation coefficient; s the standa rd de viation of th e correlation and F is the F

sta tistic. The figures provided in par entheses are 95% con fidence limits.

The hal f-lives for th iol ytic c1eavage of the agents, in the presence of excess

2-mercaptoethanol, were determined by u.v. spectro photometry, as descr ibed earlier

(Cain, Wilson & Bagule y, 1976).

Agent-DNA binding was measured by spectrofluorimetric monitoring of agent

-ethidium competition for DNA sites by the methods developed earl ier (Baguley &

Falkenhaug, 1978; Cain, Baguley & Denny, 1978). m-AMSA, its analogues and a

large range of simple 9-anilinoacridines show no signjfica nt predilection for the

various binding sites provided by the commerciall y a~ail able synthetic, doublestranded DNAs, pol y d[(A-T)], pol y [d(G-C)], polydA:polydT, po lyDG:polydC, as

weil as calf thym us DNA. The lowe st correlation coefficient (r) observed for a linear

correlation between mea sured drug binding con stants for any two ofthese DNAs was

0.95. T hese agen ts th erefore demonstrate no significant sequence -selectivity of

binding to the DNA s examined . Equ ivalent regression equations result ifthe binding

values for any ofthe above DNAs is employed. Association consta nts (K) for agent

binding to pol y [d(A-T)] alone are provided (Ta ble I) and are those employed for the

regression anal yses descr ibed .

Regression approach method

The underlying mathemat ical formalism to QSAR is that of linear free energy

relationships (Purcell, Bass & Clayton , 1972). By thi s formalism, the logarithm ofthe

mol ar do se which provides a sta ndard biological response, in this case P.D40 (- logD40;

log(I/D40)), is considered for exa mina tion as the dependent variable (Hansc h, 1971).

Rate of drug mo vem ent to site -of-action is normally modelI ed by functions oflog P

or , as in the present case , by substitution of log P values wit h the equivalent Rm

figures, With a set of congeners displaying a lim ited range of Rm values, drug

mo vement ma y someti mes be approximated by a linear function of Rm. When a

sufficiently broad set of Rmvalues are embraced in the dat a-set, then equation terms

in Rm2 usually become significa nt. Employment of such regression equation terms

can be ju stified by kin etic arguments (Penniston, John, .Beckett, Bentl ey & Hansch,

1969), as weil as by a fund of pragmatic experience (Hansch , 1971 ; Denny & Ca in,

1978).

If thiolytic c1eavage attenuat es the conce ntrations of an alogues reaching site then,

by the nature of linear free energy relationsh ips, regression equation terms in log

(thiolytic reaction rate ) should prove significant. The th iolytic c1eavage reaction

(Scheme I) is, as mea sured, of frrst ord er kinetic rate and log (ra te) is then proportional to log (reac tion half-life) and half-lives may then be conve niently considered

directl y in regression analyses employing logT'I,values.

In like fashion, the logarithms of the measured agent-DN A association constants

(log K) provide relati ve measures of the free energies of agent bindin g to DNA and

therefore serve as accep tabl e terms for use in QSAR studies.

All pertinent agent properties employed in the regression ana lyses are provided in

Table I.

AMINOACRIDINE STRUCTURE - ANTITUMOU R CORRELATES 441

Results

A cross correlation matrix (Table 2) demonstrates that the Rm and Rm2 values for the

data-set examined (Table I) show unacceptable covariance and these terms were not

then included together in any single equation. Remaining variables show satisfactory

limited covariance.

Employing the usual stepwise procedures of multiple regression analysis to the

dependent variable log (1/040) provided the individual development steps ofTable 3.

The F statistic for sequential introduction of the independent variables showed that

each entered significantly at the P< 0.01 probability level.

Further computation demonstrated that logT'I, would only be accepted into

regression equations when the statistical acceptance criteria were dropped to P< O.t.

A significant dependence of dose-potency on measured agent susceptibility to

thiolytic c1eavage could not therefore be demonstrated for the set ofagents examined.

The final regression equation derived, with associated 95% confidence limits on

coefficients added in parentheses, is:-

log (1/040) = 1.28 (±0.32) log K -1.71 (±0.60) Rm2- 2.14

n=61, 1'=0.78, s=0.42, F2,59=44.I

Conclusions

As with the results of all such regression analyses the fmdings must be accepted with

certain qualification as pertinent but as yet unexamined variable(s) may prove

highly covariant with those actually considered.

Oespite qualification the final regression equation obtained is very similar in form

to successful examples obtained by previous workers when modelling in vivo gained

biological data. There is the often seen parabolic dependence on measures oflog P, in

the present case provided by an equation term in Rm2• The analyses suggest that agent

stability to thiolytic c1eavage in vi\'(), in so far as this is adequately furnished by the in

vitro measured Tv, values, is not a significant factor for dose-potency in this drug

series.

The novel employment of log K, purported to represent analogue binding to siteof-action, shows this equation term to account for more of the variance in the

biological da ta (38%: 1'2) than any other single term. This fmding provides additional

supportive evidence for the view that ONA is the target site for these agents. This is

the first recorded instance of the demonstration of a quantitative relationship

between agent-ONA binding and antitumour potency for a range of anticancer

agents.

The da ta contained in Table I, coupled with the regression equation derived,

permit the prediction of agent structures which will likely be more dose-potent than

existing examples. These predictions are currently under active examination.

One major advantage of such a regression analysis is that it draws attention to

outliers, those agents which have potency values predicted from the final equation

c1early at variance with those actually observed, for example, agents 18, 21 and 57

(Table I). In hydrodynamic assays of the drug induced unwinding of PM2 bacteriophage closed-circular, superhelical ONA, a consequence of intercalative drugbinding, these latter three agents provide lower unwinding angles than the parent

agent 2 (unpublished observations). Such observations suggest that these agents eithcr

bind in somewhat different fashion to ONA from the more biologically active parent,

or the conformational changes in the ONA substrate following ONA intercalation

differ. A more extended application of such unwinding assays to members of this

series, coupled with followed regression analysis, might provide evidence for the

hypotheses that the changed drug-binding orientations or ONA conformations which

442 B. F. CAIN, B.C. BAGULEY, W. A. DEN NY &G. J. ATWELL

contribute to the measured unwinding angles, may play a significant role in the

antitumour activity ofthese agents.

Acknowledgements

This work was supported by the Auckland Division, Cancer Society ofNew Zealand

(lnc.) and, in part, by the Medical Research Council for New Zealand.

References

Baguley, B. C. & Falkenhaug, E. M. (1978). The interaction of ethidium with synthetic doublestranded polynucleotides at low ionic strength. Nucleic Acids Res., 5, 161-171 .

Cain, B. F. & Atwell, G . J. (1974). The experimental antitumor properties ofthree congeners of

the acridylmethanesulphonanilide (AMSA) series. Eur.1. Cancer, 10,539-549.

Cain, B. F. & Atwell , G . J. (1976). Potential antitumour agents. 20. Structure-activity-site

relationships for the 4'-(9-acridinylamino}-aklanesulfonanilides. 1. med. Chem .. 19,

1409-1416.

Cain, B. F., Baguley, B. C. & Denny, W. A. (1978). Potential antitumour agents. 28 .

Deoxyribonucleic acid polyintercalating agents. J. med. Chem ., 21, 658--{)68.

Cain, B. F., Wilson, W. R. & Baguley, B. C. (1976). Structure-activity relationships for thiolytic

c1eavage rates of antitumour drugs in the 4'-(9-acridinylamino) methanesulphonanilide

series . Mol. Pharmac., 12, 1027-1035.

Denny, W. A., Atwcll, G. J. & Cain , B. F. (1979). Potential antitumour agents. 32 . Role ofagent

base strength in the quantitative structure - antitumour relationships for 4'-(9-

acridinylamino) methanesulfonanilide analogues. .f. mcd. Chcm .. 22, 1453-1460.

Denny, W. A. & Cain, B. F. (1978). Potential antitumour agents, quantitative structure -

Antileukemic (L121O) activity relationships for the w-[4-(9:"'acridinylamino)phenyl]

alkanoic acids . J. med. Chetn.. 21,430-437.

Hansch, C. (1971). Quantitative structure - activity relationships in drug design . In Drug Design ,

vol. I, ed . Ariens, E.J. pp , 271-333. New York: Academic Press.

Legha, S. S., Blumenschein. G . R" Buzdar, A, U" Hartobagyi, G , N, & Bodey, G , P, (1979),

Phase 11 study of 4'-(9-acridinylamino}-methanesulfon-m-anisidide (AMSA) in

metastatic breast cancer. Cancer Treat. Rep., 63, 1961-1964.

Penniston, J. T ., Beckett, L., Bentley, D. L. & Hansch, C. (1969). Passive permeation oforganic

compounds through biological tissue: a non-steady state theory. Mol. Pharmac ., 5,

333-341.

Purcell, W. P., Bass, G . E. & Clayton, J. M. (1972). Linear free energy - related models: theory

and description. In Strategy ofDrug Design, pp . 21-86. New York: Wiley-Interscience.

Von HolT, D. 0 " Howser, 0 ., Gormley, P., Bender, R. A., Glaubiger, 0 ., Levine, A. S. &

Young, R, C. (1978). Phase I study of methanesulfonarnide, N-[4-(9-acridinylamino}-3-

methoxyphenyl] - (m-AMSA) using a single-dose schedule. Cancer Treat. Rep., 62,

1421-1426.

Waring, M. J. (1976). DNA - binding characteristics of acridinylmethanesulphonanilide drugs:

Comparison with antitumour properties. Eur. J. Cancer, 12,995-1001.

Wilson, W. R., Cain, B. F. & Baguley , B. C. (1977), Thiolytic c1eavage of the antitumour

compound 4'-(9-Acridinylamino}-methanesulphon-m-anisidide (m-AMSA: NSC 156

303) in blood. Chem-Biol.Lnteractions, 18,163-178.

Therapy of Tropical Diseases

Chairmen:

A. O. LUCAS, Switze rland

N. D. W. L10NEL, Sri Lanka

THERAPY OF TROPICAL DISEASESSCHISTOSOMIASIS

J. M. KOFI EKUE

WHO Tropical Disease Research Cerure.

PO Box 71303. Ndola, Zambia

Introduction

In recent years the World Health Organization (WHO) and a pharmaceutical firm

have been collaborating on studies with schistosomicidal drugs. The methods used in

the studies have been reasonably simple and standard. Common protocols were

designed for each phase ofthe studies (Davis & Wegner, 1979).

Of particular importance was the way in which pre- and post-treatment

schistosome egg counts were carried out using standard techniques. Attempts have

usually been made to include techniques to fmd out whether eggs excreted by the

patients were viable or not. Results obta ined in this way are reasonably reliable and

comparable.

Studies have been done , or are in progress, at the WHO Tropical Disease Research

Centre, Ndola , Zambia (TDRC). These studies have used this common

methodology. The results so far have shown the remarkable effectiveness and

tolerance of the new schistosomicidal drug praziquantel (Davis, Biles & Ulrich,

1979; Davis, 1980).

For example, a Phase 11 Trial was completed in 151 schoolchildren in 1979. The

detailed results are reported elsewhere (Davis, 1980). The doses of praz iquantel were

30 mg kg-1 once dail y, 40 mg kg-I once daily and 20 mg kg-1 orall y twice daily. The

two doses of 20 mg kg-1 were given at 4 hourl y intervals. Pre- and post-treatment egg

counts were done using the miracidial hatching technique (Davis, 1968). Follow-up

was at one month, three months, six months, one year and two years. There were no

significant differences between the schistosomicidal effects of the different doses of

praziquantel, At one month there were only four parasitological failures. Table I

gives a summary ofthe results.

Table 1 Praziquantel Phase I1btrial

At I month

At 3 months

At 6 mon ths

At I year

At 2 years

Number examined

151 (100%)

149

144

131 (87%)

117(78%)

Number positive

4

10

37

43

51

Probably cured

97%

92%

74%

67%

56%

446 J. M. KOFf EKUE

Using our experience of clinical trials of schistosomicidal drugs observations on

the use of metrifonate in Schistosoma haematobium infections were made.

Metrifonate has been used as an effective schistosomicide in man for many years. It is

an organophosphorus cholinesterase inhibitor.

Patients and methods

140 schoolchildren from one school on the Copperbelt Province ofZambia took part

in the study. Consent for inclusion in the trial was obtained both from the parents of

individual children and from the leaders of the community - government officials,

party leaders, village headmen and school teachers.

Preliminary parasitological surveys of urine were conducted for case collection.

Urine collections was always between 09 .00 and 14.00 h. Stools were examined

by the Kato technique and blood films were stained with Giemsa for examination for

malarial parasites.

All patients who had Plasmodium falciparum parasitaemia were treated with

chloroquine. Patients with hookworm and ascaris infestations were given pyrantel

pamoate.

Before the administration of drugs to the patient volunteers, two 10 ml sampies of

urine were examined on two different days by the filtration technique. This was a

10 ml sub -sarnple oftotal bladder content. The eggs were stained with carbol fuchsin,

the sampie was filtered and all eggs which were retained on a Whatman No . 1 filter

paper were counted. All patients who had an egg count of at least 15 eggs/IO ml urine

sampie on each occasion were included in the trial so long as they had no serious

illness.

The patients were randomised into four treatment groups, A, B, C and D. The

doses ofmetrifonate administered were as folIows:

7.5 mg kg-Ibody weight at fortnightly intervals for three doses (Group B).

7.5 mg kg-l body weight followed a fortnight later by 10 mg kg-I body weight

(Groups C and D).

7.5 mg kg-I body weight followed by 7.5 mg kg-I after a fortnight and followed by

10 mg kg-I after another fortnight (Group A).

All doses ofmetrifonate were administered orally by a medical practitioner.

Electromyography was performed on a total of 81 patients before and after the

administration of metrifonate. Whole blood, erythrocyte and plasma cholinesterase

were measured (Biles, Davis, Ekue, LeQuesue & Maxwell, in preparation).

The electrocardiogram was done before and after the administration of metrifonate

in a random sampie of 54 patients.

The patients were followed up at three months and at six months. Three 10 ml

subsampies of total bladder urine were examined on three consecutive days by the

ftltration technique for schistosome ova. All cases which were positive for

schistosome ova were immediately followed up , this time using the miracidial

hatching technique ofDavis (1968).

Here , whole bladder urine content was allowed to sediment for at least thirty

minutes. An aliquot (10mI) was aspirated from the bottom using a Pasteur pipette

and centrifuged at 3,000 rev/min for frve minutes. The supernatant (9ml) was

discarded. Cooled distilled water (4ml) was added to the specimen. The whole was

mixed weil and left under a lamp for 45 min for any eggs to hatch. Absolute alcohol

(2ml) was added and staining was done by add ing a few drops of eosin. The mixture

was centrifuged and the specimen examined. All miracida, recently dead eggs and

dead eggs were counted and recorded. If miracida were present the patient was not

regarded as cured.

THERAPY OFSCHISTOSOMIASIS 447

Results

Characteristics ofthe patients

Of the 140 schoolchildren who entered the trial, 89 had only S. haematobium

infections. The remaining 53 had multiple parasitic infections as folIows:

Double schistosome infections with S. mansoni 7/53 (13%)

P.falciparum trophozoites or gametocytes 37/53 (70%)

Hookworm 8/53 (15%)

Ascaris 12/53 (23%)

The ages and weights were similar in all treatment groups. The average age was

11.8 ± 0.5 years and the average weight was 33.2 ± 1.6 kg. The sex distribution was

similar in all treatment groups except Group B where there were significantly more

boys.

Tolerance

Table 2 shows the side effects after the oral administration of metrifonate

7.5 mg kg:' in 64 children. Most of the children were found to have fasted overnight

and walked up to 6 kilometres to schoo!. Seventeen patients developed symptoms.

All subsequent administrations of metrifonate were after the patients had been given

breakfast. No symptoms were reported again.

Table2 Unwanted side effectsafter administration ofmetrifonate

Nausea

Abdominal pain

Dizziness

Weakness

'Joints shaking'

Hot sensation

Athralgia

Sialorrhoea

Total number ofpatents

Number with symptoms

4

8

4

2

3

2

3

I

64

17

ECG. EMG and cholinesterase activity

The electrocardiogram and the electromyography did not show any significant

abnormality before and after the administration ofmetrifonate. There was inhibition

of cholinesterase activity after metrifonate but this could not be correllated with

the results ofelectromyography.

Therapeutic results and porasitologicalfollow up

Table 3 shows the results of treatment with different total doses of metrifonate. The

numbers of patients available for examination at three months and at six months

were high. At three months, the percentage eure rates of 77%, 87%, 86% , 79% , in

Groups A, B, C and D respectively were similar.

Table 4 shows the geometric mean egg count in each of the treatment groups. It

also shows the percentage reduction in egg excretion rate. The geometric mean value

before treatment for Group C was significantly higher than for the other treatment

groups, The percentage reductions in egg counts at three months and six months were

similar in all treatment groups. The results show that a total dose of17.5 mg kg! is as

effective as a total dose of 22.5 mg kg-I or 25 mg kg-I in the treatment of S.

haematobium infections.

448 J. M . KOF I EKUE

Table 3 Different regimens ofmetrifonate to schoolchildren with S. haemalObil/m infections

Groups

A B C D Total

25 mg kg-I 22 .5 mg kg-I 17.5 mg kg-I 17.5 mg kg-I

Number ofpatients examined

Before treatment 32 31 39 38 140

At 3 months 30 30 35 37 132

At 6 months 28 27 31 32 118

Number (%) probabl y cured

At 3 months 23(77) 26 (87) 30 (86) 30(79) 109 (82)

At 6 months 14(50) 15 (56) 13 (42) 18 (56) 60(51)

Ta ble 4 Different regimens ofmetrifonate 10 school children with S. hael1latobil/11l infections .

Groups

A B C D

25 mg kg! 22 .5 mg kg-1 17.5 mg kg"! 17.5 mg kg-I

Mean eggcount a

Before treatment 131 120 2 19* 159

At 3 month s 1.3 0.8 1.0 1.3

% reduction in eggcount 99 99 99.5 99

At 6 mont hs 11.9 6.7 4.3 6.2

% reduction in eggcount 91 94 98 96

a The arithmetic mean ofthe adjusted individual geometrie means.

* Geometrie mean eggcount before treatme nt was higher in group C (P < 0.05) than geometrie

mean eggcounts in groups A, Band D.

Discussion

The results have co nfirrned that praziquantel is an efTective schistosomicide. It can

be administered as a single oral dose and this is a distinct advantage over other antischistosemal drugs. .

Metrifonate has been shown to be an efTective and weil tolerated schistosom icida l

drug. None of the 14Q schoolchildren who took the drug on a full sto mach had any

symptoms.

T he administration of 7.5 mg kg-I followed a fortnight la ter by 10 mg kg- 1 body

weight was just as efTective as administering the drug in three d öses at fort nightly

interva ls.

St udies have been planned to compare metrifonate and other schistosom icidal

drugs.

Acknowledgements

I am grateful to th e stafT of the T DRe, Ndo la ; Dr A. Davis, Direct or, Parasitic

Diseases Programme, W HO, Geneva and D r D . H . G . Wegner who supplied the

tablets of praziquan tel and metri fonate. This investigation received fman cial

support from th e UN D P/World Bank/WHO Special Programme for Resea rch an d

Train ing in T ro pical D iseases.

References

Davis, A., Biles, 1. E. & Ulrich, A.-M. (1979). Initial experiences with praziquantel in the

treatment of human infections due to Schistosoma haematobium. Bull. WHO. 57,

773-779.

Davis, A. & Wegner, D. H. G. (1979). Multicentre trials of praziquantel in human

schistosomiasis: design and techniq ues. Bull. WIlO. 57, 767-771.

Davis, A. (1968). Com parative trials ofantimonial drugs in urinary schistosomiasis. Bull. WHO.

38,197-227.

THERAPY OF TROPICAL DISEASESFILARIASIS

K.AWADZI

O nchocerciasis Chem otherapeut ic R esearch Centre,

Tamale Hospital, Tamale, Ghana

Introduction

The major human filar ial parasites incl ude Wuc hereria bancrofii, Brugia malayi and

B. tim ori responsible for Iymphatic filariasis, Loa loa wh ich causes loaiasis and

Onchocerca volvulus whic h ca uses onchocer ciasis. In addition, Dipetalonem a

perstans, D. streptocerca and M anzonella oz zardi infect man but ar e of doubtful

pathogeni cit y. The adults of these parasites (macrofilariae) live for man y years and

produce larvae (rnicro filariae). In lymphatic frlariasis and loaiasis the pathology is

caused by the adult worms while in onchocer ciasis, th e microfilariae are responsible.

These parasites affect millions of people in tropical and subtropica l areas and

produce chronic ilIne ss and disability. The treatment of filar iasis has recently been

reviewed by Hawking (1979). This paper will deal ma inl y with onchocerciasis,

outline the problems posed by th is disease and summa rize recent work done in the

field ofhuman onchocerciasis chemothera py.

Onchocerciasis

Genera l considerations

Onchocerciasis is a filari al disease ca used by O. volvulus and transm itted by

blood-sucking femal e blacktlies ofthe genus Simulium. It affect s 20-40 million

people in tropical Afri ca , Yernen, Mex ico, Guatemala, Colombia, Venezuela and

Brazil. The vector requires fast-flowing streams and rivers for breeding and hence

heavy infections and severe disease occur in those living in the fertile river valleys,

the Volta river ba sin in West Africa being one of the wor st endemic areas in the

world.

The adult worms are found in subcutaneous nodules although many probably lie

impalpable in deep seated location s. The adult femal e, has a life spa n of about 15

years and produces numerous microfilari ae which invade the skin and eye and ha ve a

life span oflO-20 months. Iftaken in by th e vector the y de velop into in fective lar vae

which are deposited into the skin of man. They then undergo a phase of rnigration,

maturation and differentiation into adult male and female worms thereby completing

the cycle, The infective lar vae do not multipl y in man and heavy infections are the

result ofintense transmission.

450 K.AWADZI

Clinically, onchocerciasis presents as subcutaneous nodules, a bewildering variety

ofskin changes, eye and Iymphatic changes and progressive weight loss and debility

especially in the heavily infected. The clinical expression of the disease, however,

varies from one geographical area to the other, important ditTerences existing

between the savanna and forest zones in Africa, between Central American and

African onchocerciasis and also between these and the localised form, sowda in the

Yemen. These reflect ditTerent geographical strains ofthe parasite and ditTerences in

behaviour of the vectors . In the individual patient the clinical picture is probably

determined by the interaction ofthe parasite with the immune system.

Onchocerciasis poses problems that extend far beyond those of an infected patient.

For the individual, the chronic pruritus, disfiguring skin lesions, distortion of

inguinal and genital anatomy by 'hanging groin' and herniae, the lack of energy and

chronic ill-health constitute a serious disability. The occurrence of blindness or

severe visual impairment condemns the sutTerer to a 'Iiving death,' completely

dependent on the charity of others. The association ofthe 'blackfly' with rapid watercourses had led to the desertion of fertile river valleys which historically have

attracted human habitation and given birth to early civilization. In the community,

the young and the able have deserted, leaving behind the old and the infirrn .

Individuals fleeing from the blackfly have often contributed to over-population and

erosion of the upland areas . On the national scale onchocerciasis presents governments, many of whom have an agricultural basis and are already struggling

economically, with massive socio-economic problems. It is the increased awareness

of the clinical, social, economic and national consequences of the population of the

riverine areas by Simulium that has made onchocerciasis a disease of international

concern.

Chemotherapeutic aspects

The drug treatment of onchocerciasis may be directed against the three forms of the

parasite that occur in man - the infective larvae and developing forms,

(chemoprophylactics), the adult worm (macrofilaricides); or the microfilariae

(microfilaricides) and may be on an individual or community basis (mass therapy).

At present, there is no known chemoprophylactic for O. volvulus and there is no drug

that can etTectively destroy both the adult worms and the microfilariae and hence

therapy must employ a combination ofdrugs.

Diethylcarbamazine (DEC) which is microfilaricidal and suramin, a macrofilaricide with some microfilaricidal activity, are extremely etTective but may

give rise to severe adverse reactions which may prove fatal and this limits their use to

individual patients under close supervision rather than to mass therapy. Moreover,

the use of DEC and suramin for curative treatment involves a prolonged period of

treatment and observation over at least two to three months. This, coupled with the

uncertainty of the optimum dosage schemes for these drugs, create numerous

problems in the therapy of this disabling, debilitating but essentially non-fatal

disease .

The approach to the solution of these problems has been along the following

lines: I) Attempts to reduce the severity of the reaction to DEC by the concurrent

administration of anti-inflammatory and anti-allergic agents, 2) modification of the

dosage regimes of DEC and suramin and a detailed study of their pharmacology, 3)

assessment ofother therapeutic agents for filaricidal activity.

Suppression ofthe reaction to DEC

DEC , even in high concentration, has no etTect on O. volvulus in vitro. In the heavily

infected patient, however, DEC is transformed into an agent of massive microfilarial

THERAPY OF FILARIASIS 451

migration and destruction and produces alarming and even sometimes fatal

reactions (Fuglsang & Anderson, 1974; Bryceson, Warrel & Pope 1977; Oomen,

1969). The mechanisms underlying this transformation are not yet elucidated but the

involvement of various mediators of the allergie reaction such as histamine,

5-hydroxytryptamine (5HT), kinins, SRS-A and prostaglandins, complement

activation, immune complexes and alterations in microfilarial structure or

metabolism need to be considered (Bryceson, 1976; Henson, MacKenzie & Spector,

1979). Antihistamines, anti-5HT antagonists, prostagiandin synthetase inhibitors

and steroids have been used in attempts to suppress the Mazzotti reaction

(Villamayor, 1970; Duke & Anderson, 1972; Anderson, Fuglsang & MarshalI, 1976).

However, many of these trials have not been carried out systematically and the

assessment of the effiicacy or otherwise of the agents tested have sometimes been

based on 'drop-out' rates rather than on direct observations on patients treated.

In order 10 evaluate the reactions produced by various dosage regimes of DEC, to

compare the severity of the reaction to DEC to that of new microfilaricides, and to

assess the efficacy ofvarious agents in reducing to acceptable levels the severity ofthe

Mazzotti reaction, it is imperative that a method for the quantitation ofthe reaction

be evolved. Such a method has been evolved and forms part of the routine

management of patients undergoing chemotherapeutic trials at the Onchocerciasis

Chemotherapeutic Research Centre (OCRC) in Tamale (Awadzi, 1980). The method

brings together the commonly occurring reactions to DEC and applies to them a

scoring system so that the total 'disability' experienced by the patient over a given

period can be expressed by a single figure. This permits the effects on the patient of

DEC or other filaricide to be expressed in quantitative terms. This method has

formed the basis ofthe evaluation ofindomethacin, cyproheptadine and prednisone

as anti-Mazzotti agents in a number of double-blind placebo controlIed trials in

patients moderately to heavily infected with O. volvulus (Awadzi et al., in

preparation). The results ofthese trials are summarised in Table I.

Table 1 The effect of indomethacin, cyproheptadine and prednisone in suppressingthe main

contributory factors to the Mazzotti reaction

Anti-Mazzoui grade

Feature lndomethacin Cyproheptadin e Prednisone Pruritus 0 0 0

Rash 0 0 0

Gland reaction 0 0 +HFever 0 0 ++++

Tachycardia

(postural) 0 0 ++++

Hypotension

(postural) 0 0 ++++

Joint reaction 0 0 +HMuseIe aches 0 0 +HHeadaches 0 0 +

DosesofDEC 2Agover7 days 200mgdailyx7days 200mgdailyx7days

% Reduction in

rnicrofilarial density

Active 98.8 96.3 77.7

(97.8-100) (93.5-99.0) (49.3-93.8)

Placebo 98.0 97.0 94.5

(96.8-98.9) (93.5-99.0) (79.5-100)

Key to anti-Mazzotti grade; reaction score measured by active/placebo score and given as

a percentage. Grade 0=>80%; Grade +=60-79%; Grade -1+=40-59%; Grade +H-=20-39%;

Grade ++++ =<20%.

452 K.AWADZI

Indomethacin and cyproheptadine did not significantly reduce the severity of the

Mazzotti reaction nor did they interfere with microfilarial destruction. Prednisone,

however, was very effective in reducing the severity ofmost ofthe components ofthe

Mazzotti reaction with the exception of the itching and the rash. Prednisone also

interfered with microfilarial destruction by DEC - mean percentage reduction was

77.7 (range 49.3 - 93.8) in the prednisone group as compared with 94.5 (range 79.5-

100) in the placebo group. It is necessary to carry out further trials in order to find a

dose regime of prednisone and DEC which will produce adequa te microfilarial

destruction without severe react ions. It is also necessary to find an agent which will

eliminate the itch after DEC. The evaluation ofother candidate drugs is also essential

in order to find a safe anti-Mazzotti agent.

Modification ofthe dosage schemes ofDEC and suramin

DEC

The dose of diethylcarbamazine citrate recommended for the treatment of

onchocerciasis has varied considerably and the following are some of the regimes

recommended:

Duke (1971) recommended 50mg on day one ; 50mg three times a day on day two;

100mg three times a day on day three and then 200mg three times a day on days four

to ten.

Duke & Anderson (1972) suggested 50mg on day one; 100mg twice daily on day two;

and then 200mg twice daily for the next five days. These doses were to be reduced

proportionately in persons weighing under 40kg. This regime, however, produced

severe reaction s in heavily infected patients (Fuglsang & Anderson, 1974). The

regime was then modified as folIows, 25mg on day one: 25mg twice dail y on day two;

50mg twice daily on day three and then stepwise increments by 50mg dail y to a

maximum of 200mg twice daily to be continued for up to 14 days (Duke &

Anderson, 1975).

Rollo (1975)recommended 0.5mg kg-i on day one ; 0.5mg kg-1twice daily on day two;

1.0mg kg! three times dail y on day three , then 2mg kg-I three times daily for a total of

21days.

The Extra Pharmacopoeia (Mart indale, 1977) recommends an initial dose of 50mg

increased to 150- 500mg daily and the U.S. Pharmacopoeia, 2 - 4mg kg-I three times

daily for one to four weeks.

These dosage schemes varying from a total dose of2.25 - 7g over periods of one to

four weeks highlight the difficulties and uncertainties in the use of this very efficient

microfilaricide which has been in use for over 30 years. The aim of all these regimes

is the elimination or destruction of most of the initial microfilarial load . However,

since the adult worms are not killed, repopulation ofthe skin by microfilariae occurs

after some weeks and DEC has to be repeated either in periodic short courses or given

intermittently in small doses.

In an attempt to avoid the severe reactions that occur in heavily infected persons

with the elimination ofthe initial microfilarial load , Rougemont, Boisson, Borges da

Silva & Zinder (1976) recommended a schedule ofsmall weekly doses as opposed to

progressive doses 'whose side effects cannot be foreseen.' Sowa & Sowa (1978)

investigated the use ofminimal doses (12 .5 and 25mg) ofDEC given daily over 7 - 20

weeks in schoolchildren 5 - 15 years of age. Th ere was a high default rate due to

severe reactions, especiall y in those receiving 25mg. The y recommended that the

initial dose should not exceed 12.5mg for children up to 30kg weight. They obtained a

good clinical result and histological evidence of damage to the adult female worms,

although no controls were used. The misadventures of some ofthe atternpts at mass

therapy with DEC and the hurried alterations in planned dosage schedules indicate

THERAPY OF FILARIASIS 453

that such inadequate information exists at the moment on the reactions to therapy of

the individual patient, on the optimum dose and duration of treatment, and on the

frequency and at what dose level DEC should be administered, that mass therapy of

onchocerciasis with DEC is probably premature.

In order that DEC therapy be established on a rational basis the following factors

have to be taken into account. I) DEC given in an adequate continuous dosage

schedule produces 'a single peak reaction' in which areaction occurs, which may be

severe, in the first few days of therapy and thereafter declines rapidly despite

increasing dosage (Awadzi & Gilles , 1980a). 2) This reaction may be produced by a

small dose in the heavily infected patient and an initial small dose does not

necessarily protect against a severe reaction. 3) The administration of small

intermittent doses may lead to a 'multiple peak reaction' and though the severity may

diminish with repeated dosage, the regime may be less acceptable than one which

produces a 'single peak reaction.' 4) In the absence of a macrofilaricide, DEC will

have to be repeated at intervals, the timing of which should be such that only mild

discomfort is produced.

There is an urgent need for the formulation of dose schedules of DEC which take

into account the factors outlined above.

At the Onchocerciasis Chemotherapeutic Research Centre (OCRC), Tamale, the

following strategy has been evolved: It is assumed that severe reaction will occur in

heavily infected patients irrespective of the starting dose of DEC (unless the dose is

too small to have any microfilaricial effect). A continuous dose scheme of DEC is

employed to produce a 'single peak reaction.' The total dose employed must be

adequate to destroy most or all of the initial microfilarial load. An adequate dose is

defmed as one which in a patient with a minimum aggregate microfilarial count of

100, as assessed at the outer canthus, scapula, iliac crest and calf using the OCRC

method (Awadzi, Roulet & Bell, 1980b), reduces the microfilarial count by at least

90%; such a degree of reduction being maintained for at least one month. The

Table 2 Microfilarial destruction by seven dosage schedules ofDEC in a total ofl54 patients.

Mean results are shown with the range in brackets.

Dosage schedule

Mean initial microfilarial density

Mean % reduction in microfilarial

density

At oneweek Atone month

I) 100mgsingle dose 438 61.6 51.4

(20 patients) (183-1446) (25.1-93 .7) (5.0-91.8)

2) 200mg single dose 495 66.2 62.6

(22 patients) (166-1347) (42.5-93.4) (32.2-92.9)

3) 200mg dailyx3

(24 patients) 431 92.6 86.4

Total dose: 0.6g (165-1187) (77.1-98 .2) (64.6-96.7)

4) 200mg twice dailyx3

(27 patients) 409 94.0 87.8

Total dose: 1.2g (138-801) (86.4-99.7) (68.7-95 .7)

5) 200mg dailyx7

(21 patients) 370 94.9 93.2

Total dose: l.4g (172-873) (81.8-100) (84.7-100)

6) 2.4g over 7 days 385 96.2 95.5

(20 patients) (149-1043) (87.9-99.8) (83.6-100)

7) 6.6g over 14days 319 98.9 97.7

(20 patients) (133-518) (96.5-100) (94-99.6)

Ta ble 3 Pattern of reconstitution ofskin microfilariae by seven dosage schedules of DEC in a total of 154 patients. The results are shown as the mean ,

with the range in brackets.

+:>-

VI

+:>-

Mean initial Mean microfilarial dens ity as %ofinitial

Dosage schedule m icrofilarial density

At 3 months At 6 mon ths At 10mo nth s

I) 100mg single dose 438 49 .7 75.2 81.4

(20 patients) (183- 1446) (21-96.9) (11.1-212.3) (26.2-155.6)

2) 200mg single dose 495 43 .0 48.7 61.2

(22 pati ent s) (166-1347) (13 .2-99.4) (11.2-95.9) (25.1-92.2)

r

3)200mgdailyx3 p

(24 patients) 43 1 21.1 29. 1 48.6 p

Total dose: 0.6g (165-1187) (6.1-46 .9) (4.0--59.9) (7.2-153 .1) 0t::

4) 200mg twice dai ly x 3

(27 patients) 409 19.7 33.4 55.5

Total dose: I.2g (138-801) (5.8-34.2) (12.2-8 7.7) (19.1-130.5

5) 200mg dai ly x 7

(21 patients) 370 13.5 24.0 44.9

Total dose : l.4g (172-873) (2.7-38.9) (6.2-109.5) (10.3-92.3)

6) 2.4g ove r 7 da ys 385 7.4 21.6 37.3

(20 patients) (149-1043) (0--12.5) (1.3-48.6) (0.7-89.4)

7) 6.6g over 14days 319 3.1 12.3 21.2

(20 pati ent s) (133-5 18) (0--11.2) (0.7-55 .8) (2.1-47.1)

THERAPY OF FILARIASIS 455

reaction to this dose can then be suppressed by ernploying anti-Mazzotti agents

which in carefully controlled trials have been found to be efTective. Further doses of

DEC can then be given at a time when the build-up ofskin mi crofilariae has not yet

attained a level above which significant reactions are expected.

In pursuance ofthis strategy, seven do sage schemes ofDEC have been evaluated as

regards the severity of reaction produced, their levels of m icrofil aricidal potency

(LMP) determined one week and one month after completion of therapy, and the

pattern of reconstitution of skin microfilariae over aperiod of ten months. The

results are summarised in Tables 2 and 3. Adequate LMPs are achieved by dosage

schedule 5, 6 and 7. With schedules 5 and 6 it will be necessary to repeat the regime

aft er three months; with schedule 7 it is probably adequate to repeat DEC after six

months, since the average microfilarial density at this time is below 50 and

mi crofilarial densities below this, determined as described above are associated with

mild reactions (Awadzi , personal ob ser vation). A sim ilar approach has been adopted

by Prod'hon, Moreau & Mongin (l979) and Prod'hon, Sainte-Marie, Moreau &

De sfontaine (l979). They concluded that DEC, given in a dail y dose of 200mg

combined with levamisole 120mg for 14 days and after one year DEC 200mg daily

with levamisole 60mg given for five days, ofTered the optimum regime for mass

therapy. However, further work is needed to clarify the situation.

Suramin

Rougemont, Delmont, Ranque, Ducam & Prost (l979) evaluated five dosage schemes

of sura m in varying from a total do se of 2.0g to 3.8g in a hyperendemic village in

Mali. lt was concluded that the regime consisting of'regularly increasing weekly doses

of 0.2, 0.4 , 0.6, 0.8 and l.Og followed by a dose of 0.8g proved to be effective,

producing a 90 % decrease in the parasite load as asse ssed eight months after therapy.

Side efTects were acceptable and no deterioration in vision occured. A sim ilar do se

regime is currently been evaluated at the aCRC, Tamale.

Pharmacology 0/radiolabelIed DEC and suram in

The manipulations of the do sage schemes of DEC and surami n can be greatl y

facilitated by a detailed study of their pharmacology. Initial studi es using radiolabelIed compounds ha ve been carried out at Tamale in a total of 12 patients and

results will soon be ava ilabl e (Edwards et al., in preparation).

Assessme nt 0/potentialfilaricides

Metrifonate

Salazar-Mallen, Gonazalez-Barranco & Jurado Mendoza (l97Ia) administered

metrifonate to a total ofl9 patients in Mexico who recei ved the drug fortnightl y after

breakfast. Two patients received three doses of 7.5mg kg-1 and then one dose of

IOmg kg-I

. Nine patients received 1O-15mg kg" for four do ses and eight patients

received 1O-15mg kg! repeated 5-16 times. There was a significant reduction in

microfilarial counts, especially in those who received at least six doses. There was

al so clinical and histological evidence of macrofilaricidal activity. Subsequently,

Sal azar-Mallen , Gonzalez-Barranco & Dei Carmen Montes (l97Ib) administered

metrifonate at IOmg kg-I dail y combined with atropine sulphate for six consecutive

da ys to seven patients and obtained negati ve skin biopsies in six patients which

persisted at one month in five patients.

Duke (l972) evaluated metrifonate against a West African forest strain of O.

volvulus but found little microfilaricidal activity and no evidence for a macrofilar icidal action. The do se was later increased to the limits of tolerance in a

chi mpanzee , obtaining a significant microfilaricidal but no ma crofilaricidal efTect

(Duke, 1974a). Fuglsang & Anderson (l977) in vestigated the efTects ofa single do se of

456 K. AWADZI

metrifonate 10mg kg-1 in 15 patients moderately to heavily infected with O. volvulus.

This study was later extended to three groups of patients receiving IOmgkg-1 as three

single doses at fortnightly intervals, frve single doses on five consecutive days and frve

to six doses at intervals determined by the patient (Fuglsang & Anderson, 1978a).

They concluded that metrifonate possessed modest microfilaricidal activity,

produced less severe reactions than DEC and had some beneficial effect on anterior

segment lesions . They suggested that metrifonate may have a role to play in the

initial treatment of very heavily infected patients by making a subsequent course of

DEC and suramin more acceptable. Abaru & McMahon (1978) gave metrifonate to a

total of 24 adults who received three dose regimes of 2.5mg kg-1

, 7.5mg kg-I and

IOmgkg! daily for three consecutive days. A partial microfilaricidal effect was

observed only in the group receiving IOmgkg" . There was no evidence of a macrofilaricidal effect and side effects were common.

In 18 patients with mild to moderate infections with O. volvulus Awadzi, Haddock

& Gilles (l980a) evaluated metrifonate given in three dosage regimes each of

IOmg kg! body weight, either as a single dose, as six consecutive daily doses or every

two weeks for three doses. Significant microfilarial destruction (more than 90%)

occured in patients receiving multiple daily doses and in most patients on the intermittent dose regime. The multiple daily dose regirne produced severe gastrointestinal

effects and a reversible proximal muscle paralysis. Each dose in the intermittent dose

regime produced areaction although with diminishing severity. There was no

evidence ofa macrofilarical effect.

Subsequent evaluation involved a comparative single blind study of the intermittent dose regime with DEC in a total dose of 6.6g over 14 days and also in the

evaluation of metrifonate 10mg kg-1 body weight given daily for three or six days

(Awadzi & Gillcs 1980a; Awadzi & Gilles 1980b). The following conclusions were

drawn from these trials: I) Metrifonate is a partial microfilaricide with no macrofilaricidal activity. 2) There is a wide variation in the level of microfilarial

destruction in patients receiving the same dose. 3) Three doses of IOmg kg-1 body

weight give on average the same degree of microfilarial destruction (approx. 75%)

whether given intermittently or daily . 4) There is no justification for giving more than

three daily doses, as nicotinic effects begin to appear and microfIlarial destruction is

not enhanced. Improvement in the microfilaricidal effect of metrifonate can be

achieved only by a more prolonged intermittent dose regime. 5) Metrifonate does not

present a serious challenge to DEC as the 'reference' microfilaricide,

Levamisole

Duke (unpublished observations) treated ten adult males infected with the West

African forest strain of O. volvulus with levamisole 120mg given on three

occasions at weekly intervals and detected no macrofilaricidal activity. High doses in

a chimpanzee were both micro- and macrofilaricidal but toxic effects made it

unlikely that the drug could be used at high dosage in man .

Mebendazole

This has been assessed by Duke (I974b) in an experimentally infected chimpanzee

and found to be ineffective. Maertens & Wery (1975) in a double-blind trial treated a

total of 34 patients with mebendazole 100mg twice daily for 14 days (13 patients),

mebendazole 100mg twice daily for 30 days (six patients), mebendazole 100mg twice

daily with levamisole 50mg daily for 14 days (nine patients). A control group of six

patients were given placebo tab lets twice daily for 14 days . None of these dosage

schemes was found to be effective against O. volvulus. Trials of mebendazole,

levamisole and their combination are currently proceeding at Tamale.

TH ER APY OF FILARIASIS 457

Nifurtimox

Fuglsang & Anderson (l978b) administered nifurtimox in a dose of 15-20mg kg-I

bod y weight dail y for five da ys to 14 onchocerciasis pat ients in the savanna area of

the Cameroon. There was no evidence ofa microfilaricidal efTect. Assessment ofskin

microfilarial count eight months after therapy in six pat ients showed that the count

had decreased in three patients, was unchanged in two and increased in one patient.

A possible efTect on the adult worms was suggested and further trials recommended.

Nitro/ urans

In the experimental chemotherapy of filaria sis, nitrofurantoin was shown to have

high acti vity against the adult parasites of Litomosoides carinii infection in the

multimammate rat Ma stom ys natelensis though the chemotherapeutic index was

low (Foster, Pringle, King & Par is 1969). Nitrofurantoin has also been shown to have

chemoprophylactic activity against nearl y all larval stages of the parasite (Lammler

& Wolf, 1977). Furazolidone, another nitrofuran , has been shown to have considerable macrofil ari cidal and microfilaricidal activity in the same experimental

system with a high chemotherapeutic index (Lammler, Sanger & Wegerhof, 1978).

In three separate studies Awadzi et al., (to be published) evaluated furazolidone

and nitrofurantoin for filarial activity in human onchocerciasis. In study 1

furazol idone was administered to nine patients in a dose of 100mg every six hours for

ten days. After a rest period of one week, DEC was administered in a total dose of

6.6g over 14 days. In stu dy 2, eight patients received furazolidone combined with

DEC in the dosage stated abo ve. In study 3. nine patients received nitrofurantoin in a

dose of 100mg six hourl y for ten days. DEC was administered as described for

furazol idone.

Th e microfilaricidal efTect of furazolidone, nitrofurantoin and DEC was assessed

one week after completion of the appropriate schedule. The macrofilaricidal efTect

was assessed indirectly by eliminating skin microfilariae with DEC , studying the

pattern of repopulation and comparing thi s with that of a group of patients treated

previously by an identical DEC dose regime . Neither furazol idone nor nitro furantoin

showed an y microfilaricidal acti vity. Furazol idone had no macrofilaricidal acti vity.

The build up of microfilariae was slower than expected in seven out of nine patients

treated with nitrofurantoin, suggesting a possible macrofilaricidal effect. The results

are encouraging enough to warrant furth er trials.

Other drugs

Pyrantelpamoate, oxamniquine, metronidazole and tinidazole have been evaluated

by Kaie (1978) and found to possess no significant antifilarial acti vity,

Local therapy ot onchocerciasis

Onchocerciasis afTects mainly the skin and eye and it is logical that local applications

of antifilarial agents to these sites be attempted in order to avoid severe reactions that

result from systemic therapy.

Langharn, Traub & Richardson (1978) advocate the application ofDEC lotion (I or

2%) to the skin. Good parasitological results were obtained with only mild reactions.

Most oftheir patients were , howe ver, onl y Iightly infected (mean microfilarial counts

between 5.5-12.8). Hutchinson , El-Sheikh, Jones, Anderson, Fuglsang &

MacKenzie (1979) applied this 'transepidermal approach' to 30 moderately to

heavily infected patients (156 ± 27 microfilariae per snip) in the Sudan savanna.

Local and system ic reactions were very severe and more prolonged than after oral

DEC.

LocaI application of DEC eye drops has been studied (Ben-Sira, Aviel, Lazar,

Lieberman & Leopold, 1970; Anderson & Fuglsang, 1973; Jones, Anderson & Fugl-

458 K.AWADZI

sang, 1978a) but the efficacy of this method had yet to be established. Jones,

Anderson & Fuglsang, (1978b) applied increasing concentrations oflevamisole and of

mebendazole to one eye in groups of four patients with ocular onchocerciasis in

northem Cameroon. No effect resulted from up to 3% mebendazole suspensions but

3% levamisole solutions were microfilaricidal. It was recommended that this

approach be pursued in the search for potential filaricides,

Conclusions

Diethylcarbamazine and suramin remain after several decades, the only drugs currently available for the treatment of onchocerciasis. They are etfective but have

serious disadvantages. A number ofstrategies have been developed to tarne these two

drugs in order to make them applicable to community therapy. The search for safer

filaricides continues and is actively supported by the WHO Special Programme for

Research and Training in Tropical Diseases.

Acknowledgements

This work received su p port from th e Filariasis Component of th e United Nations

Developrnent Programmc/World Bank/World Health Organization Special

Programme for Re search and Training in Tropical Diseases.

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Clinical Pharmacology of

Fertility Regulation

Chairmen :

A. KESSLER, Switzerland

R. CHAUDHURY, India

DILEMMAS IN CONTRACEPTIVE

DEVELOPMENT: VARIATION IN

ENDOCRINE RESPONSE TO

LOW DOSE PROGESTOGENS

E. DICZFALUSY & B.-M. LANDGREN

Reproductive Endocrinology Research Unit

and Department of Obstetrics and Gynaecology,

Karolinska sjukhuset, Stockholm. S weden

Introduction

Since the pioneering studies reported by Martinez-Manautou, Cortez, G iner, Aznar,

Casasola & Rudel (1966) and Martinez-Manautou, Giner-Velasquez, CortesGallegos, Aznar, Gutierrez-Najar & Rudel (1967), a vast amount of literature has

accumulated on the contraceptive use of various form s of proge stogen-only

'minipills' , and different aspects of the problem have been reviewed recently

(Rin ehart, 1975; Moghissi, 1976; Fotherby, 1977; Landgren & Diczfalusy, 1980). The

fund amental principle ofthis method is the continuous daily administration ofsmall

dose s of progestogens without interruption. The major advantage of this approach is

that the majority of biochcmical and metabolic changes induced by combined oral

contraceptivcs (Briggs, 1977) as weil as several adverse effects, for instance on blood

coagulation and lactation, do not seem to represent a problem in women treated only

with progestogens (Rinehart, 1975; Fotherby, 1977), not even when administered in

mass ive doses (Benagiano, 1977).

In spite of the se advantages, progestogen-only 'minipills' did not ac hieve an y

greater popularity or wider acceptance, probably for two major reason s: a) lower

efficacy , and b) higher frequency of bleeding irregularities (mainly intermenstrual

bleeding) when compared to various combined (oestrogen-progestogen) formulations

(Moghissi, 1976; Fotherby, 1977). The exact nature of hormonal changes associated

with this type of contraception is incompletely understood. It is generally agreed,

however, that 'rninipills' do not invariably inhibit ovulation, although the exact

frequency ofthis is not known and the figures reported vary widely (between 14-82%)

(Landgren & Diczfalusy, 1980).

Since the efficacy of various formulations of 'rninipills' is much better than the

above figures would suggest (generally 1-3 pregnancies per women year) (Fotherby,

1977), it is obvious that the contraceptive efficacy ofminipills cannot be attributed to

ovulation inhibition alone. Among additional mechanisms of contraceptive action,

effects on sperm migration in the cervical mucus, on tubal transport ofthe fertilized

ovum, and/or on implantation have been considered. For obvious reasons, it is very

difficult to assess the importance ofthese factors separately.