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PARASITOLOGICAL REVIEWS
EXPERIMENTAL PARASITOLOGY 12, 2 74-322 (1962)
Chemotherapy and Chemoprophylaxis of Africa11
Trypanosomiasis*
James Williamson
National Institute for Medical Research, The Ridgeway, Mill Hill, London, N.W.7
1. Introduction ..........................................................
1.1. General .........................................................
1.2. Historical ......................................................
2. Chemotherapy and Chemoprophylaxis ..................................
2.1 (a) Trypanosomiasis in Man ....................................
(b) Trypanosomiasis in Animals ..................................
2.2. Neutral Aromatic Arsenicals and Antimonials ....................
2.3. Melaminyl Arsenicals and Antimonials ..........................
2.4. Acridine Derivatives ...........................................
2.5. Diguanidines and Diamidines ....................................
2.6. 6-Aminoquinaldine and -cinnoline Derivatives ....................
2.7. Phenanthridinium Derivatives ...................................
2.8. Carboxylated Aromatic Arsenicals and Antimonials ................
2.9. Sulfonated Naphthylamine Derivatives ...........................
2.10. Nitrofurans ....................................................
2.11. Antibiotics .....................................................
2.12. Miscellaneous ..................................................
I. INTRODUCTION
1.1 GENERAL
This review of the chemotherapy of African trypanosomiasis will be limited mainly to developments which have occurred in the last decade, as the last major synoptic review of the subject was made by Findlay in 1950. Useful surveys of current progress have been
* Due to space requirements, this review will be published in two parts. The first part appears in this issue (Sections 1 and 2). The second part (Sections 3 and 4 and the Reference List) will be published in the October number (12, No. 5) of Experimental Parasitology.
Page
274
274
277
283
283
285
287
292
298
298
304
308
314
315
317
318
321
made subsequently by Browning (1954), Goodwin (1952), Goodwin and Rollo (1956) and Goodwin (1958); several reviews have also appeared which deal with more specific aspects, such as the physiological basis of chemotherapy (von Brand, 1951a, b, 1952), diamidine prophylaxis of sleeping sickness (Gall, 1954), treatment of Gambian sleeping sickness (Neujean and Evens, 1958), drug resistance (Schnitzer and Grunberg, 195 7 ; Bishop, 1959), and the chemotherapy of animal trypanosomiasis (Davey, 1957; Marshall, 1958). To these may be added recent and graphic pictures of the overall disease problem by Davey (1958), Nash
274
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 275
(1960) and Morris (1960), and the biennial reports of the International Scientific Com- mittee for Trypanosomiasis Research may also be cited for valuable information on cur- rent trends both in the laboratory and in the field.
The continuing importance of trypano- somiasis control in Africa may be indicated very simply by a few basic considerations. Some 4% million square miles of tropical Africa, an area 1% times that of the U.S.A., is occupied by the tsetse tly, Glossinu, the insect vector of the disease (Swynnerton, 1936). As a result, as Hornby (1952) has stated, trypanosomiasis has the unique dis- tinction of being the only disease which, by itself, has denied vast areas of land in Africa to all domestic animals except poultry. The traditional wheel-less “hoe and head-load economy” of Africa is a direct consequence of the failure of ox-drawn transport to survive in the tsetse areas, which, in addition, prob- ably limited the southward migration of horse-borne Islamic culture from the north, and equally, and more recently, the north- ward advance of immigrant farmers from the Cape across the Vaal and Limpopo rivers (Nash, 1960). The impact of trypano- somiasis on imported animal transport was again evident during the first World War; as Hornby (1919) reported, “I am writing this in Portuguese East Africa, where during the past three months, I have watched two thousand donkeys contract and die of nagana . . . .”
A more recent example of the dynamic aspect of the tsetse-fly problem is the critical situation which exists today in south-east Africa where “an area of perhaps as much as fifty thousand square miles is in process of invasion” (Ford, 1960). This advance, on a hitherto unprecedented scale requiring inter- national action, has been taking place for some years in Mozambique and the Federa- tion of Rhodesia and Nyasaland and now threatens the Union of South Africa (Ford, 1960; de Sousa, 1960). The gravity of the situation lies in the speed with which the tsetse fly is being restored in areas which it occupied before the great rinderpest epizootic of 1896. This disease decimated ungulates, and thus killed off their dependent fly, within
the vast East African tsetse belt known to exist before that date from Somaliland to the Transvaal.
Before the advent of drug treatment, and consequent on the population migration as- sociated with the slave trade, exploration, military operations and the colonial develop- ment of inland communications, epidemics of human sleeping sickness occurred on a vast scale; a mortality of half a million has been estimated in the Congo during the years 1895 to 1905 (Scott, 1939), and in the great epidemic round Lake Victoria in 1902-1905, some 200,000 people, or about two-thirds of the population., died in one province alone (Buxton, 1948). As Lotte (1952) has emphasized, “. . . sleeping sickness is the only social illness with [a] lethal and ir- reversible incidence on demography”; the typical scquelae of death, sterility, increased infantilz mortality and consequent popula- tion displacement are discussed in detail by Morris (1952). In 1935, 84,000 cases of sleeping sickness were diagnosed in Nigeria, representing an infection rate of about 20% ; twenty years later the corresponding figure was 0.2%, a dramatic reduction which can largely be ascribed to organized and effective chemotherapy (see sections 2.1, 2.5).
It is important to distinguish here the two commonly differentiated forms of sleep- ing sickness, that due to Trypanosoma gambiense which produces a chronic and relatively asymptomatic infection, and the more acute and virulent T. rhodesiense infec- tion; the biological basis of this dichotomy has been discussed in detail by Ashcroft (1959), and in ecological terms by Morris (1951). The relative numerical importance of these two infections, their geographical distribution (illustrated with great clarity by Leeson, 1953) and their response to chemo- therapy, may be assessed from the figures compiled by van Oye (1958), covering the period 1948-1957. For T. gambiense, in the West, in the areas of French Equatorial and West Africa, French Cameroons, Angola, Belgian Congo, Gambia, Ghana, Sierra Leone, Nigeria, Portuguese Guinea, Togo, Liberia and Ruanda-Urundi, new cases declined from 53,000 for the year 1948 in a total population of 77 million, to 15,000 for the year 1957 in
276 WILLIAMSON
a total population of 91 million; mass chemo- prophylaxis’ with pentamidine (section 2.5) has been largely responsible for this decline.
With the numerically less important T. rhodesiense infection in the East, the picture is rather different, as chemotherapy of this disease has been ineffective until very recently (see section 2.3). In the areas most affected, Kenya, Tanganyika, Nyasaland and the Rhodesias, Uganda, Ruanda-Urundi and Mozambique, new cases annually remained stable at a level of about 1,000 for a total population which rose from 29 million in 1948 to 38 million in 1957.
Although the disease in man can now be said to be under control, total eradication is a long way off, and epidemic outbreaks still occur (Scott, 1960). The current context of emergent African nationalism, the rapid development of modern transport and the resultant increase and shift of large popula- tions, underline the absolute importance of maintaining the high standards of vigilance in sleeping sickness control set up by British, French, Belgian, and Portuguese authorities. A relaxation of these control measures would almost certainly restore human trypano- somiasis in Africa to its former lethal poten- tial (Nash, 1960; Morris, 1960) ; as Nodenot ( 1958) aptly remarks, “. . . l’incendie semble Cteint mais les braises couvent SQUS la cendre. . . .”
As a result of the effective control gained over sleeping sickness, animal trypanosomi- asis or nagana (Bruce, 1896) (a corruption of the Zulu word “ngana” meaning feeble or weak (Rornby, 1952)), and due mainly to T. congolense and T. vivax, is now of greater economic and even medical importance; it is now recognized as ‘I. . . the most important single limiting factor for livestock production in many parts of Africa” (1st Session of the F.A.O. Subcommittee on Research Freedom from Hunger Campaign. Bull. Of. Intern. Epizoot. (1961) 55, p. 543). Effective chem- otherapy of this infection is of more recent origin, but with the rapid development in infected cattle of resistance to the few active trypanocides available, and in the absence of a reliable long-lasting prophylactic drug, the disease now constitutes a majo,r block to the provision of an adequate intake of animal
protein for the bulk of the tropical African population (Buxton, 1948; Davey, 1948; Nash, 1960).
T. evansi, the “surra” parasite, is trans- mitted mechanically by biting flies and has thus a wider geographical distribution than the tsetse fly. It causes an infection of camels and was enzootic in a belt across the northern areas of Africa between the 13th and 15th parallels of north latitude, with an incidence of more than 50% in some areas (Bennett, 1933) ; this has been effectively controlled by suramin treatment since 1925 (Knowles, 1925; Bennett, 1933; Evans, 1956; Larrat, 1958) and more recently also by quinapyramine (antrycide) . Some 63,000 camels in Mauritania were treated in 1959 with quinapyramine and “une u&e sym- metrique trypanocide” (suramin? ) (BUZZ. Ofl. Intern. Epizoot. (1961) 55,551). In the Sudan Republic, Leach (1961) indicates that quinapyramine is effective in the treatment of suramin-resistant strains in camels. ‘LDourine”, the trypanosome infection (T. equiperdum) of equines, which is trans- mitted, not by tsetse or other biting flies, but by coitus and has thus, like T. evansi, a much wider geographical distribution, cannot be considered as a purely African trypanosomi- asis ; effective control with quinapyramine has been instituted in many areas, especially in North Africa (Vaysse and Zottner, 1950; Zottner, 1952).
Overall figures for the incidence of tryp- anosomiasis in African cattle are difficult to assemble satisfactorily in the same form as the data on sleeping sickness collected by van Oye ( 1958)) but some local examples may suffice to illustrate the ravages of the animal infection, and its major importance in the African economy. In general, the dis- tribution of cattle and of the tsetse fly are, as Buxton (1948) pointed out “. . . to a con- siderable extent the converse of one another”, and the number of cattle relative to the vast areas involved is small. Very approximate figures derived from cattle population esti- mates in the Symposium on Animal Tryp- anosomiasis (Inter-African Advisory Com- mittee on Epizootic Diseases, Luanda, 1958) and from Buxton (1948), indicate that for the African areas considered by van Oye
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 277
(1958) with a total population of about 124 million, the total cattle numbers are of the order of 30 to 40 millio’n, about half of which are held in Kenya, Tanganyika, Nyasaland, Uganda and the Rhodesias. Only small num- bers of dwarf shorthorn breeds, relatively resistant to infection, can be kept in Sierra Leone or Ghana, areas almost totally dominated by tsetse fly; Davey (1948) has estimated that the number of cattle slaugh- tered each year in Sierra Leone could provide only “. . . little more than one pound of beef per head annually”. The cattle population of the relatively fly-free areas of Northern Nigeria has been estimated as being between 4 and 6 million (Davey, 1948; Wilson, 1956), but the seasonal migration south of over a million head in search of dry-season grazing in the Southern Guinea zone, involves passage through tsetse areas in which losses from trypanosomiasis run into thousands. Again, in Southern Rhodesia, where (‘. . . ap- proximately 16,000 square miles of land is rendered unsuitable for cattle rearing on account of tsetse fly . . .” (Mackinnon, 1956), losses in one cattle reserve exposed to fly en- croachment were over 70 per cent during a period of four years; in another (Urungwe), where 2,000 out of 7,000 cattle died from trypanosomiasis in 1950-52, and the re- mainder were to be transported for slaughter, over half of these were (‘. . . too weak to walk to the collecting point for transport and were slaughtered in the reserve” (Lawrence & Bryson, 1958). Some fifteen years ago, in the southern Sudan, during the peak period of a large-scale outbreak of bovine tryp- anosomiasis, losses were estimated as about 10,000 head each month (Evans, 1956). Larrat (1958), and Finelle (1958), review- ing the position in the territories of Senegal and Oubangui-Chari respectively, have esti- mated an annual financial loss from live- stock mortality directly attributable to tryp- anosomiasis as about 500 million francs in the former area and about 60 million francs in the latter. In animal disease in Africa, trypanosomiasis clearly constitutes a problem of the first magnitude.
T. congolense and T. vivax are the tryp- anosomes mainly responsible for the mor- tality of African cattle and sheep, but a
uniquely virulent, rapidly fatal and drug- refractory infection of pigs is caused by T. simiae, “. . . the lightning destroyer of the domestic pig” as Bruce described it (Hoare, 1936), which is responsible for “. . . epizoo- ties t&s meurtrieres” (Finelle, 1958). This is the only trypanosome which can produce in a large domestic animal the kind of rapidly fulminating septicaemic infection typically seen in laboratory rodents infected with syringe-passaged T. brucei-group strains. The infection has been recorded in many parts of Africa (Tanganyika, Northern and South- ern Rhodesia, Belgian Congo, Nigeria, French Guinea, Ghana and Sierra Leone) and con- stitutes a considerable menace to the future development of African pig-farming as an additional source of animal protein.
The considerations affecting chemothera- peutic control of animal trypanosomiasis in tropical Africa are possibly even more com- plex than those determining control of the infection in man. Discussion of these is largely outside the scope of this review, but the contribution of such varied factors as trypanosome species and strain variation, fly density and infection rate and tsetse bionomics generally, in relation to control systems aimed at the insect vector (relevant vegetation clearance methods and insecticide application), human settlement and its at- tendant administrative problems, enumera- tion and examination of nomadic cattle herds, soil erosion caused by over-stocking, game reservoirs and their destruction, selective or otherwise, the nutritional status and breed of the cattle affected, intercurrent infections, the operation of an immune response and so forth, may indicate that drug treatment of trypanosomiasis, although it is at present the most effective form of control, cannot be considered in isolation (cf. Shortt, 1951; Whiteside, 1958a, b) ; the disease, both in man and animals, as Nash (1960) empha- sizes, is an intricate and fascinating biological complex.
1.2. HISTORICAL
With the biological background of the disease sketched in briefly, some details of the historical evolution of its chemotherapy may be indicated in order to set more
TABL
E 1’
H
isfor
iml
Dev
clogm
cnl
of
Tryp
anos
omio
sis
Chem
othe
rapy
(1
830-
1920
)
(i)
(ii)
Perio
d (ii
i) Ba
cker
ound
he
alth
pr
oble
ms
inno
vatio
ns
in
tech
niqu
e Ch
emica
l di
scov
erie
s
1817
.65:
As
iatic
ch
oler
a pa
ndem
ics
in
Euro
pe
s (M
acNa
mar
a,
1892
) 18
30:
List
er:
achr
omat
ic le
nses
fo
r co
mpo
und
m,
mic
rosc
ope
d 18
34:
Rung
e:
anilin
e fro
m
coal
ta
r
1 18
34:
Purk
inje
: m
icrot
ome
(Gar
rison
. 19
14)
1848
: 1s
t U.
K.
Publ
ic He
alth
Ac
t 18
40:
Fritz
sche
: an
iline
from
in
digo
Food
pr
eser
vatio
n by
ca
nnin
g (A
pper
t, 18
10)
1817
.65:
As
iatic
ch
oler
a pa
ndem
ics
1854
-56:
Cr
imea
n wa
r: dy
sent
ery
mor
tality
18
56:
Perk
in:
synt
hesis
of
m
aw&e
fro
m
ani-
line
sulp
hatv
(in
at
tem
pted
qu
inin
e sy
n-
ISbl
-70:
Pa
steu
r: m
icroo
rRan
ismr
as
cnm
mnn
th
esis)
(P
erkin
. 18
%)
8 m
d %
1870
-71:
Fr
ance
-Pru
ssia
n an
aest
hetic
su
rger
y
fact
or
in
ferm
enta
tion,
de
cay
and
infe
ctio
n (P
aste
ur.
1556
, 18
66,
1870
) 18
37.6
3:
Hofm
an,
A.
W.:
triph
enylm
etha
ne
dyes
(H
ofm
ann,
18
63)
1858
: G
riess
: di
azo
reac
tion
1863
: Bi
cham
p:
1st
arom
atic
arse
nica
l (la
ter
1867
: Li
sLer
: an
tisep
sis
know
n as
At
oxyl)
fro
m
use
of
arse
nic
as
war:
peak
se
psis
Iro
m
oxid
ant
in
fuch
rrn
dye
prod
uctio
n
1874
: Ba
yer.
eosin
.~
. Fr
ance
: wi
ne
ferm
enta
tion
prob
lem
s,
silkw
orm
:
dise
ase
and
shee
p lo
sses
(a
nthr
ax)
1876
: Ca
ro:
mel
h+ne
bl
ue
Colo
nial
de
velo
pmen
t in
In
dia
and
Afric
a;
Liv-
in
gsto
ne
(185
8)
desc
ribes
na
gana
tre
atm
ent
1817
: Ab
bC:
oil
imm
ersio
n ob
ject
ive
and
illum
i- na
tion
appa
ratu
s fo
r m
icros
cony
8 18
80-9
0:
Med
ical
and
vete
rinar
y in
vest
igat
ions
in
In
dian
an
d .4
frica
n co
loni
al
terri
torie
s Ex
pans
ion
of
Ger
man
sy
nthe
tic
dye
and
phar
- 18
81:
Skra
up.
quin
olin
e sy
nthe
sis
?
mac
eutic
al
indu
stry
19
84:
BBtti
ger:
cotto
n-su
bsta
ntive
ar
e dy
es
s e
1902
.05:
La
ke
Vict
oria
sl
eepi
ng
sickn
ess
out-
brea
k;
UK.
Colo
nial
O
ffice
an
d Ro
yal
So-
1902
: La
vera
n an
d M
esni
l: lry
pano
sam
es
in
vilro
ciety
rn
vest
ipat
ory
com
miss
ion
1904
’ va
n W
einb
erg
and
Ullm
ann:
try
pan
red
(Ehr
lich
and
Shig
a,
1904
)
1906
’ M
esni
l an
d N
icolle
. try
pan
blue
, Af
ridol
vio
kl Is
ymm
etric
al
urea
de
rivat
ive)
0 2 19
07:
Ehrli
ch
and
Berth
eim
; M
oore
, Ni
eren
-
‘: ;it
sf:‘,
ia a
nd
Todd
: Al
oxyl
cons
titut
aon
esta
b-
8 52
1909
’ Br
rml
and
Sicr
anet
cin,
1st
arom
atic
an-
1914
.18:
1s
t W
orld
W
ar;
casu
alty
se
psis
; eq
uine
lim
unia
l
trans
port
loss
es
from
try
pano
som
iasis
in
19
12:
Bend
a:
Tryp
afla
vine
(acr
iflavin
e)
Afric
an
cam
paig
n
1916
, Hc
yman
n,
Koth
e,
Drew
1 an
d O
ssen
beck
: Ba
yer
205
(sur
amin
) (F
ourn
eau
cl
a[.,
1924
) 19
19:
Jaco
bs
and
Hei
delb
ergw
try
pars
amid
e
TABL
E I
(Con
tinue
d)
(iv)
(v)
(vi)
Biol
ogica
l-med
ical
appl
icatio
n of
ne
w co
mpo
unds
Pa
thog
enic
para
site
disc
over
ies
Expe
rimen
tal
infe
ctio
n
w ” 18
37:
Donn
C:
spiro
chae
te
from
sy
philit
ic le
sion
d ? 18
41-4
3:
Vale
ntin
: G
luge
; G
ruby
: Tr
ypam
. aw
na
in
fish
and
amph
ibia
1862
: Be
neke
: an
iline
dyes
as
hi
stol
ogica
l st
ains
(C
on”,
1953
) 18
69:
Hoffm
an,
H:
fucb
sin
as b
acte
rial
stai
n
P 18
72:
Dava
ine:
an
thra
x in
ra
bbits
s 18
75:
Wei
gert:
m
ethy
l vio
let
as
tissu
e co
cci
stai
n 6 a
1877
: Ko
ch,
Ehrli
ch:
use
of
thin
film
, fix
atio
n 18
77:
Man
son:
in
sect
ve
ctor
in
fila
riasis
d
and
stai
ns
1878
: Le
wis:
try
pano
som
e (T
.&wi
n’)
in
Indi
an
rat
1880
: Ev
ans:
try
pano
som
c (T
. ev
ansi)
in
In
dian
ho
rse
infe
ctio
n (“S
urra
”)
1881
-91:
Eh
rlich
: m
etby
lene
bl
ue
as
haem
ato-
lo
gica
l an
d in
travit
al
stai
n,
anal
gesic
an
d an
timal
aria
l (E
hrlic
h,
1881
, 18
86;
Ehrli
ch
and
Lepp
man
n,
1890
; G
uttm
ann
and
Ehr-
8 lic
h,
1891
)
e 18
93:
Ling
ard:
po
tass
ium
ar
senj
te
trypa
nocid
al
d in
“S
urra
”
P 18
96:
Bruc
e:
arse
nic
and
arwn
ite
trypa
nocid
al
1896
: Br
uce:
ts
etse
-bor
ne
trypa
noso
mes
as
ca
use
in
“aga
na
of
naga
na
1896
: R
ouge
t: try
panc
wme
(T.
cqui
pcrd
um)
ar
caus
e of
“D
ourin
e”
in
hors
es
1902
: Bl
umen
thal
: At
oxyl
phar
mac
olog
y
1898
: Ne
pveu
: 1s
t de
mon
stra
tion
of
trypa
no-
1898
: Ka
ntha
ck:
Durh
am
and
Blan
dfor
d:
T.
som
e in
m
an
bruc
d in
la
bora
tory
an
imal
s
1901
: Du
tton:
T.
gom
bien
rc
in
ma”
19
02:
Lave
ran
and
Mes
nil:
arwn
ite
trypa
nod-
da
1 in
ex
perim
enta
l ro
dent
in
fect
ions
an
d in
vit
ro
1905
: Th
omas
: T.
gom
bien
se
in
labo
rato
ry
anf-
mal
s
1904
: Eh
rlich
an
d Sh
iga:
@
pan
red
trypa
noci-
19
04:
Brod
en:
T. c
ongo
knsc
da
1 in
ex
perim
enta
l in
fect
ions
19
05:
Thom
as:
Atox
yl try
pano
ddal
19
05:
Ziem
ann:
T.
viva
19
05-0
7:
Thom
as
and
Brei
nl;
Koch
; Ko
ch,
Beck
an
d Kl
eine
: At
oxyl
treat
men
t of
sl
eepi
ng
R
sickn
ess
z $
1905
.07:
Fr
anke
an
d R
oehl
: try
pano
cidal
dr
ug
resis
tanc
e _
1906
: N
icolle
an
d M
esni
l:’ try
pa”
blue
an
d Af
ridol
vio
let
trypa
nocid
al
1908
: Pl
imm
er
and
Thom
son:
ta
rtar
emet
ic
trypa
nocid
al
1909
: Br
einl
an
d Ni
eren
stei
n:
p-am
inop
heny
l- st
ibin
oxid
e try
pano
cidal
19
09:
Beva
n:
use
of
tarta
r em
etic
in
Af
rican
ca
ttle
trypa
noso
mfa
sis
(Bev
an,
1928
). 19
10:
Step
hens
an
d Fa
ntha
m:
T.
rhod
esicn
se
1917
: Br
owni
ng,
Gul
bran
sen
and
Thor
nton
: 19
12:
Bruc
e d
al.:
T. s
imbx
in
pi
gs
acrif
lavin
e as
an
tisep
tic
TABL
E II
His
toric
al
Dev
elop
men
t of
Tr
ypan
osom
iasi
s C
hem
othe
rapy
(1
920-
1960
)
Perio
d (9
(ii
) Ba
ckgr
ound
he
alth
pr
oble
ms
Inno
vatio
ns
in
tech
niqu
e (ii
i) C
hem
ical
di
scov
erie
s
1925
-42:
T.
ga
mbi
ense
pa
ndem
ics
in
Wes
t (N
ash,
19
60)
Dev
etop
men
t of
ts
etse
su
rvey
an
d co
ntro
l od
s in
Af
rica
Cl
;h”
Anim
al
indu
stry
de
velo
pmen
t in
Af
rica
192.
3: V
oegt
lin
et a
l.:
mod
e of
ac
tion
of
arse
nica
l dr
ugs
Afric
a 19
26:
Behr
ens;
G
addu
m;
Blis
s:
dose
-resp
onse
an
alys
is
1927
-31:
Q
uast
el:
drug
s &
bact
eria
l m
etab
olis
m
met
h-
1929
: Yo
rke
el
al.:
Afric
an
trypa
noso
mes
vi
able
24
hou
rs
in
vitro
at
37
” C
19
30-3
2:
York
e et
al
.: try
pano
cida
l dr
ug
resi
st-
ance
st
udie
s 19
31:
Mor
gan
& W
alls
: im
prov
ed
synt
hesi
s of
ph
enan
thrid
ine
deriv
ativ
es
1938
: Fu
lton
& C
hris
toph
ers:
dr
ugs
and
lryp-
an
osom
al
met
abol
ism
19
38:
Haw
king
: re
vers
ibilit
y of
in
itial
up
take
of
ar
seni
cals
by
try
pano
som
es
1924
: U
hlen
huth
et
al.:
An
timos
an
1926
: Br
owni
ng
et a
l.:
styr
ylqu
inol
ines
19
26:
Roe
hl:
Pam
aqui
n (a
ntim
alar
ial
%am
ino-
qu
inol
ine
deriv
ativ
e)
1933
: M
auss
&
Mie
tsch
: At
ebrin
(a
ntim
alar
ial
amin
oacr
idin
e de
rivat
ive)
19
35:
Dom
agk,
M
iets
ch
& Kl
arer
: Pr
onto
sil
(Dom
agk,
19
35)
1937
-39:
Ki
ng
et
al.:
arom
atic
di
amid
ines
19
37:
Jens
ch:
Surfe
n C
19
38:
Brow
ning
et
al
.: ph
enid
ium
(p
hena
nthr
i- di
ne)
1939
-45:
2n
d W
orld
W
ar:
loss
of
ac
cess
to
Ger
- 19
39-4
7:
Albe
rt:
drug
ac
tion
and
elec
troch
emic
al
man
an
timal
aria
ls;
loss
of
Ja
va
quin
ine
prop
ertie
s in
ac
ridin
e se
ries
(Alb
ert
1951
a,
stoc
ks
b)
1925
-42:
T.
ga
mbi
ense
pa
ndem
ics
in
Wes
t Af
rica
1940
: W
oods
; Fi
ldes
: p-
amin
oben
zoic
ac
id-s
ul-
fani
lam
ide
anta
goni
sm
; “ra
tiona
l ap
proa
ch”
chem
othe
rapy
1942
: Ki
ng
& St
rang
eway
s:
corre
latio
n of
try
p-
anoc
idal
ac
tion,
re
sist
ance
be
havi
or
& ph
ysi-
cal
prop
ertie
s of
ph
enyl
arse
noxi
des
1943
-48:
U
.S.
rese
arch
on
mal
aria
pa
rasi
te
met
ab-
olis
m
and
its
inte
ract
ion
with
an
timal
aria
ls
(see
McK
ee,
1955
) 19
45:
Pete
rs
et a
l.:
deto
xica
tion
of
arsc
nica
ls
with
2,
3-di
mer
cdpt
opro
pano
l (B
AL)
1947
: Ko
pac:
di
amid
ine-
nucl
eopr
otei
n in
tera
c-
tion
1940
: Fr
iedh
eim
: try
pano
cida
l m
elam
ine
arse
ni.
cals
1944
: D
odd
& St
illman
: ni
lrofu
ran
1945
: Ea
gle:
bu
tars
en
1945
: C
urd
et
al.:
prog
uani
l (p
alud
rine)
(b
igua
- ni
de
from
an
timal
aria
l py
rimid
ine
serie
s)
1947
: W
alls
: di
mid
ium
(p
hena
nthr
idin
e)
TABL
E II
(Con
thue
d)
Perio
d (9
(ii
) Ba
ckgr
ound
he
alth
pr
oble
ms
Inno
vatio
ns
in
tech
niqu
e (ii
i) C
hem
ical
di
scov
erie
s
1949
-50:
Tr
ypan
osom
iasi
s in
re
sear
ch
cent
ers
set
up
in
Briti
sh
East
&
Wes
t Af
rica
8 2 d x
Incr
easi
ng
impo
rtanc
e of
an
imal
try
pano
som
iasi
s as
sle
epin
g si
ckne
ss
is b
roug
ht
unde
r co
ntro
l
1946
48:
Che
n &
Gei
ling;
M
arsh
all:
phos
phor
y-
lativ
e gl
ycol
ytic
cy
cle
in A
frica
n try
pano
som
es
1951
: vo
n Br
and:
co
rrela
tion
of
trypa
noso
me
met
abol
ism
&
syst
emat
ics
1951
: G
uim
arae
s &
Laur
ie:
sura
min
-pen
tam
idin
e co
-pre
cipi
tatio
n 19
51:
Orm
erod
: an
tryci
de-n
ucle
ic
acid
in
tera
c-
tion
in
trypa
noso
mes
19
52:
Willi
amso
n &
Rol
lo:
Afric
an
trypa
noso
me
nutri
ent
anal
ysis
1955
: So
ltys:
ch
emop
roph
ylax
is
& im
mun
ity
1956
-61:
D
esow
itz;
Solty
s;
Wei
tz;
Gra
y:
rein
- ve
stig
atio
n of
‘tr
ypan
osom
al
imm
unity
w
ith
mod
ern
tech
niqu
es
1957
-60:
N
ewto
n;
Fern
ande
s:
trypa
noci
dal
ac-
tion
and
nucl
eotid
e m
etab
olis
m
1949
: Fr
iedh
eim
: M
el
B (m
elar
seno
xide
-BAL
) 19
49:
Cur
d &
Dav
ey:
quin
apyr
amin
e (a
ntry
cide
) (c
hlor
ide
as d
epot
pr
ophy
lact
ic)
(qui
nolin
e/
pyrim
idin
e)
1952
: W
atki
ns
& W
oolfe
: ho
mid
ium
(e
thid
ium
) (p
hena
nthr
idin
e)
1952
: Po
rter
et a
l.:
Styl
omyc
in
(ach
rom
ycin
, pu
rom
ycin
) ;
1st
effe
ctiv
e try
pano
cida
l an
ti-
biot
ic
1955
: Je
nsch
: Be
reni
l (d
iam
idin
e)
1956
: W
atki
ns
& W
oolfe
: Pr
othi
dium
(n
on-d
epot
pr
ophy
lact
ic)
(phe
nant
hrid
ine/
pyrim
idin
e)
1956
: W
illiam
son
& D
esow
itz:
sura
min
ates
of
qu
ater
naly
am
mon
ium
try
pano
cide
s as
dep
ot
prop
hyla
ctic
s 19
58:
Wra
gg
et
al.:
Met
amid
ium
an
d its
su
r- am
inat
e (p
hena
nthr
idin
e/di
amid
ine)
19
60:
Berg
: is
o-M
etam
idiu
m
(act
ive
isom
er
of
Met
amid
ium
)
TABL
E II
(Con
tinue
d)
z
Perio
d Bi
olog
ical
-med
ical
ap
plic
atio
n of
ne
w
com
poun
ds
Expe
rimen
tal
infe
ctio
n
1920
: M
ayer
&
Zeis
s:
prop
hyla
ctic
ac
tivity
of
su
ram
in
1921
: Yo
rke:
T.
rh
odes
iens
e in
m
an
treat
ed
with
su
ram
in
1925
-30:
An
timos
an
in
cattl
e try
pano
som
iasi
s (S
chw
etz,
19
33)
1925
: Pe
arce
: try
pars
amid
e ac
tive
in
late
-sta
ge
slee
ping
si
ckne
ss
1935
: vo
n Ja
ncso
&
von
Janc
so:
synt
halin
try
p-
anoc
idal
1943
: H
ornb
y et
al
.: ph
enid
ium
in
ca
ttle
tryp-
an
osom
iasi
s 19
44:
van
Hoo
f et
aZ.
: la
rge
scal
e ch
emop
roph
y-
laxi
s of
sl
eepi
ng
sick
ness
w
ith
pent
amid
ine
1944
: C
arm
icha
el
8: B
ell:
dim
idiu
m
in
cattl
e try
p-
anos
omia
sis
1946
: D
odd:
Fu
raci
n try
pano
cida
l 19
49:
Wils
on:
quin
apyr
amin
e ch
lorid
e pr
ophy
- la
ctic
in
T.
sim
iae
infe
ctio
n in
pig
s 19
49:
Gob
le:
antim
alar
ial
g-am
inoq
uino
lines
ac
- tiv
e in
ex
perim
enta
l T.
cr
usi
infe
ctio
ns
1953
-54:
W
ilde
& R
obso
n;
Ford
et
al
.; W
ilson
et
al
.; U
nsw
orth
: ho
mid
ium
in
ca
ttle
tryp-
an
osom
iasi
s 19
.55:
Bau
er;
Mim
e et
al.:
Be
reni
l cu
rativ
e in
cat
- tle
try
pano
som
iasi
s 19
57:
Apte
d:
Mel
B
cura
tive
in
late
-sta
ge
T.
rhod
esie
nse
slee
ping
si
ckne
ss
1957
: Br
ownl
ie
et a
l.:
prot
hidi
um
prop
hyla
ctic
in
ca
ttle
trypa
noso
mia
sis
1958
: Fa
irclo
ugh:
m
etam
idiu
m
in
cattl
e try
p-
anos
omia
sis
1958
: W
hite
side
: cr
oss-
resi
stan
ce
anal
ysis
an
d dr
ug-in
duce
d im
mun
ity
in
prop
hyla
xis
of
cattl
e try
pano
som
iasi
s 19
58:
Wat
son
& W
illiam
son:
ex
perim
enta
l th
erap
y an
d ch
emop
roph
ylax
is
of
T.
sim
iae
infe
ctio
ns
in
pigs
19
58-6
0:
Fern
ande
s et
al
.: co
mbi
ned
styl
omyc
in-
prim
aqui
ne
treat
men
t cu
rativ
e in
ex
peri-
m
enta
l T.
cr
uzi
infe
ctio
ns
1952
: U
nsw
orth
&
Nes
bitt;
D
esow
itz
& W
atso
n:
T. v
ivax:
ad
apta
tion
to l
abor
ator
y ro
dent
s
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 283
recent drug developments in perspective. The provenance of new drug types has been, and continues to be, largely empirical, but in some cases at least, it is possible to discern, albeit tenuously, an evolutionary pattern which may assist both the interpretation of current innovations and the prediction of future trends. Protozoological adherents of the Woods-Fildes “rational approach” (Fildes, 1940) may take comfort from the fact that both the first and the latest dem- onstrations of anti-protozoa1 chemotherapy were derived on logical grounds; Guttman and Ehrlich in 1891 from a histological study of the selective uptake of methylene blue by the malarial parasite, proceeded to show that the dye was active against tertian malaria in man, and in the last two years, Fernandes and his collaborators (Fernandes and Castellani, 1958, 1959; Silva, Yoneda and Fernandes, 1959; Fernandes, Pereira and Silva, 1959; Moraes, Faria and Fernandes, 1960) have devised an effective treatment of experimental T. cruzi infections, based primarily on analysis of the interaction of known drugs with the parasite’s nucleotide metabolism. The earlier evolution of the antimalarial Daraprim (pyrimethamine) (Russell and Hitchings, 1951; Falco et al., 1951; Hitchings 1960) represents another and perhaps the most complete example of a clinically effective antiprotozoal drug derived primarily from a “metabolite-an- tagonist” basis.
However, no effective parasiticidal drug has yet been synthesized from first principles, “tailored” as it were, to the vulnerable metabolic areas of an infective organism; in the case of African trypanosomes, the paucity of relevant information on parasite metab- olism and its interaction with existing drugs (see 3.1, 3.2 below) has severely limited an understanding of trypanocidal drug action and contributed virtually nothing to the design of new trypanocides.
Ehrlich’s pioneer investigations of the action of chemically defined substances in infectious disease represent the exploitation by an individual of exceptional genius, of the convergence of a number of 19th century technical developments (cf. Walker, 1948 ; Rhoads, 1954). Only the elements of a de-
sirable treatment of this aspect of chemo- therapy can be indicated here in the skeletal form of a time chart (Tables I and II) ; a more extended version has been attempted elsewhere (Williamson, 1949).
In this time chart, the main headings indicate the principal determinants of the evoluti0n of synthetic parasiticidal drugs in general. For relevance, the sequence is restricted as far as possible to trypanocides, the early development of which, in any event, is synonymous with the beginnings of chemo- therapy.
2. CHEMOTHERAPY AND CHEMOPROPHYLAXIS
2.1 (a) TRYPANOSOMIASIS IN MAN
The effective drug control of human sleep- ing sickness during the last twenty years has rested essentially on three trypanocides: suramin (2.9), tryparsamide (2.2) and pen- tamidine (2.5) (see Tables I and II), and the only major innovation in treatment in the last ten years has been the demonstration of the curative activity of Friedheim’s mel- aminyl-substituted aromatic arsenicals (2.3), (especially Mel B) in late-stage T. rho- desiense sleeping sickness (Apted, 1953, 1957) and in tryparsamide-resistant late- stage T. gambiense infections (Friedheim, 1948, 1951a). The extent of this advance in the treatment of Rhodesian sleeping sickness may be gauged by reference t,o reviews as recent as that of Laufer (1955) in which suramin appears as the main, indeed the only practicable drug, curative only if given not more than three weeks after the onset of acute symptoms (Willett, 1955); cases with nervous system involvement were virtually doomed within a matter of months. This major chemotherapeutic advance and its possible consolidation with compounds still under trial, such as Mel W, a water-soluble derivative of Mel B (Friedheim and De Jongh, 1959), and furacin derivatives active against Mel B-resistant cases, are discussed in detail below (sections 2.3, 2.10).
During the last decade, ample evidence has accumulated (Gall, 1954; Demarchi, 1958) to evaluate the policy of mass pentamidine prophylaxis of Gambian sleeping sickness,
284 WILLIAMSON
vigorously and efficiently pursued by French, Belgian and Portuguese colonial health authorities especially, and in many areas with striking success, following its advocacy by van Hoof, Henrard and Peel in 1944. The use of pentamidine in this way has been a major revolution in T. gambiense control, particularly in areas of arsenic-resistance, and the scale on which mass treatment was introduced may be illustrated by figures for French West Africa given by Le Rouzic and Koch (1949) which show the relegation of tryparsamide to a secondary role in favor of pentamidine :
Consumpticn Nos. receiving Pentamidine Tryparsamide prophylactic
(kg.1 (kg.1 treatment
1945 0.19 805.6 1
1946 2.06 608.9 1,512
1947 3 .oo 398.0 1,489
1948 88,299
Similar figures for French Equatorial Africa are given by Kernevez and Chassain ( 195 1) ; prophylactic injections of diamidines rose from 261 in 1946 to 80,751 in 1949 and to 489,507 in 1953 (Demarchi, 1958). The massive scale of treatment in French, Belgian and Portuguese territories is apparent from the overall figures given by Demarchi (1958).
Angola Belgian Congo French Equatorial
Period 1949-1957 1945-1937
No. of
prophylactic treatments
4,433,177 3,637,868
Africa 1946-1957 2,787,810
Spectacular reduction of disease incidence by as much as a hundredfold has resulted (Demarchi, 1958), especially in epidemic areas (Le Gac, 1951; Le Gac and Ziegler, 1952; Le Gac and Mulet, 1953), but as Lotte (1953) and Neujean and Evens (1958) have stressed, treatment of the active endemic form must be tenacious and prolonged and the advantages of mass prophylaxis against the residual endemic form are open to discus- sion. Apart from the serious source of danger which lies in the inactivity of pentamidine against the later stages of infection, involving the central nervous system. which are often
difficult to diagnose and may be masked by mass prophylaxis (Harding and Hutchinson, 1950; Scaillet and Haddad, 1950; Jonchkre, 1951; Gall, 1954; Deroover, 1958), the complex ecology of the disease (Morris, 1951, 1952; Hutchinson, 19.53, 1954), and its variable effects in man (Duggan, 1959), make assessment of the relative merits of mass survey and treatment of active foci, or of mass prophylaxis, very much a matter of individual judgment for any given area. For example, mass prophylaxis is not applicable to populations with a large migrant element (Scott, 1957; Baudart, 1951), an increasingly important factor in the face of growing popu- lation movement in emergent Africa. Coupled with this, the very success of prophylaxis tends to induce a false sense of security in the indigenous peoples so treated, with con- sequent absenteeism on occasions of retreat- ment (Demarchi, 1958). Elementary but vital considerations, such as staff shortage and the balancing of claims on public health expenditure, also contribute to the overall difficulties of deciding on the best method of control (McLetchie, 1948; Ross, 1960) ; as Demarchi (1958) has indicated 51 est certain que l’immense armature sanitaire mise en oeuvre pour lutter contre la maladie du sommeil est terriblement coateuse.”
Total eradication of Gambian and Rho- desian sleeping sickness in man by elimina- tion of the insect vector alone is impracti- cable, but as human settlement is the most efficient means of reducing the incidence of tsetse fly, there is much to be said for mass prophylaxis as a means of decreasing the “circulating virus” to a level below which it would no longer sustain a dangerous infection rate in the fly (Demarchi, 1958). In other words, the man-fly-trypanosome cycle could be broken for long enough to allow new settlement in fly areas to be built up. This approach to Rhodesian sleeping sickness is complicated mainly by the difference in habitat of the Glossina morsitans type vector from that of the G. palpalis type vector of T. gambiense, by the inadvisability of mass pentamidine treatment of the virulent T. rhodesiense infection, except in the earliest stages of the infection to prevent an epidemic (Silva and Caseiro, 1957)? and by the ex-
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 285
istence of an animal reservoir (He&h, McMahon and Manson-Bahr, 1958).
Obviously there is an urgent requirement for a cheap, simply administered and well- tolerated drug which would be as effective a prophylactic as Pentamidine, active thera- peutically against all stages of the infection in both Gambian and Rhodesian sleeping sickness, incapable of inducing drug resistance and active also against strains with acquired resistance to other drugs. It is possible that the two requirements of prolonged tissue retention (for prophylaxis) and ability to penetrate into the cerebrospinal fluid are mutually exclusive; further research on the so-called “blood-brain barrier” is clearly required (see section 3.1).
2.1 (b) TRYPANOSOMIASIS IN ANIMALS
During the last decade there have been no revolutionary advances in drug control of animal infections in the field comparable to the pentamidine prophylaxis of T. gambiense and the Mel B treatment of late-stage T. rhodesiense infections in man. Antrycide (quinapyramine) (section 2.6) (Curd and Davey, 1949) came into use in Africa about 1950, and in the form of the “pro-salt” (con- taining the sparingly soluble chloride salt which has prophylactic properties) was the first active prophylactic drug available for cattle infections. Although its protective powers are not generally considered to extend beyond two months, and its use, like that of all other “cattle” trypanocides except tartar emetic, has been dogged by acquirement of drug resistance, it is still a mainstay in field practice.
The early years of its use in Africa co- incided with the eclipse of phenidium and dimidium (section 2.7) (the phenanthri- dinium drugs first introduced into field use in 1943-45, and later discarded because of re- sistance development, photosensitization and delayed toxicity), and the appearance of ethidium (homidium) (Watkins and Woolfe, 1952), a much less toxic version of dimidium. The impact of antrycide and ethidium on cattle treatment may be indicated by figures such as those of Larrat (1958) for the Haute Volta region of French West Africa where the
number of prophylactic antrycide treatments rose from 6,206 in 1953 to 140,782 in 1957, and of Wilson (1958) for Northern Nigeria where the number of cattle treated with antrycide and ethidium rose from 45,000 in 1951-52 to 641,000 in 1957-58. In the latter area, dimidium was never released for field use, and tartar emetic treatment con- tinued up to 1950 when 34,200 head were treated, compared with 2,200 in 1930 and 11,088 in 1938. In the absence of other active drugs, tartar emetic held sway for forty years because, despite its toxicity (about 6% mortality in treated animals), deaths in untreated animals might be as high as 50% (Wilson, 1958).
These rising figures reflect the expansion of cattle industry associated with increasing economic development and improved health services, but mass cattle treatments have been followed rapidly by extensive drug re- sistance (Wilson, 1958; Nash, 1960; White- side, 1958a, b; Whiteside, Fairclough and Bax, 1960; Williamson, 1960) (see section 3.3). The few active “cattle” trypanocides which have become established in the field or have survived preliminary field trials are tartar emetic, ethidium (2.7), antrycide (2.6), berenil (a diamidine) (2.5), pro- thidium (2.7), and metamidium (2.7). Of these, only tartar emetic and berenil have not readily given rise to drug resistance; this corresponds to laboratory experience where artificial production of trypanosome strains resistant to tartar emetic or to the aromatic diamidines is difficult or impossible (Yorke, Murgatroyd and Hawking, 1932 ; Fulton and Grant, 1955). Antrycide and the group of the aminophenanthridinium drugs, ethidium, prothidium and metamidium, can all give rise to cross-resistant strains in cattle (White- side, 1958a, b) and the tendency is increased by the current trend of what may be de- scribed as “cannibalization-synthesis”, where portions of active drug molecules are re- combined to give active variations on a rather restricted theme; prothidium for ex- ample is essentially the early phenanthridine drug phenidium (section 2.7) with the pyrimidyl moiety of antrycide as a substit- uent, and metamidium is a form of ethidium with a substituent consisting of the major
286 WILLIAMSON
portion of a m-amidino-substituted variant of berenil (sections 2.5, 2.7). Further ex- amples of this tendency to hybridization will be given later, but in the absence of any fruitful lead from metabolic work on the parasite (section 3.2)) synthetic organic chemists in the pharmaceutical industry and elsewhere are restricted of necessity to in- tensive exploitation of the few heterocyclic structures with demonstrated trypanocidal activity.
Pentamidine, which is active against tryp- arsamide-resistant trypanoso’mes, was in- troduced into sleeping sickness therapy at a time when arsenical-resistant strains of T. ganzbiense had developed on a menacing scale in many areas of West Africa (e.g. van Ho’of, 1947); cross- resistance problems in sleeping sickness in man have thus never become so crucial as they now are in animal trypanosomiasis, es- pecially as pentamidine has shown no tendency to induce drug-resistance, and the new melaminyl drugs are also active against tryparsamide-fast strains. A remote, but potential, danger exists nevertheless in the use of these two types of drug, as resistant strains experimentally produced in the laboratory by treatment with either a diami- dine-type or melaminyl-type drug are cross- resistant, not only to members of each group, but also to a wide variety of other trypano- tides ( Rollo and Williamson, 195 1; William- son and Rollo, 1952, 1959).
A few of the innumerable factors which complicate both the initial evaluation and the ultimate application of an active “cattle” trypanocide have been indicated earlier (sec- tion 1.1)) and are discussed analytically in a valuable review by Whiteside (1958). There is an apparent crude difference in the relative virulence of T. vivax and T. congolense in West and East Africa; T. vivax is considered to be the more acutely lethal infection in the West and T. congolense is the more virulent in the East; as Herin (1958) says of T. vivax “la virulence de ce parasite parait varier avec la longitude.” Differential vir- ulence may obviously affect drug suscepti- bility, but evidence of this is, so far, only suggestive and probably insignificant. Wilson (1958) notes that over many years in North-
ern Nigeria “T. z&ax was considered more amenable to treatment by tartar emetic than T. congolense,” and at least one new tryp- anocide of the antrycide type (Cinnoline 528, section 2.6) was less active against West African T. vivax than against East African T. congolense and was therefore rejected for field use.
Comparisons of this type based on field practice or even on apparently adequately controlled field trials are difficult to establish (Marshall, 1958) mainly because of the variations which can occur (a) in the tsetse fly (species, habitat, infective inoculum, infection rate, density, frequency of bite, etc.), (b) in the trypanosome (species and strain), (c) in the host animal (age, breed, sex, colour, weight, condition, nutritional status, intercurrent infection, management and herding) and (d) in the area (climate, season, vegetation (e.g., incidence of poison- ous plants) and the interaction of these with (a) and (c) above). In attempts to control this variability, Smith and Rennison (1958) have defined trypanosome “challenge” as “the number of infective bites from a tsetse which a host receives in unit time”, and have stressed the necessity for refining methods of assessing ‘Lchallenge” in the light of field experience and in particular of the work of Whiteside (1955), who showed that in cattle, for a given prophylactic drug, the greater the challenge the shorter the period of prophy- laxis obtained. Whiteside also defined an index of trypanosome challenge for field use as the product of the number of non- teneral male and female tsetse per 10,000 yards (the Apparent Density), and their in- fection rate.
Attempts such as these to analyse, reduce and control the variables in the tsetse-tryp- anosome-animal complex are of the utmost importance for a satisfactory assessment of drug performance; many recent drug trials are models in this respect, and demonstrate the additional and more precise information that can be extracted from carefully con- trolled experimentation. The work of Smith (Smith, 1959; Smith and Brown, 1960) and of Stephen (1958) for example, conducted with the smaller-scale laboratory resources of the East and West African inter-territorial
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 287
trypanosomiasis research institutes respec- tively, illustrates the application of these principles; the use of rotational application of “Bruce” boxes containing known numbers of tsetse flies with a known infection rate, as in Stephen’s trials, show how the ?hallenge” variables may be controlled, given the necessary resources. An overriding require- ment for satisfactory assessment of drug per- formance is repetition of trial in as many different areas as possible, and recommenda- tions to this end, and to the necessity that field trials should take note of variables such as those listed earlier, have been made by the Inter-African Advisory Committee on Epi- zootic Diseases at their Symposium on Animal Trypanosomiasis held in 1958.
In Africa, where the resources for adequately controlled experiment in cattle are so variable, a simple but highly desirable way of ensuring some measure of compa- rability among a number of therapeutic or prophylactic trials carried out in different areas and under different conditions of challenge, is to incorporate at least two sets of control animals, one set untreated and in- troduced regularly into the experiment and the other treated with the well-tried drug antrycide pro-salt as a standard yardstick; both sets are exposed to the same challenge as the experimental animals.
These procedures, from the small-scale laboratory-controlled trial to the large-scale field trial in open country in fly areas, are costly and laborious, the more so in the case of prophylactic drug trials; to conform even to minimal scientific requirements, they re- quire extensive technical staff and resources, but set against the real and potential value of the African animal industry, the cost of this kind of vital research can be considered as very marginal indeed.
So far, none of the prophylactic drugs de- veloped in the last decade has yet come into generally approved use as a means of pro- tecting cattle for longer than the two-month period afforded by antrycide prosalt. As in sleeping sickness in man, the prime require- ment is for a long-lasting prophylactic drug, useable in conjunction with fly-eradication measures, to promote land reclamation and agricultural settlement, or to protect cattle
during prolonged seasonal trekking through fly belts in search of grazing. An alternative approach to prophylaxis, originating in the work of Soltys (1955) suggests the possibility of drug-stimulated immunity as a method of protection. Antrycide and ethidium have been shown to’ be active in this respect (White- side, 19584 b; 1960) but the work, though promising, is still embryonic (see section 3.3).
In the sections which follow (2.2 to 2.9), individual trypanocidal drugs are grouped on the basis of their ionic character at blood pH, a division which has some relevance to possible modes of drug action (see sections 3.1, 3.3 and Table VI).
2.2. NEUTRAL AROMATIC ARSENICALS ANL ANTIMONIALS
Although the synthesis and use of aromatic arsenicals continue unabated for such diverse purposes as motor fuel antioxidants, wood preservatives, phenol resin manufacture, ana- lytical precipitants, additives to anti-fouling paint and dentifrices, growth promoters in animal feeds, and as artificial haptens in experimental immunochemistry, the develop- ment and examination of new therapeutic compounds has largely been restricted to ex- perimental tubercular, amoebic, coccidial and filarial infections.
In this class of aromatic arsenic and anti- mony compounds carrying substituents which are only weakly ionized at blood pH, no new trypanocide has been developed to replace
bH.CH,.CONH,
0 I \ / HO- As -0Na
II 0
(22.1) Tryparsamide
either tryparsamide (2.2.1) (sodium p- glycineamidophenylarsonate) or an equiv- alent compound orsanine (2.2.2) (so- dium 2-hydroxy+acetamidophenylarsonate)
288
NH.COCH, I
WILLIAMSON
HO-is-ONa
II 0
(2.22) Orsanine
equally favoured by many French workers in Gambian sleeping sickness. The compounds to be described here have shown activity, but have not come into general use.
A new type of trypanocidal phenylarsonate devised by McGeachin (McGeachin and Cox, 1951; Cantrell and McGeachin, 1951) was based on the work of Jacobs and Heidel- berger (1919b) which produced tryparsa- mide. Only the methyl and ethyl esters of 4-arsonophenylglycine had been prepared by the latter workers, and McGeachin and Cox (1951) therefore investigated ester formation with a number of polyalcohols. The most active compound was a- [N- (p-arsonophenyl) glycyl J monoglyceride ( 2.2.3). In tests on
NH.CH,.COO.CH, I I
t~.01=4
I CH, .OH
(2.2.3)
mice infected with T. equiperdum, this com- pound (2.2.3) was more active than the corresponding acid, but much less active than tryparsamide; a single intraperitoneal injection of 750 mg/kg cured only one mouse in five, with an estimated LD 50 of about 2000 mg/kg (Cantrell and McGeachin, 195 1) .
The discovery of British Anti-Lewisite (BAL) (dimercaprol) (2,3-dimercaptopro- panol) (2.2.4) by Peters, Stocken and Thompson (1945) and its ability to de- toxicate trivalent arsenic by the formation of a highly stable ring compound led to the
H, .C.SH
I H, .&SH
I CH,.OH
(2.2.4) BAL (British Anti-Lewisite)
combination of BAL with a number of phenyl- arsenoxides such as oxophenarsine (m-amino- p-hydroxyphenylarsenoxide) (2.2.5) to pro- duce the corresponding cyclic thiol derivative, in this case BAL-OX0 (2.2.6) (Peters and Stocken, 1947; Friedheim and Vogel, 1947).
OH
b I \ NH* + BAL
/ -
As II 0
(2.2.5) Oxophenarsine
OH
b I \ NH* /
S/A=\S L Hz /
-CH.CH,OH (2.2.6) BAL-OX0
Several compounds of this type, prepared by Friedheim, were tested against sleeping sickness in man by Le Rouzic (1949b) ; they included tartar emetic-BAL (Detox) and BAL compounds with the arsenoxide form of tryparsamide (TPB), stovarsol (STB) (2.2.7) and carbarsone (CBB) (2.2.8). (BAL compounds of Friedheim’s p-melami- nylphenylstibinoxide (MSbB) and p-mela-
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 289
OH
NH.COCH,
NH.CH,.CONH, I
Q I \ / /As\ 3 ;
H2C-CW.CH20H (2.2.8) CBB
minylphenylarsenoxide (Melarsen oxide) were also tested (see sectio’n 2.3) ; the latter BAL product has since become well-known as Mel B (melarsoprol B.P., or Arsobal). The failure rate of Detox, TPB, STB and CBB in later-stage cases was apparently fairly high, and none of them has subsequently come into general usage.
Sweet and Tillitson (1951) and Tillitson ( 195 1) have claimed trypanocidal activity for a series of dithioarsenites derived from oxophenarsine (2.2.5) (or its 4-hydroxy- ethoxy analogue) ; in the dithioarsenite group- ing, -As(S.CH~.CO.R)~, R = -OH, -0 -alkyl ( 1 to 12 C atoms) or -NHz, the alkyl ester with 6 to 12 C atoms being the most active. Dithioarsenites of this kind have been frequently prepared (Cohen, King and Strangeways, 1931; Doak and Eagle, 1951) and in general are less toxic and less active than the free arsenoxide parent form, and usually more easily dissociable than the cyclic dithiol products of BAL condensation.
A product “Trepolysin (4000 M),” ap- parently a camphorsulphonate of oxophen- arsine, active in nervous syphilis, has been tested in sleeping sickness cases in French Equatorial Africa by Ceccaldi, Trinquier and Arnoult (1953) but was not considered suit- able. Although active against blood and lymph parasites, it had no advantages in this respect over existing drugs, and in ad- dition it was poorly tolerated by old cases, inactive against cerebrospinal fluid (CSF) infections and, in fact, appeared to hasten the onset of nervous involvement.
Although a number of heterocyclic arsenic compounds, with arsenic as a heteroatom have been prepared (Mann, 1950), such as arsacridine (Hewett, Lermit, Openshaw, Todd, Williams and Woodward, 1948) and arsaphenanthridine derivatives (Cookson and Mann, 1949; Mann, 1958) including quater- nary arsonium salts, little or no information is available on the biological activity of these compounds. The “bioisosteric” analogy (Friedman, 1951) whereby the positive charge, tetrahedral configuration and some of the biological activities (e.g. Peyron, De- Pierre, and Jacob, 1954) of the quaternary ammonium group are also common to ar- sonium and stibonium groups, might be ex- pected to apply to such metal-containing analogues of trypanocidal acridinium and phenanthridinium drugs; one difficulty as Schatz (1960) indicates, may be that in this series of isosteres, which also includes sulpho- mum and phosphonium groups, the usual tendency is for biological activity to decrease, and toxicity to increase in the order: R4Nf, R$f, R,P+, R4As+ and R&Sb+.
An interesting attempt to produce an on- colytic metabolite ana.logue containing arsenic was made on these lines by Angier, Gazzola, Semb, Gadekar and Williams ( 1954) who synthesized pteroic acid analogues in which an arsenic acid group was substituted for the carboxyl of the terminal p-aminobenzoic acid moiety. The possibilities of applying this kind of approach to trypanosomal chemotherapy have yet to be explored.
Some examples of attempts to combine the therapeutic activity of aromatic arsenicals with those of other types of compound are provided by Gailliot and Baget (1955) who
290 WILLIAMSON
prepared salts of tetracycline antibiotics with phenylarsonate derivatives (no record of trypanocidal activity), and by Bose and Bose (1952). The latter, impressed by the tryp- anocidal activity of aromatic diamidines (section 2.5), tested (i) p-amidinophenyl- arsenoxide and (ii) a number of related phenylarsenoxides with a variety of sub- stituents in the p-amidino group, against T. equiperdum and T. evansi infections in mice. The group of compounds (ii) were much more toxic and less trypanocidal, though more bacteriostatic in vitro, than (i). Insuf- ficient data are given to estimate the curative power of (i) but the therapeutic index was obviously low. One danger of this type of “hybridization” approach to synthesis is that the hybrids may produce strains rather readily cross-resistant to the different drug types represented in the molecule. The melaminyl- phenylarsonate “Melarsen” for example, in- duces resistance both to aromatic arsenicals and to the diamidines (Roll0 and William- son, 1951) (sections 2.3, 3.3). Schmidt and Kikuth (1950) have described a number of compounds containing both arsenic and anti- mony, either as an -As=Sb- linkage in an arsphenamine-type molecule, or as sub- stituents on the same benzene ring; an arseno- phenylglycine analogue was active against T. lewisi and an unspecified arsenical-resis- tant trypanosome, but the trypanocidal ac- tivity of the second group of compounds was not remarkable.
The trypanocidal action of oxophenarsine in mice infected with T. equiperdum is said to be potentiated by 4’-chloro-3,4-dihydroxy- chalcone which is itself slightly trypanocidal (Moss, Carley, Beiler, and Martin, 1952) ; the bromo-substituted analogue and several other halogenated derivatives of similar struc- ture had no action.
Toxicity and Pharmacological Studies
Apart from the routine investigations re- ported for new substances such as those listed above, the toxic properties of established trypanocides continue to be examined. In particular, medication with tryparsamide and other pentavalent arsenicals has long been known to be responsible for ocular complica- tions and even to have been implicated as
an important source of blindness in the Bel- gian Congo (Debeir, 1953, 1954), where the incidence of ocular damage after treatment was of the order of 10 to 20%; to some extent this risk is now being diminished by mass pentamidine prophylaxis and the use of Mel B.
The pathology of central nervous lesions in advanced sIeeping sickness has not been extensively investigated (and lesions in the peripheral nervous system, considered to be at least as important (Janssen, van Bogaert and Haymaker, 1956; van Bogaert and Jans- sen, 1957)) have received even less atten- tion), but it is generally believed that the ocular lesions in sleeping sickness are not due primarily to the infection but to treat- ment with pentavalent arsenicals (Ridley, 1945; Pieraerts, 1952; Debeir, 1954). Loss of vision is attributed to degeneration of the ganglionic cells of the retina possibly as a result of vasoconstriction in the retinal blood supply (Ridley, 1945) ; a characteristic feature of the lesion is the pallor of the optic disc. As a remedy Debeir (1953) recom- mended intravenous vasodilator administra- tion; BAL treatment is of little use in late cases. More recent investigations in monkeys and rabbits by Hurst (1959) show that tryp- arsamide, in addition to producing hemor- rhagic and necrotic lesions at a number of sites in the central nervous system, character- istically produce further lesions in the cerebel- lar white matter and the optic tracts. The latter lesions involve considerable prolifera- tion of glial cells.
The apparent restriction of optic atrophy to pentavalent arsenicals could be taken as a reflection of the inability of phenylarsen- oxides to pass from the blood into the cere- brospinal fluid, but the results of Hurst (19591, who also examined p-melaminyl- phenylarsenoxide and an analogous pyrimi- dylphenylarsenoxide (see section 2.3), both of which are capable of penetrating the CSF but do not produce optical lesions, suggest that this may be an over-simplification.
The experimental detoxication of penta- valent arsenicals by p-aminobenzoic acid (PABA) and related compounds (Sandground and Hamilton, 1943 ; Sandground, 1944) does not seem to have had any clinical application
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 291
in trypanosomiasis presumably because the effect is demonstrable only by pre-treatment with the detoxicant. PABA has however been used successfully in this way to reduce the toxicity of growth-stimulant phenylarsonate additives in poultry feed (Wharton, Fritz, Schoene and Smidt, 1959).
New work on the distribution of organic arsenicals and antimonials is not extensive despite the possibilities offered by the use of isotopically-labelled drugs, such as the ( 76As) -atoxyl (sodium p-aminophenylarso- nate) and ( 76As) -arsphenamine (3,3’-dia- mino-4,4’-dihydroxyarsenobnzene dihydro- chloride) prepared by Buu-HoY et al. (1949). ( lz4Sb) -1abelled sodium antimony tartrate, sodium antimony thioglycollate and sodium antimony thiosalicylate have been used in distribution experiments on T. gambiense- infected rats (Ito, Kirita, and Tomatsu, 195 5)) which showed that although maximal uptake of all compounds in any organ oc- curred in one hour, the Sb concentration in any given organ depended on the compound administered. With all three compounds, the highest and lowest Sb concentrations were found in the kidney and pancreas respec- tively; rats infected with T. gambiense showed a more rapid Sb excretion and a lower concentration of Sb in the organs than uninfected rats. The three compounds dif- fered however in their effect on the rate of disappearance of trypanosomes in mice in- fected with standardised T. gambknse in- ocula. Depending on the drug, linear, uni- molecular or sigmoid rate curves were obtained which may have reflected differ- ences in mode of action.
A study in rats of the rapid inactivation of oxophenarsine given intravenously (Peters and Wright, 1951) and using a biological assay of trypanocidal activity in parallel with a chemical analysis of arsenic content, has shown that although the former activity is decreased in the blood 4 hours after drug injection and is lost in 24 hours, the blood arsenic level, after an initial fall, is in fact higher in 24- and 48-hour samples than in 8- and 12-hour samples; at 8 hours the trypanocidal activity in vitro on an arsenic basis, is equivalent only to 1% of the level found by chemical analysis. Although blood
cells have a strong affmity for arsenic, in- activation by rat blood in v&-o occurs much more slowly, and extravascular tissues are therefore implicated as a major source of in- activation in vivo.
Empirical inquiries into the action of aro- matic arsenicals, including some of tryp- anocidal interest, on other kinds of cell, such as tumour (Leiter, Downing, Hartwell, and Shear, 1952) or mammalian cells in vitro (Savchuck, Loy, and Schiaffino, 1960), in general have confirmed the superior cyto- toxicity of phenylarsenoxides over phenyl- arsonates, and the most extensive structure- activity correlation in this series has been carried out by Doak and Eagle ( 195 1) (see also Eagle and Doak, 1951). Their study of a large series of substituted phenylarsen- oxides extended and confirmed the earlier observations of King and Strangeways (1942) and of Gough and King (1930), and in their examination of acidic substituents, which in general reduce the parasiticidal activity of the parent phenylarsenoxide, they record the unique activity of the y-(p-arsen- osophenyl) butyric acid derivative, reported earlier as ‘Butarsen’ (Eagle, 1945) (see sec- tion 2.8).
Only a few generalizations on monosub- stituted derivatives could be made, such as that (a) -CHa, -NOB, -Cl, -NH2, -OH, or -F groups did not markedly alter the toxicity or parasiticidal activity from that of the parent phenylarsenoxide, although (b), acidic substituents in general markedly de- creased both toxicity and activity, especially if ionized at blood pH, and (c), as noted by Gough and King (1930), amide substituents caused a marked decrease in toxicity with only a slight effect on activity. Little guid- ance was available to predict the behavior of di-substituted compounds, and in view of the exceptional behavio,ur of compounds such as melarsen oxide, oxophenarsine and butar- sen, the authors rightly emphasize “the un- reliability of generalizations with respect to the effect of a given type of substituent on the biological activity of arsenosobenzene.”
A valuable feature of this study was the extended evidence demonstrated for a rela- tionship between the systemic toxicity of arsenicals and the degree to which they were
292 WILLIAMSON
bound to erythrocytes, and for a similar correlation between parasiticidal activity and the amount of drug bound by the parasite, in confirmation of the earlier work of Yorke, Murgatroyd and Hawking (1931b), Reiner, Leonard and Chao (1932) and Hawking ( 1937). No correlation was found between toxicity or parasiticidal activity and the hydrolysis constant of the corresponding thioarsenites; the high selectivity of arsenical action was considered to be most plausibly explained by differences in cell permeability. Alternative assumptions, and also the unsatis- factory nature of the authors’ explanation of the effect of pH on the trypanocidal ac- tion of butarsen are discussed elsewhere (section 3.3).
2.3. MELAMINYL ARSENICALS AND
ANTIMONIALS
This novel class of phenyl-arsonates, -arsenoxides, -stibonates and -stibinoxides in which melamine (2,4,6-triamino-s-triazine) (2.3.1) is coupled in the p-position to the
2
(2.3.1) Melamine
metal-containing substituent, originated with Friedheim’s publication in 1940 of the tryp- anocidal activity of melarsen (disodium p- melaminyl-phenylarsonate) (2.3.2).
(2.3.2) Melarsen
Friedheim’s original synthesis (1942), in which cyanuryl chloride (2.3.3) was con- densed with atoxyl (sodium p-aminophenyl- arsonate) in alkali, followed by reaction with ammonia, was found unsuitable for large-scale
Cl
A N \N
CIAN& (2.3.3) Cyanuryl chloride
production by Banks, Gruhzit, Tillitson and Controulis (1944), who modified the synthe- sis by condensing atoxyl with 2-chloro-4,6- diamino-s-triazine in acid solution. The cor- responding arsenoxide (melarsen oxide) is obtainable by reduction with sulfur dioxide in the presence of hydriodic acid (Friedheim, 1944). (A useful synoptic diagram of these and other related syntheses of organic arsenicals, which indicates the pivotal utility of atoxyl, is given by Doak and Freedman (1960).)
Of the antimony analogues, the pentavalent compound corresponding to melarsen (Fried- heim and Berman, 1946; Friedheim, Vogel and Berman, 1947), can exist as a crystalline monomer, or as an amorphous polymer (MSb) which, unlike other polymeric stibanilates, shows increased trypanocidal activity and decreased toxicity, and in this form also has remarkable prophylactic ac- tivity (Mayer and Brousseau, 1946; Rollo, Williamson and Lourie, 1949). The trivalent analogue (MSB3) corresponding to melarsen oxide does not polymerize and has no prophy- lactic activity; its trypanocidal activity is comparable to that of melarsen oxide (Rollo, Williamson, and Lourie, 1949).
Following earlier experiments (Friedheim and Vogel, 1947), both melarsen oxide and MSb 3 have been coupled with BAL (2,3- dimercaptopropanol) (2.2.4) to give Mel B (arsobal) (2.3.4) and MSbB respectively (Friedheim, 1949, 1953b,c; Le Rouzic, 194913).
A variety of forms of these six basic com- pounds has been described in patent form by Friedheim (1945a, 1945b, 1945c, 1947a, 194713, 1947c, 1953c, 1956), but despite their toxic side-effects, only melarsen and Mel B, because of their activity against tryparsamide-resistant T. gambiense infec- tions and particularly against late-stage T.
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 293
(2.3.4) Mel B
rhodesiense infections, have come into field use.
Activity against tryparsamide-resistant T. gambiense is of considerable importance. Van Hoof (1947) quoted almost 100% incidence of tryparsamide-resistant strains in certain areas of the Belgian Congo by 1947, and Friedheim (1949) states that in 1946 in French West Africa, 80% of all second-stage cases of Gambian sleeping-sickness were tryparsamide-resistant ; the gravity of these figures was emphasized by the fact that of 50,000 new cases discovered in 1946, 60% were already in the second stage of the infec- tion. The activity of melarsen oxide against tryparsamide-resistant trypanosomes demon- strated both in the field (van Hoof, 1947) and in the laboratory (Williamson and Lourie, 1948), and its ability to affect the cerebrospinal fluid infection in man, have led to development, first of the less toxic BAL- compound Mel B, and more recently to an apparently even less toxic and more water- soluble derivative, Mel W (Friedheim and De Jongh, 1959; De Jongh and Friedheim, 1959). This compound, which can be given intramuscularly in saline or glucose, unlike Mel B which is given intravenously in propylene glycol, is half as toxic as Mel B in mice treated intraperitoneally, and so far appears to be well tolerated in man; its con- stitution has not yet been announced.
(i) Melarsen (2.3.2). Although less atten- tion seems to have been paid to melarsen than to Mel B (van Hoof (1947), for ex- ample, recommended that it be abandoned because of its toxicity), the favourable im- mediate results obtained with it by McLetchie (1948) in Nigeria have been confirmed by Duggan and Hutchinson (1951), and on the recommendation of the International Scientific Committee for Trypanosomiasis Research, further melarsen trials with adequately standardised products have been
undertaken (Butler, Duggan and Hutchin- son, 1957; Hutchinson, 1956). The toxicity of Mel B requires that treatment be admin- istered under strict medical surveillance or in hospital. This is often difficult or im- possible in the majority of cases of sleeping sickness, ;ts these occur in undeveloped country where treatment conditions must necessarily be crude. Melarsen is much less toxic than Mel B, is water-soluble, easy to administer and in Gambian sleeping sbckness, a recommended course (8 injections of 15 mg/kg at S-day intervals) is shorter than, and at least as effective as an equivalent tryparsamide course ( 12 injections of 3 g at S-day intervals) ; it also has the advantage of being more active than tryparsamide in late-stage cases and of being active also against tryparsamide-resistant cases. The main factor militating against its more wide- spread use would appear to be its higher cost. Ross (1960), referring to treatment of sleeping sickness in Northern Nigeria, (an area equivalent to that of France), states that melarsen is used only for cases which have relapsed after suramin-tryparsamide or pentamidine-tryparsamide treatment, or for far-advanced cases, mainly because pen- tamidine-tryparsamide treatment gives good results with early cases, but also because melarsen is much more expensive.
Duggan and Hutchinson (1951) recorded results of treatment in over 200 cases of sleeping sickness, followed up for two years. Treatment courses of 15 mg/kg for 8 or 12 days at S-day intervals gave 100% cure in early cases, over 80% cure in intermediate cases and over 50% cure in late cases, but toxic effects were greater than with tryp- arsamide. Butler et al. (19.57), however pointed out that before 1951, when standard- ized products became available, adequate assessment of melarsen was complicated by the possibility of batch variation, and under- took a careful trial of four different treat- ment courses. They showed that the in- dividual dose was more important in pro- ducing toxic effects than the length of the course, and recommended a maximum of 15-20 mg/kg for field use in Nigeria; no significant differences were observed in 8- or 12-day courses at this dose.
294 WILLIAMSON
292 patients were treated with melarsen, of whom 244 were followed up for at least two years. Twenty-four advanced cases who received a higher dose of 30 mg/kg given under medical supervision showed a re- markable cure rate of 7 1% ; the corresponding rate in 25 cases given 20 mg/kg was 56%. This compares very favourably with the normal expectation of about 30% cure or less after tryparsamide treatment. Although melarsen and control courses of suramin- tryparsamide or pentamidine-tryparsamide were all equally effective against early cases (cure rates 96%, 91% and 78% respectively), melarsen was also effective in the treatment of intermediate (70% cure) and advanced cases (38 s cure) which had relapsed to up to eight previous treatment courses, including tryparsamide.
On this basis, melarsen would seem to be preferable to Mel B, and to have most, if not all of the advantages claimed for Mel W. Its possibilities in the treatment of T. rho- desiense sleeping sickness do not appear yet to have been explored as extensively as those of Mel B; Apted (1957) states that it has been used “in a small series of cases with good results comparable with those obtained with Mel B”.
(ii) Melarsen oxide (2.35). This triva- lent form of melarsen was described by Friedheim (1944) following an announce-
(2.3.5) Melarsen oxide
ment of its activity against sleeping sickness in West Africa (Friedheim, 1941) . Although it has been superseded by its BAL derivative, Mel B, early trials indicated its value in late-stage Gambian sleeping sickness and its activity against tryparsamide-resistant in- fections, despite the drawback of considerable toxicity (Findlay, 1950). As late as 1953, when extensive Mel B trials were already under way, Jonchere, Gomer and Reynaud noted that melarsen oxide could “cure a
considerable number of patients who would not be saved from death by other drugs,” but that its toxicity would preclude its gen- eral use in the field.
(iii) Mel B (Arsobal) (melarsoprol B.P.) (2.3.4). During the last decade, Mel B has come into considerable prominence, to the virtual abandonment of its precursor, melarsen oxide. Friedheim ( 1949), in his first ac- count of its activity in sleeping sickness in man, recommended a treatment course of daily intravenous injections of 3.6 mg/kg for 4 days, followed by an interval of one week and a further course of four similar injections. A 3- to 4-day interval between injections was advised for very advanced cases to avoid Herxheimer-type reactions due to sudden massive destruction of parasites in the central nervous system. In this first trial 50 inter- mediate T. gambiense cases, of whom 20 showed trypanosomes in the cerebrospinal fluid, were treated and followed up for 7 months; parasites disappeared from the body fluids of all cases after the first injection with a return, in varying degree, to normal of the pathological cerebrospinal fluid values (cell number and protein content). In a later trial in the French Cameroun, Friedheim treated 393 cases of tryparsamide-resistant T. gambiense sleeping sickness with Mel B, and 50 similar cases with a related product RP 3826 (2.3.6). All cases showed nervous system involvement and were “the doomed ‘left overs’ of mass treatment.” On the basis of observation periods extending up to 15 months, Friedheim claimed that Mel B treat- ment, of the type recommended in his earlier trial ( 1949)) could ensure a significant chance of cure for about 97% of tryp- arsamide-resistant second stage cases,
The compound RP 3826, which was much less effective than Mel B, is melarsen oxide dithiomalate (2.3.6), and its inferior activity “demonstrates clearly the advantage of cyclic over open chain arsenic dimercaptide structures” (Friedheim, 1951a) ; thiomalate had in fact been used earlier by Ceccaldi, Trinquier, Pellissier and Arnoult (1949) to reverse the toxic effects of melarsen oxide in man.
Some 3% of the tryparsamide-resistant cases were also resistant to Mel B, and this
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 295
CH,.COONa
resistance was enhanced Il-fold by pretreat- ment with the dithiomalate compound. The development of resistance to melaminyl drugs is discussed elsewhere in relation to possible cross-resistance with the diamidines (section 3.3) but this early demonstration of resist- ance in the field is of considerable impor- tance in view of the increasing use of mel- aminyl trypanocides.
The overall impression of Mel B treat- ment of both Gambian and Rhodesian sleep- ing sickness is that it is undoubtedly active on all stages of the infection, but that its toxicity requires hospital treatment and precludes treatment of ambulant patients, and that cases of Mel B-resistant infection, although as yet fairly rare, can and do occur.
In the early stages of its trial in Africa, Mel B gave encouraging results from a number of areas. In Ruanda-Urundi, in an area with a high incidence of arsenical-resist- ant trypanosomes, Mel B was effective in treating 187 new cases uncovered in 1950, of whom 180 were in the nervous stage, possibly as a result of mass pentamidine prophylaxis during the preceding four years (Vincent and Lietar, 195 1). In similar situations in other Congo areas, Mel B has also proved valuable (Sterckx, 1953 ; Burke, 1957), and Neujean and Evens (1958) confirm this in an extended study of 410 cases.
In French Equatorial Africa, Ceccaldi (1952) found Mel B treatment of advanced cases 50-600/0 successful and was able to cure lO/ll early cases with a single injection of 4 mg/kg, an effect also demonstrated by Pinto (19.54). Out of 73 cases, there were five toxic encephalopathies of which two were fatal, but useful information on optimal treatment courses was obtained and later
applied successfully (Ceccaldi, 1953) in a series of 105 drug-refractory cases. In this series more than 40% were cured using two separated courses of three or four daily in- jections of not more than 3.6 mg/kg. In West Africa, Ferreira, de Almeida, and Pires (1950) reported favorable effects on ad- vanced T. gambiense infections in Portuguese Guinea, and Richet, Lotte, and Foucher (1959) have reviewed extensive trials of Mel B in the North Dahomey area in French territory. It was first used in 1948 and in the succeeding 10 years, 795 cases were treated, of whom 19 died following treat- ment ; in 148 cases treated since 1952, when more rigorous treatment precautions were introduced, no deaths ensued. For safety, Mel B treatment was not advised for young patients, first-stage infections or for those in poor condition or with psychic changes, and BAL was recommended as a helpful anti- dote. Its curative activity in advanced cases was nevertheless considered remarkable.
Similar dramatic effects have been obtained in otherwise hopeless cases of Rhodesian sleeping sickness in Belgian territory (Marneff, 1955; Adriaenssens, 1960)) in Portuguese territory (Silva, Caseiro, Carmo and de Basto, 1954; Silva, 1957), and in British East Africa (Apted, 1953, 1957; Taube and Nixon, 1958), but even with the recommended doubIe course of treatment, toxic side effects have been serious in many cases. Marneffe (1955) recorded four deaths in 23 treated cases, and of 55 cases treated by Adriaenssens (1960)) 15 devel- oped drug complications; 10 of these were fatal encephalopathies. Similar fatalities (11/165 and 27/339 treated cases) were recorded by Silva et al. (1954) and Silva (1957) respectively. In the interval between
296 WILLIAMSON
the two Mel B courses, Taube and Nixon (1958) have used suramin treatment, which often has a tonic effect on advanced sleeping sickness cases, as a means of improving the condition of the patient and thus reducing the chances of toxic side effects of Mel B.
There is no doubt however, that despite the toll of toxic deaths and injuries, Mel B has saved the lives of many advanced cases of Rhodesian and tryparsamide-resistant Gambian sleeping sickness who would other- wise have inevitably died (Apted, 1957; Neujean and Evens, 1958).
(iv) ikls6. This interesting antimony analogue of melarsen (2.3.2) (sodium p- melaminylphenylstibonate) , (Friedheim and Berman, 1946; Friedheim, Vogel and Ber- man, 1947; Friedheim (patents) 1947d, 1948, 1951b), exists as a polymer which tends to precipitate in a sparingly soluble form in the tissues ; polymerization probably occurs through the stibonic acid radical (Friedheim, 1953b). For this reason it is only weakly toxic and has a high therapeutic index and re- markable prophylactic activity, either orally or parenterally, in experimental trypanosome infections (Mayer and Brousseau, 1946; Rollo, Williamson, and Lourie, 1949). The monomer, although of equivalent therapeutic activity, has no prophylactic activity.
The potential value of MSb in clinical prophylaxis in French Guinea was indicated by Le Rouzic (1949a) but apart from an ex- tended trial of its therapeutic activity against Gambian sleeping sickness in this area (Friedheim, 1953b), no further clinical ap- plication has ensued. Production batch variation was considered to be an important cause of conflicting results in the experi- mental clinical trials of the melaminyl drug series (Lourie, 1952) and to be especially important in a polymerized antimonial like MSb. Effective biological standardization
was urged (Lourie, 1952), and as a result, international biological reference preparations of both Mel B and MSb were set up in 1954 (W.H.O., 1960). A standardization proce- dure for MSb samples in terms of toxicity and therapeutic and prophylactic activity against T. equiperdunz infections in mice was also described at this time (Edge, Hill and Stone, 1954), and indicated that batch variation was not perhaps so serious as had been previously thought. In view of the unique properties of this compound, which, of all trypanocides so far synthesized, would seem to correspond most closely, at least ex- perimentally, to Ehrlich’s ideal of a therapia sterilisans magna, it is surprising that its clinical development has not been pursued, particularly as it also shows prophylactic activity against experimental filarial infec- tions (Kershaw, Williamson, and Bertram, 1949).
Friedheim (195313) treated 12 first-stage T. gambiense sleeping sickness cases with MSb given intramuscularly as a micronized suspension of the free acid in oil; aqueous solutions of the sodium salt were not rec- ommended as they gave rise to painful in- filtrations. Nine t’o twelve daily doses ranging from 6 to 22 mg/kg, effected cure as judged by subsequent observation over 27 to 30 months. Four cases (3 early and 1 advanced stage), were treated orally with 5 to 6 daily doses of 10 to 20 mg/kg but curative results, although apparently effective, were more slowly achieved.
(v) MS6B and MSb3. MSbB (2.3.7) a trivalent analogue of MSb containing the cyclic dithiastibina radical formed by com- bination with BAL, and thus representing the antimony equivalent of Mel B, was found to have comparable trypanocidal activity to Mel B (Friedheim, 1953b). Given orally in combination with MSb free acid, effective
(2.3.7) MSbB
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 297
cure (70-1000/o) of 98 first stage, and 19/26 second stage cases was achieved. The ad- vantages claimed for this form of treatment were that treatment time could be cut down to 3 to 7 days, and that the risks of arsenical encephalitis which attend melarsen and Mel B usage were obviated. Further confirma- tion of these results would be desirable, in view of Le Rouzic’s report (1949b) of failure rates of about 20% in first- and second-stage cases treated with a similar drug combination.
The antimony equivalent of melarsen oxide, MSb3, has been used experimentally in the form of the dithioglycollate (Fried- heim, 1947e) but it is less stable and more toxic than the corresponding arsenoxide; this deficiency is said to be overcome by formation of the cyclic BAL derivative MSbB (2.3.7).
(vi) 4-(2,6-diaminopyrimidyl-4-amino) phenylarsenoxide (I.C.I. compound 12,065). At this point, a new pyrimidylphenylarsen- oxide (2.3.8) of similar structure and prop-
“&JJAs40 (2.3.8) I.C.I. compound 12,065
erties to melarsen oxide may legitimately be considered, although it is not strictly a mem- ber of the melamine drug series. This was the most active compound (Ainley and Davey, 1958) of a series of arylarsen- oxides prepared by Ainley (1955), and could be used either as an insoluble carbonate addition salt or as the soluble isethionate; both had similar curative activity in T. rho- desiense infections of mice. Baker (1958), in a more intensive study of its properties, showed that the intramuscular LDSO in mice was about 22 mg/kg, with a CD50 of 6 and 2 mg/kg respectively, against two recently isolated strains of T. rhodesiense; this cura- tive activity was much less than that origi- nally reported for an old syringe-passaged strain (0.25 mg/kg). The toxicity and
therapeutic ratios for compound 12,065 and Mel B were very similar in mice (4 and 6 respectively), but there was some evidence that the pyrimidyl compound was more toxic in man.
Toxicity and Pharmacology
In Friedheim’s original study (1944) of a series of melarsen analogues, the im- portance of intact amino groups in the 2,4- positions of the triazine ring is indicated by the sharp increase in toxicity (up to 3O-fold) which follows progressive substitution of one or both groups by alkyl substituents to form secondary and tertiary amino groups. This structural specificity is no doubt signifi- cant for such unique properties of the melamine drugs as their ability to act on trypanosomes resistant to other types of or- ganometallic drug, and to show cross-resist- ance to non-metallic drugs such as the diamidines, but most of the central and peripheral nerve toxicity to which the mela- mine arsenicals can give rise, appears to be essentially similar to that caused by other arsenicals (Michielsen and Triest, 1954; Hurst, 1959). The typical symptoms of Mel B toxicity have been graphically de- scribed by Neujean and Evens (1958).
So far, no optical lesions have been re- ported, in contrast to tryparsamide which shows a characteristic pattern of nervous damage in experimental animals (Hurst, 1959). Kidney damage and resultant albu- minuria are common symptoms of arsenical toxicity and have been reported for Mel B (Michielson and Triest, 1954) ; in experi- mental animals, melarsen oxide and its pyrimidyl analogue, compound 12,065, produced kidney lesions similar to those caused by organometallic diuretic com- pounds, and compound 12,065 was in fact found to be diuretic. In man, Mel B has also been found to produce myocardial dam- age and hypertension in a number of cases (Michielson and Triest, 1954), and as a further instance of the care necessary in Mel B treatment, Janssens, Van Bogaert, Michiels, and Van de Steen (1960) have shown that Mel B treatment is capable of aggravating encephalitis due to an inter- current viral infection, especially in sleeping
298 WILLIAMSON
sickness cases where the patient’s resistance is usually low.
Urinary excretion studies have shown that another contributory factor in Mel B toxicity is arsenic retention, which appears to occur particularly in long treatment courses (Mon- net and Baylet, 1951a). These authors have also shown (1951b) that storage instability under tropical conditions is unlikely to con- tribute to Mel B toxicity, and demonstrated in mice the interesting point that, on the basis of equivalent arsenic content, Mel B was somewhat more toxic than the parent melarsen oxide. This effect has been noted with other arsenoxide-BAL compounds and is discussed by Findlay (1950). The in- creased lipid solubility which the cyclic dithiol radical confers (Friedheim and Vogel, 1947) is probably responsible, and may ac- count also for the ability of this type of drug to penetrate into the cerebrospinal fluid (see section 3.1) .
Cross-resistance with diamidine drugs might suggest that the melamine ring opens in vivo by hydrolysis to a corresponding biguanide-type compound. This appears unlikely, as p-biguanidophenylarsonic acid (2.3.9) (corresponding to an “open ring” melarsen), has been synthesized by Banks, Controulis, and Holcomb (1946) and found by McGeachin (1953) to have no tryp- anocidal activity; the toxicity appeared to be similar to that of melarsen.
yJ2
,,N6\
2.4. ACRIDINE DERIVATIVES
Trypanocidal acridine derivatives such as acriflavine, are mainly of academic interest
in relation to modes of trypanocidal drug action and drug resistance, and are discussed in this context in sections 3.2 and 3.3. No new active trypanocidal compounds of this type have been produced in the period under review.
2.5. DIGUANIDINE AND DIAMIDINE
DERIVATIVES
In human trypanosomiasis, as outlined in section 2.1, pentamidine (4,4’-diamidinodi- phenoxy-1,5-n-pentane) (2.5.1) continues to be the only diamidine compound in clinical use.
The salts in general use are the di-isethio- nate, where X = P-hydroxyethanesulfonic
CH20H acid, ( , or as in the French
CH2. SOaH equivalent lomidine, the dimethanesulfonate, where X = methanesulfonic acid, CHs*SOaH. The molecular weights are respectively 592.7 and 532.7, and for comparative therapeutic purposes, the drug weights which contain 1 mg of pentamidine base are 1.74 mg and 1.56 mg respectively. Occasionally the dihydro- chloride may be used, of which 1.21 mg con- tains 1 mg of pentamidine base.
No new diamidine or diguanidine tryp- anocide has been developed for human tryp- anosomiasis, but a new aromatic diamidine with a diazoamino linkage (Berenil, 2.5.8), has been introduced effectively into cattle trypanosomiasis therapy. The use of di- amidines is not however restricted to tryp- anosomiasis, and their activity against leish- mania1 and fungal infections and also against tumour cells, has yielded information which in some cases is relevant to the trypanocidal and prophylactic activity of the group.
Stilbamidine (4,4-diamidino-stilbene) (2.- 5.2), is known to have a selective action on neoplastic cells and its use in multiple myelomatosis was initiated according to Karnofsky and Burchenal (1949) “on the
HN‘?~O-($H$ -0
H,N’ 2 (25.1) Pentamidine
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 299
(2.5.2) Stilbamidine
rather fanciful rationale that since hyper- globuiinaemia occurs in kala-azar and in multiple myeloma, and since stilbamidine is effective against kala-azar, it might also be effective against multiple myeloma.” Its ef- fectiveness may be doubtful (Bichel, 1952), but a considerable amount of work by Snapper and his colleagues has concentrated attention (a) on its ability to combine with nucleic acid (Snapper, Schneid, and Kurnick, 1950; Snapper, Schneid, McVay, and Lieben, 1952), (b) on the properties of a less toxic derivative, 2-hydroxystilbamidine (2 5.3) (Ashley and Harris, 1946), and (c), on the retention of diamidines in human tissues for long periods; the evidence from (c) has been useful in understanding why the prophy- lactic activity of pentamidine is high in man but negligible by comparison in rats and mice.
R” ‘\,H I
(2 .S .3) 2 -Hydroxystilbamidine
2-Hydroxystilbamidine (Ewins, Ashley, Self, and Harris, 1946; Ewins, 1950) does not give rise to the trigeminal neuropathy which stilbamidine causes in man, possibly because the latter is much less stable to ultra- violet irradiation (Snapper et aE., 1952). It also shows a characteristic shift in the fluorescence spectrum when combined with ribonucleic acid (RNA) and this property has been used by Ormerod (1951b) in studies on trypanosomes resistant to an- trycide and stilbamidine (see section 3.3) which suggest that such combination with RNA-containing structures occurs in normal but not in resistant parasites.
The fungistatic properties of aromatic diamidines ( Heilman, 19 5 2 ; Taschd jian,
1954; Mackinnon, Artagaveytia-Allende, and Garcii-Zorron, 1958) may be related to their leishmanicidal activity, and in fact a relation between fungistatic and leishmanicidal prop- erties is suggested by McMillan (1960), who showed that, among a number of anti- biotics, only those with antifungal activity were active also against Leishmania spp. in vitro. If this is so, then pentavalent aromatic antimonials which are active in leishmaniasis might be expected to be good antifungal agents.
Pentamidine
The use of pentamidine (2.5 .l ) in sleep- ing sickness in the field has been outlined earlier (section 2.1). Mass prophylaxis is generally based on a single intramuscular injection of 2.9-3.3 mg pentamidine base/kg (Gall, 1954), and a standard therapeutic course for first stage cases is a series of injections, on consecutive or alternate days, of 3-4 mg base/kg to a total of 25-30 mg base/kg (Willett, 1955; Neujean and Evens, 1958). Oral prophylaxis is inadequate (Gall, 1954). A short combined pentamidine and tryparsamide curative course (Duggan and Hutchinson, 1951; Butler, Duggan and Hutchinson, 1957; Ross, 1960) has ad- vantages in the survey and treatment method of control of Gambian sleeping sickness, but should be restricted to early cases (Ross, 1960).
For early cases of Rhodesian sleeping sick- ness, Gelfand and Alves (1954) stressed that pentamidine treatment had the advan- tages of brevity and ease of administration compared with suramin; favourable results from Mozambique have also been reported by Silva (1956a). The value of large-scale pentamidine prophylaxis in this area, and in Ruanda-Urundi on the occasion of epidemic outbreaks of T. rhodesiense infection, is stressed elsewhere by Silva (1956b, 1958) and Silva and Caseiro ( 1957).
An interesting facet of mass prophylaxis is the possibility that among indigenous Africans where children are often suckled for up to two years or longer, excretion of pentamidine in the mother’s milk may be sufficient to confer protection on the child. Launoy ( 1955) has tested this experi-
300 WILLIAMSON
mentally in rats and shown that there is in- sufficient excretion by this route to protect suckling rats from T. gambiense infection.
Stilbamidine (2.5.2)) although of equiv- alent activity to pentamidine, did not come into extensive field use, mainly because of its instability in aqueous solution to light, pH and temperature, with formation of toxic products (Findlay, 1950). A trial of its prophylactic activity has however been car- ried out in the Congo by De Scheitz and van Hoye (1953) during a mass pentamidine prophylaxis survey. Six hundred and ninety people were treated and the protective results were considered equal to those obtained with pentamidine. In this survey the authors also found some indication of sensitization to repeated prophylactic pentamidine injections, even with the long intervals of at least 6 months which separated the treatments.
Toxicity and Pharmacology
The toxic effects of pentamidine are mainly due to a combination of vasodepressor effect, histamine liberation and a hypoglycaemic action (Gall, 1954). To reduce the in- cidence of these side-effects, sleeping sickness cases are preferably treated in a prone posi- tion and rested in the shade for some hours after each injection (Neujean and Evens, 1958; Gall, 1954; Demarchi, 1958); ad- renaline is usually kept at hand throughout the administration, and calcium has also been recommended (Gasq and Lapeyssonnie, 1949). Gross hypoglycaemic effects have been relieved by glucose, intravenously or by mouth (Klinkhamer, 1958; Demarchi, 1958). Transitory hyperuremia has recently been suggested to be a more constant feature of diamidine treatment than previously sus- pected (Payet and Sankale, 1960) ; it was not caused by infection, fever, lysis of tryp- anosomes or renal damage.
A number of isolated experimental studies with diamidines and diguanidines has some bearing on the clinical toxicity of pentami- dine. Stilbamidine shows curare-like action in nerve-muscle preparations, and in low concentration, inhibits acetylcholinesterase (Bergman, Wilson and Nachmansohn, 1950) ; its known ability to release histamine may be related to its tendency to localize on mast
cells where histamine production may occur (Fell, 1957). The hypoglycaemic activity of guanidines as a group has been recognized since the discovery of synthalin (2.5.4) (l,l’- decamethylene diguanidine) which was used as a synthetic substitute for insulin, and
“\ /=
//“” -i-(CHJ,()-L-C
\ -NH,
(2.5.4) Synthalin
subsequently found to have trypanocidal action ; it was in fact the progenitor of the trypanocidal diamidines (see Findlay (1950) and section 1.2).
Synthalin produces a specific hydropic degeneration of the u-cells of the pancreatic islets in both the rabbit (Davis, 1952) and the fowl (Beekman, 1956), an effect ob- viously related to its hypoglycaemic activity; its inhibition of certain leukaemic and malig- nant ascitic cells, which is not related to a hypoglycaemic effect (Mihich, Mulhern, and Hornung, 1960) also suggests some form of specific cytotoxicity. Steiner and Williams (1958) included synthalin in their inquiry into the mode of action of a number of hypo- glycaemic guanidine derivatives including phenethylbiguanide, which is a highly active oral hypoglycemic agent. All the compounds, and especially synthalin, were strong inhib- itors of cytochrome oxidase, but this inhi- bition was not considered to be solely re- sponsible for the hypoglycaemic effect in the intact animal.
Experiments on the tissue retention of diamidines are of more immediate relevance to the prophylactic action of pentamidine in man. Snapper, Lieben, Greenspan, and Schneid (1951) found that about one-third of injected stilbamidine and Z-hydroxystil- bamidine was recoverable from tissue de- posits in the liver, kidney and adrenals in man and experimental animals ; the bulk of the deposit was in the liver in man and rab- bits, and in the kidneys in mice. Liver and adrenal deposits in man were retained for extended periods; in one case appreciable amounts were found 23 months after the
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 301
last injection, and in another, marked traces were still present after three years and three months. Stilbamidine has been found in sheep liver up to 3 months after injection, and in rabbits, 2 months after injection, all organs were found to retain stilbamidine, which was also present in skin (Henry, Man- sour, Watson and Zaki, 1952). In these studies, estimation methods based on the fluorescent properties of stilbamidine were used, but Reid and Weaver ( 195 1)) using ( 14C) -1abelled stilbamidine in mice, showed that although 70% was excreted within 4 days of intravenous injection, traces were detectable in many tissues six months later. Fulton and Mathew (1959) obtained similar results with [ 14C] -stilbamidine in rats, and showed that appreciable amounts (0.16%, 0.18% and 0.17% of injected drug in liver, kidneys, and lungs respectively) were detectable 230 days after intravenous injection; similar amounts in the kidneys, and traces in the lung and heart, were present up to 280 days after injection.
Synthesis of new trypanocidal diamidines continues with varying success. Insoluble salt formation between pentamidine and peni- cillic acid has been used to promote prophy- laxis in rats (Launoy, 1949) ; a more in- teresting and fruitful variation has been the demonstration that insoluble salt formation with suramin decreases the toxic action of pentamidine without affecting its tryp- anocidal action (Guimaraes and Lourie, 1951). (This will be dealt with more ex- tensively in section 2.9.) In the direction of “hybrid” synthesis, trypanocidal amidine derivatives of phenanthridine have been ex- amined intensively, especially in the laborato- ries of Messrs. May & Baker Ltd. (Barber, Gregory, Major, Slack and Woolman, 1947; Libman and Slack, 1951). Simple substitu- tion of amidino groups for amino groups in the active compounds phenidium and dimid- ium (section 2.7) had a dystherapeutic effect (Libman and Slack, 1951), but later work on these lines has led to the highly effective metamidium, described in section 2.7.
2-Hydroxypentamidine has been prepared in the hope that chemotherapeutic activity would be increased as in 2-hydroxystilbami-
dine, but the product, although of comparable curative activity to pentamidine, was more than twice as toxic (Davis, 1958a). At- tempts have also been made to improve activity by varying the linkage between the amidinophenyl moieties, for example, by using a substituted amidine group (Crund- well, 19.56), or by demonstrating that the activity of phenamidine (4,4’-diamidinodi- phenyl ether) (2.5.5) can be retained in the
(2.5.5) Phenamidine
corresponding tricyclic form as 3,6-diamidi- nobenzofuran (2.5.6) (Moffatt, 1951). No outstanding activity was however achieved in either case.
(2.5.6)
The most interesting attempt of this type has been the development of 2-amino-4,6-bis (p-amidinoanilino)-s-triazine (M. & B. 2242) (2.5.7) (Ashley and Berg, 1954, 1959; Ashley, Berg and MacDonald, 1960), where the inter-ring linkage is melamine, as in Surfen C (section 2.6) and in Friedheim’s mel- aminyl trypanocides (section 2.3).
This compound was prepared on the basis that the 4-aminoquinoline moieties of Surfen C (2.6.2) could be regarded as vinylogues of 2-aminoquinoline, which is itself a cyclic amidine derivative; M. & B. 2242, with two amidinoanilino groups joined by an amino- triazine ring would thus be equivalent to Surfen C. M. & B. 2242 is in fact an active trypanocide especially against T. congod- ense ; it is less active than antrycide or dimidium but also much less toxic. Com-
302 WILLIAMSON
(2.5.7) M & B. 2242
parative figures for subcutaneous injection in mice are :
LD 50 CD 50 (~~/k) (w/W
M. & B. 2242 Antrycide
4700 37.5
dimethylsulphate 30 1.5 Dimidium chloride 60 0.6
The compound was warranted sufficiently active to be tested in cattle; one published report (Whiteside, Fairclough, and Bax, 1960) indicates that at the maximum dosage of 10 mg/kg it was seldom effective against any resistant infections, and was surpassed in this respect by both berenil and met- amidium.
Berenil
The only new aromatic diamidine which has come into accepted field use, in this case for animal trypanosomiasis, is berenil (2.5.8), N - 1,3 - diamidinophenyl- triazene diaceturate (M.W. (base) = 201.2, M.W. (salt trihydrate) = 489.4) (Jensch, 1954, 1955). Though stable when dry, an aqueous solution in contact with air is stable only for 2 or 3 days; a more stable hydrazone form has been described (Jensch, Loewe, and Bauer, 1958) (4-amidinobenzal- dehyde - 4 - amidinophenylhydrazone ( 2.5.9)) but this has not so far replaced berenil in the field. (In this derivative, the triazene “bridge” is replaced by the isosteric hydra- zone -CH=N-NH-) .
(2.5.8) Berenil
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 303
The chemical origins of berenil are de- scribed in considerable detail by Jensch (1958) who shows how development pro- ceeded by systematic dissection of the Surfen C “bis-4-amino-chinaldin-Typ” molecule (2.6.2) into chemically and therapeutically equivalent portions. The first “4-amino- chinaldin-Mischtyp” series consisted of a 4-aminoquinaldine nucleus linked with a guanidino-, amidino- or guanylformylhydra- zone-substituted phenyl moiety, and in the second “guanyl-hydrazon-Typ,” the phenyl ring was first omitted without loss of activity, and then the 4-aminoquinaldine nucleus itself was found to be replaceable by a guanidino- or amidinophenyl group without loss of activity, and in some cases with improved tissue tolerance. This led logically to the syn- thesis of symmetrical molecules of the “bis- guanyl-phenyl-Typ,” in view of the known parasiticidal activity of the aromatic di- amidines. By varying the inter-ring “bridge” grouping on the basis of experience in earlier series, maximal activity was found in berenil, which consists of two amidinophenyl moieties joined by a triazene bridge.
The great interest of this work (and of the extension of it exemplified earlier in the development of compound M. & B. 2242) is the way in which biological activity was shown to be retained in chemically equiva- lent structures. As Jensch (1958) concludes: “Der Pyridinanteil in 4-amino-chinaldin ahnelt demnach in seinen chemotherapeuti- schen, sowie in mancherlei chemischen und physikalischen Eigenschaften einer Amidino-, Guanidino-, oder Guanylformylhydrazono- gruppe.”
Berenil, apart from its trypanocidal activity, is also highly effective against Babe& infections, an added advantage in veterinary usage in Africa; in the form of the dilactate salt it was, in fact, originally named “Babesin.” (High activity against T. colzgoE- en$e has been subsequently shown by Taylor, Terry, and Godfrey (1956) to correlate with activity against Babe& in a series of “cattle” trypanocides which included phenanthridin- ium derivatives and antrycide.)
The trypanocidal activity of berenil has been reviewed in detail by FussgSnger and Bauer (1958), following the original reports
of biological and clinical trials by Bauer (1955a, 1955b) and Fussggnger (1955). In small animals, berenil is more active against T. congolense than against T. brucei tryp- anosomes; the curative subcutaneous dose for T. congolense in mice may vary from 1-2.5 mg/kg up to 5 mg/kg depending on the strain (Davey, 1957).
The maximum tolerated dose in mice is 146 mg/kg (subcutaneous LD 5) ; an intra- peritoneal LD 10 of 100 mg/kg has also been recorded (Williamson and Rollo, 1959). In larger animals the dose (intramuscular) is correspondingly less, i.e., about 40 mg/kg in the rabbit, 15-20 mg/kg in the dog and 8-10 mg/kg in sheep and cattle. The last dose level is said to be well tolerated in cattle, but no systematic examination of the effect of higher doses seems to have been reported. This is of some importance for the use of berenil in West African cattle infections, where the recommended field curative dose of 3.5 mg/kg is not always effective, and the advantages of berenil as a “sanative” drug (section 3.3) for treating resistant strains are thereby offset; these advantages might be achieved by using higher doses of berenil, but more would first need to be known of its toxicity.
On the basis of extensive treatment in Tanganyika, Portuguese East Africa, the Gold Coast, the Belgian Congo and the Union of South Africa and other areas, Fuss- ganger and Bauer (1958) claim that a single intramuscular dose of 3.5 mg/kg will cure T. congolense and T. vivax infections “in allen Teilen Afrikas.” T. brucei infections are less susceptible and require a dose of 5 mg/kg; this dose in pigs is not curative for T. simiae infections.
The rapidity with which berenil is degraded metabolically and excreted, usually within 24 hours of treatment, is probably respon- sible both for its lack of prophylactic activity and its apparent inability to give rise to re- sistant strains (Whiteside, Fairclough, and Bax, 1960; Fairclough, 1960; Deom, 1960). Its activity against strains resistant to other trypanocides in current field use (see section 3.3) has proved extremely valuable in cattle trypanosomiasis therapy, especially in East Africa.
304 WILLIAMSON
Hc)J&$~cH3
3 (2.6.1) Surfen
In T. gambiense sleeping sickness in man, berenil, like pentamidine, is effective in the early stages when parasites are confined to the blood and lymph, but has so far proved insufficiently active to be considered as a clinically useful agent (Nash, 1956, 1957; Neujean and Evens, 1958; Fussganger and Bauer, 1958).
2.6. 6-AMINOQUINALDINE AND -CINNOLINE
COMPOUNDS
The most effective compounds in this group are active primarily on congolense- vivax infections of animals (antrycide (quinapyramine) , cinnoline (‘528” and Tozocide) . Their development has owed much to Jensch’s pioneer demonstration (Jensch, 1937) of activity in the bis- aminoquinaldine compounds Surfen ( 2.6.1) , ( 1,3-di (4-amino-2-methylquinol-6-yl) urea hydrochloride), and more especially, Surfen C (Congasin) (2.6.2) (di (4-amino-2-meth- ylquinol-6-yl) melamine), which was the first synthetic metal-free drug to show marked ac- tivity against T. congolense infections in the field. The 4-aminoquinoline nucleus was chosen by Jensch because of tautomeric similarities with therapeutically active derivatives of S- aminoacridine (2.6.3) (such as the antiseptic, Rivanol, and the antimalarial, atebrin (mepacrine), which can be considered as a combination of two 4-aminoquinoline struc- tures). Similar reasoning led Barrett, Curd
and Hepworth (1953) to eliminate the tria- zine bridge of Surfen C and replace one of the quinaldine moieties by an amino- pyrimidine system with similar tautomeric possibilities; the weak activity of the result- ing compound was enhanced by quaterniza- tion, and after some difficulty a highly active
NH2
(2.6.3)
bis-quaternary salt, originally considered an impurity, was isolated, characterized and later synthesized unambiguously (Ainley, Curd, Hepworth, Murray and Vasey, 1953). This salt was antrycide chloride (2.6.4) (4- amino - 6 - (2 - amino - 6 - methylpyrimidin- 4-ylamino) quinaldine-1 : I’-dimethochloride) (Curd and Davey, 1949, 19.50; Curd, 1950).
Antrycide (quinapyramine) Antrycide, either in the form of the soluble
dimethosulfate salt (CHa.S04)2, or as a mix- ture of the dimethosulfate and the sparingly soluble chloride to form “antrycide prosalt” used as a prophylactic, is the only member of this drug group to have come into general field use in Africa, and so far, is the only
CHEMOTHRRAPY OF AFRICAN TRYPANOSOMIASIS 305
‘q:H,E’jz
NH2
(2.6.4) Antrycide (quinapyramine) chloride
prophylactic drug of any type for cattle tryp- anosomiasis to have emerged successfully from the experimental trials stage.
The chloride form, like all the other halide salts prepared, is relatively insoluble in water, and after subcutaneous injection, is retained in the tissues as a “depot” from which enough of the soluble form reaches the circulation to maintain a continuous, low but protective trypanocidal blood level. As Curd and Davey (1950) showed, surgical excision of this depot from treated rabbits abolishes the prophylactic effect. In field practice, the depot becomes encapsulated with fibrous and eventually calcified tissue, the blood supply is thus cut off, and prophylaxis is limited to about two months. That sticient active drug is still present in the depot has been demon- strated by experiments in cattle in which a fibrosed capsule has been excised, and its con- tents, after crushing and re-injection into another animal, have been shown to be capa- ble of conferring protection for another ex- tended period. Attempts to exploit this by crushing the encapsulated depot in situ have been unsuccessful (Wtiteside, Fairclough, and Bax, 1960).
Antrycide dimethylsulfate. Following Davey’s original field trials in the Sudan and East Africa (Davey, 19.50)) extensive field trials and increasing usage in all parts of Africa have confirmed the efficacy of antry- tide dimethylsulfate as a curative drug, at the recommended field dose of 5 mg/kg (Davey, 1957). In a later review, Marshall (1958) states that “several million cattle have been treated with it, with excellent results, generally in T. congo&nse cases, but with slightly less success in T. vivax cases.”
The activity of antrycide, unlike that of
its ancestor, Surfen C (2.6.2)) is not re- stricted to the congolense-vivax group, and extends equally to T. ezransi, T. equinum and T. equiperdum and to a lesser degree, to the brucei-group trypanosomes (Curd and Davey, 1950); it appears to have little effect on T. vivux in the tsetse fly (Davey, 1957). T. evansi infections in camels, even when sura- min-resistant, have been successfully treated with antrycide (Evans, 1956; Davey, 1957; Leach, 1961) ; T. ewnsi and T. equiperdum infections in horses have also responded well (Davey, 1957)) although equines in general appear more susceptible to antrycide toxic- ity (Whiteside, 1958a).
In T. simile infections of pigs, cures have been recorded at doses of 3 to 5 mg/kg (Wilson, 1949a, b) but these were blood- induced infections, possibly more susceptible to drug action; subsequent experience with tsetse-infected pigs (Unsworth, 1952 ; Wat- son and Williamson, 1958) showed that although high doses (up to 50 mg/kg) of antrycide chloride may be curative, the di- methylsulfate is not.
The most recent, and one of the most promising applications of antrycide dimeth- ylsulfate treatment is found in the work of Smith (1958) and of Whiteside and his col- leagues (Whiteside, 1958b; Whiteside, Fair- clough and Bax, 1960) on the extensive residual immunity which was claimed by Soltys (1955) to develop in cattle after antry- tide prosalt prophylaxis. Residual immunity of this type had in fact been observed many years earlier with tartar emetic (Bevan, 1928). Whiteside et al. (1960) have shown that with autrycide prosalt, this phenomenon may not be so much an acquired immunity as a residual prophylaxis due to depots of the
306 WILLIAMSON
drug. To overcome this objection, they used curative regimes (antrycide dimethylsulfate and ethidium) and showed that even in an area of high trypanosome challenge, tem- porary immunity of 5 to 12 months duration followed a curative regime lasting 7 months. A prophylactic effect of antrycide dimethyl- sulfate treatment has also been noted in the Congo by Deom (1960). In some cases the immunity observed (Whiteside, 195813) was apparent after as few as two antrycide treat- ments and one ethidium treatment. There is some evidence that the nature of the drug used affects the results, but an important point was that the infections which ultimately appeared were not resistant to the drugs previously used. This work is still in the experimental stage, but further work on the combined effects of chemotherapy and the immune reaction will clearly be profitable (see section 3.3).
Antrycide prosalt. On the basis of Davey’s first field trial experience (Davey, 19.50), it became clear that neither the dimethylsulfate nor the insoluble chloride alone would be useful for prophylaxis. The former was ex- creted too rapidly and the latter, although persistent, did not give blood levels high enough to cure an established infection quickly; these low blood levels appeared to be independent of dose size (Spinks, 1950b) and, moreover, were likely to encourage formation of resistant trypanosomes. A mix- ture of the dimethylsulphate (3 parts) and the chloride (4 parts) was therefore issued commercially by Imperial Chemical (Phar- maceuticals) Ltd., and named antrycide pro-salt; a standard dose of 11.7 mg/kg con- tained 5 mg/kg of the dimethylsulphate. More recently, a cheaper revised formulation has been issued for trial, in which the chloride content has been reduced by one-half. Pre- liminary results (Marshall, 1958; Whiteside et #aZ., 1960; Deom, 1960) indicate that the new form is at least equivalent to the old form; in Kenya, a three-monthly treatment with either form is effective in low fly chal- lenge, a two-monthly treatment is needed in medium challenge, but in high-challenge areas, this regime sometimes fails to give continued protection. In the Congo, Deom (1960) stresses that the new form seems as
active as the old, and has the added advan- tages of not causing persistent local reac- tions and of costing less.
Antrycide pro-salt is curative for, and has some prophylactic activity in, T. simiae in- fections of pigs (Wilson, 1949a, b; Watson and Williamson, 1958), but better results are obtained either with high doses of antry- tide chloride alone (50 mg/kg) or with antrycide-suramin complex at 40 mg/kg (Watson and Williamson, 1958; Stephen and Gray, 1960a) (see section 2.9). Protection periods of about six months or longer may be possible with either drug in mature pigs, but further extended field trials will be necessary to confirm this.
Antrycide toxicity and pharmacology. An- trycide, as a his-onium compound, has curare- like properties (Ormerod and Paton, 1950; Garner, 1950) both in small animals and in cattle and horses. Toxic symptoms such as excessive salivation, sweating, uncontrolled tremors and occasional temporary collapse, occur in a proportion of cattle treated with the recommended dimethylsulfate dose of 5 mg/kg. (Unsworth and Chandler, 1952; Davey, 1957), and these may be increased to the point of fatality if animals are treated while under stress from heat, fatigue, mal- nutrition, intercurrent infection or any of the numerous complicating factors which occur so commonly in African field conditions (Lawrence and Bryson, 1958). Toxicity may also be increased by a reduction of the in- jected dose volume, as noted in mice by Lourie and Walker ( 1951). Severe kidney damage has been a characteristic feature of animals examined post martem after antry- tide poisoning (Davey, 19.5 7).
Antrycide chloride, because of its low solubility is virtually non-toxic. The egg-like unabs’orbed deposits which remain after repeated prosalt treatment are unsightly, and for that reason injections are usually made into the extensive dewlap of African Zebu cattle to avoid damage to meat or hide; the deposits do not however give rise to any symptoms of general or chronic toxicity.
Using a method of analysis based on the high fluorescence of an antrycide-eosin salt Spinks (1950a) was able to measure antry- tide in plasma, tissues, and urine at levels
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 307
down to 0.20-0.04 pg/ml. He showed that in mice, rabbits and also in a calf, antrycide was localized in liver and kidney tissue and maintained there for some weeks after in- jection; within 24 hours of dimethylsulfate injection, plasma levels fell from a maximum of 4 pg/ml to less than 0.01 pg/ml (Spinks, 1950b).
CinnoZine “528”
The development of this compound ( N1: N3- his (4’-aminocinnolyl-6’) -guanidine dimeth- iodide) (2.6.5) (Lourie, Morley, Simpson and Walker, 1951, 1954; Morley and Simp- son, 19.52) arose from a study of quaternary amino-cinnolines, -quinazolines and -quino- lines as simplified models of phenanthridinium salts (2.7.1)) the chemotherapeutic activity of which was considered to be influenced by requirements such as cationic resonance and molecular size and symmetry (Keneford, Lourie, Morley, Simpson, Williamson and Wright, 1948, 1952).
Experiments with “528” in T. congolense- infected mice showed activity similar to antrycide dimethyl sulphate; the therapeutic indices (LD lo/CD 90) were 11.6 and 12.5 respectively, for corresponding subcutaneous LD 10 and CD 90 values of 51.5 and 4.45,
and 24.5 and 1.95 mg/kg respectively (Lourie et al., 1951).
Field trials in West Africa (Chandler, 1957) were however disappointing. Although T. congolense-infected cattle could be cured at doses of 051.0 mg/kg, the maximum tolerated dose of 2 to 2.5 mg/kg was unable to cure cattle infected with the virulent West African T. v&ax.
Tozocide
The origins of this compound (6’- (4-quin- aldylamino) hexyl - 4 - amino - quinaldinium iodide hydriodide) (Austin, Collier, Potter, Smith and Taylor, 1957) (2.5.6) are similar in some respects to those of antrycide and cinnoline “528” in that all three were dis- covered as active impurities in syntheses de- signed to produce related compounds of structure similar to the Jensch Surfen C type (2.6.2). The starting point here was a syn- thetic curarizing agent, decamethylene-bis- (iso-quinolinium bromide), which was found to have some trypanocidal activity; this activity was markedly enhanced in the bis- aminoquinaldinium analogue and further variation led to Tozocide (Austin, Potter and Taylor, 1958) which has an unsym- metrical molecule. The two symmetrical
(2.6.5) Cinnoliie “528”
(2.6.6) Tozocide
308 WILLIAMSON
isomers, in which the hexamethylene bridge linked either the two 4-amino groups or the two quinaldinium nitrogen atoms, were both less active against T. congolense, although the former was more active against 5”. rkodes- iense than the unsymmetrical Tozocide. The chloride salt of Tozocide had some prophy- lactic activity in mice, and this protective effect was doubled by formation of an in- soluble “complex” with suramin, in agreement with similar observations by Williamson and Desowitz (1956) on the suramin complexes of ethidium and antrycide (see section 2.9). Trials in African cattle have shown some activity against 7’. congolense infections in Kenya but not in Nigeria (Marshall, 19.58); the drug is also inactive against Ay-trans- mitted T. sinaiae infections in pigs (Stephen and Gray, 1961).
Several trypanocidally active synthetic variants on the Tozocide pattern of two aminoquinaldine moieties linked by a poly- methylene bridge have been reported by Jensch (1957), Ashley and Davis (1957)! and Schock (1957, 1959), but so far none has shown sufficient activity for further development. Two Surfen C variants with trypanocidal action have been described, one with quinoline replacing quinaldine (Ochiai and Morishita, 1956) and the other a quaternary quinaldinium analogue (Societe des usines chimiques Rhone-Poulenc, 1955b).
Other types of his-aminoquinaldine com- pounds have been studied extensively by Goble (1950) and Goble and Hoppe (1952). These have been essentially bis-aminoquin- aldyl amides or ethers of the series in which Jensch (1937) first showed high activity against T. cruzi of a derivative containing a diallylmalonyl bridge (Bayer 7602 AC). In general, compounds with branched chain bridges were only weakly active against brucei-group trypanosomes, but showed more activity against T. cruzi; the converse effect was shown by compounds with an un- branched chain bridge. None was active against T. congolense. Quaternization of the diamide bridge derivatives increased activity against T. rhodesiense, and further com- pounds of this type have been described by Hepworth ( 1954).
2.7. PHENANTHRIDINIUM DERIVATIVES
This important class of trypanocides in- troduced by Morgan and Walls (193 1) and Browning et al. (1938), shows high activity against vivax-congolense infections of African cattle. The newer drugs, ethidium (homid- ium) (2.7.2), prothidium (2.7.3) and meta- midium (2.7.5) are lineal descendants of the first two products which came into field use, phenidium (7-amino-9- (p-aminophenyl) - lo-methylphenanthridinium chloride) (2.7.1: X = H, Y = NH2) and later the more active dimidium (Walls, 1947a, 1947b) (2,7-di- amino - 9 - phenyl - 10 - methylphenanthridin- ium bromide) (2.7.1; X = NH-. Y = H).
(2.7.1)
The choice of the phenanthridine nucleus seemed opportune, as it is isomeric with acridine and contains both quinoline and iso- quinoline rings, all of which are associated with compounds of high biological activity; the advantages of quaternization for tryp- anocidal activity, already recognized in the aminoacridine series, were quickly established also in the phenanthridine group. The pro- duction of phenidium and dimidium demon- strated the association of high trypanocidal activity with phenylphenanthridinium salts containing two primary amino groups, prefer- ably in the 2- and 7-positions; 3-amino sub- stituted compounds are less active. This was confirmed by later synthesis and biological tests of a comprehensive series of related derivatives (Brownlee, Goss, Goodwin, Woodbine, and Walls, 1950; Walls and Whittaker, 1950a, 1950b), which also showed that trypanocidal activity was greatly reduced by modification of the primary amino groups, for example by acetylation or replacement with amidine groups; replacement with nitro groups did not reduce activity so markedly and 7-alkoxy derivatives were also especially
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 309
active against T. congolense. Trials of four of the most promising compounds in West African v&ax-infected African cattle (Good- win and Unsworth, 1952; Goodwin and Chandler, 1952) showed that the triamino compound (2.7.1; X = Y = NHZ) was active but toxic. Another experimentally ac- tive 2,7-diamino derivative containing a 2- thienyl substituent in place of phenyl in the 9-position, was tested against T. congolense- and T. v&ax-infected cattle in the Sudan (Neal and El Karib, 1954) but the results were similar to those in West Africa (Good- win and Unsworth, 1952), and Thienpont (1953) reported unsatisfactory results with this compound in Ruanda-Urundi.
Dimidium has been used very extensively in the field in East Africa, but by 1952, as a result of mass treatment, dimidium-resistance was a problem in the Rhodesias where the higher doses then introduced in an effort to overcome the drug-fastness led only to heavy mortality, delayed toxicity and photosensiti- zation (Burdin and Plowright, 1952a, b; Davey, 1957; Marshall, 1958; Shaw, 1958; Lawrence and Bryson, 1958) ; this experience has been general in a number of other African areas where dimidium was used extensively (e.g., Finelle, 1958; Deom, 1960).
Ethidium Bromide (Homidium) The first, and so far, the best established
improved substitute for dimidium in the field, has been ethidium bromide ( 2.7.2) which differs from dimidium bromide only in pos- sessing an ethyl instead of a methyl group on the quaternary nitrogen heteroatom (Watkins and Woolfe, 1952; Watkins, 1952; Woolfe, 1952; Short, Peak and Watkins, 1953). As Davey (1957) remarks: “The difference in
(2.72) Ethidium bromide (homidium)
biological properties is therefore remarkable; it is equally remarkable that 14 years went by before the attributes of the ethyl quaternisa- tion group were discovered.”
In experiments on mice (Watkins and Woolfe, 1952; Woolfe, 19.52) with a homol- ogous series of quaternary N-alkyl derivatives of dimidium, the therapeutic activity varied enormously. The ethyl homologue was 10 times and 50 times more active against T. congolense and T. gambiense respectively, than the parent dimidium, and the propyl homologue was 35 times more active against T. brucei, but this access of activity fell off sharply with higher homologues. The effect on toxicity was small by comparison, and subsequent field trials in cattle have con- firmed that ethidium is not only somewhat more active than dimidium against T. con- golense but also much less toxic; extensive use of ethidium in the field has not produced the liver lesions and photosensitization which accompanied large-scale dimidium treatment.
Similar enhancement of activity by chang- ing the quaternary alkyl group was dem- onstrated experimentally in a number of other phenanthridinium derivatives, including phenidium (Woolfe, 1956a, 1956b), but none of the compounds was sufficiently active to displace ethidium. Ethidium bromide is readily soluble only in hot or boiling water, but this practical disadvantage has been overcome by the use of the chloride (novidium) (Wragg, 1955) which is soluble in cold water.
On the basis of trials in the Sudan, Kenya, Tanganyika, and Nigeria against a variety of strains of T. congolense and T. vivax (Forde, Wilmshurst, and Karib, 1953a, 1953b; Wilson and Fairclough, 1953; Wilde and Robson, 1953; Unsworth, 1954a, 1954b), a therapeutic dose of 1.0 mg/kg has been recommended. Intramuscular injection is advised by the manufacturers in order to reduce the local reaction which can occur at this dose given subcutaneously, because ethidium, like most active quaternary am- monium-type trypanocides, with the possible exception of antrycide, has dermonecrotic properties.
The importance of the nature of the chal- lenge in assessing the performance of tryp-
310 WILLIAMSON
anocides in cattle infection (sections 2.1; 2.6) is illustrated by the following reports on ethidium. In Ruanda-Urundi, ethidium at 1.0 mg/kg can cure about 90% of congolense- vivax infections in cattle not exposed to re- infection, but in fly areas the relapse and re- infection rate is high (Deom, 1960). On the other hand, in Kenya, in areas of high infec- tion incidence, curative ethidium treatment seems to induce a certain residual prophy- lactic effect (Whiteside et al., 1960), possibly as a result of stimulation of the immune re- action. Experimentally, some prophylactic activity has been observed in cattle given high doses of ethidium bromide (up to 5.0 mg/kg) and subsequently challenged with either blood-induced T. vivax and T. congol- enSe infections (Leach, Karib, Ford, and Wilmshurst, 19.55) or experimental tsetse- transmitted T. vivax infections (Desowitz, 1957), but the variability of the results has restricted ethidium to curative use only. Ex- tended prophylaxis in cattle has been achieved by formation of a complex with suramin (section 2.9) but considerable local reaction has so far precluded its adoption for field use.
Ethidium is not curative for T. simiae in pigs (Watson and Williamson, 1958) but at 1.0 mg/kg in the horse, it was well tolerated and curative for a tsetse-transmitted T. vivax infection (Stephen and Mackenzie, 1958).
Protlzidium
Having produced one successful new phenanthridinium derivative by the simple and original expedient of changing the alkyl quaternary group, Watkins and Woolfe
(1956) then proceeded to effect an equally novel synthesis by substituting the pyrimidyl moiety of antrycide (2.6.4) into the ‘l-amino group of 2,7-diamino-9-p-aminophenyl-lo- methylphenanthridinium chloride, a com- pound resembling phenidium ( 2.7.1) , and one of the four active compounds tested by Goodwin and Unsworth ( 1952). The resulting compound, prothidium (RD 2801) (2-amino- 7- (2-amino-6-methyl-4-pyrimidylamino) -9- p - aminophenylphenanthridine 10 : 1’ - di- methobromide) (2.7.3) (Watkins, 1958a, 19583; Short and Watkins, 1957) had re- markable curative and prophylactic activity.
Two related compounds, RD 2787, in which a nitro group replaces the p-amino group in the 9-phenyl moiety of prothidium, and RD 2902, which is essentially ethidium with the pyrimidyl moiety of antrycide substituted into the 7-amino position, were also active and all three were tested in cattle in Africa (Robson and Milne, 1958).
In this first African trial, prothidium at a dose of 2 mg/kg protected Zebu cattle against blood-induced T. congolense infec- tions for a period of 6 months, but the com- pounds RD 2787 and RD 2902 were less active. Further trials in Tanganyika, Kenya, the Sudan and Nigeria (Brownlie, Watkins, and Woolfe, 1957; Robson, 1958a, b; Marshall, 1958; Whiteside et al., 1960; Smith and Brown, 1960; Lyttle, 1960), have shown that a 2 mg/kg dose can give at least 4 months protection in fly areas of low or medium challenge, but that this period may be decreased in areas of heavier challenge (Robson, 1961), necessitating a higher dose; toxicity at higher doses has been more
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 311
pronounced in Nigeria than elsewhere (Kirby, 1960b). Although most emphasis has been placed on its prophylactic activity, pro- thidium is also an active curative drug; doses of 0.2-0.4 mg/kg are curative for T. congol- ense (Whiteside et al., 1960).
The general acceptance of prothidium into field use has been held up by the discovery in 1959 of the unfortunate fact that glucose, used as excipient in the commercial pro- duction of tablets, was combining with, and partly inactivating, the drug component. A recent report indicates, however, that this handicap is likely to be overcome (Robson, 1961).
Metamidium
The most recent active phenanthridinium derivative, metamidium (M. & B. 4404) (Wragg, Washbourn, Brown, and Hill, 1958), is another synthetic hybrid devised on similar lines to prothidium. In this case,
a portion of the berenil molecule (2.5.8) was linked into the ‘I-position of ethidium (2.7.2) by coupling diazotized p-aminobenzamidine and ethidium chloride in the presence of sodium acetate. Two isomeric p-amidino- phenyldiazoamino derivatives were produced which had considerable trypanocidal activity, and further syntheses showed that the m- amidino-substituted product, also obtained as a mixture of two isomers, was the most promising of the series. This mixture was highly active, therapeutically and prophy- lactically, in experimental T. congolense in- fecti’ons and was named metamidium.
The two isomers have been provisionally considered as (a) 2- (m-amidinophenyldiazo- amino-) - 7 - amino - 10 - ethyl - 9 - phen- ylphenanthridinium chloride hydrochloride (2.7.4) and (b) 7 - (m - amidinophenyldiazo- amino) - 2 - amino - 10 - ethyl - 9 - phen- ylphenanthridinium chloride hydrochloride (2.7.5).
H N, NH2
Y
c1 Cl’ HCI
(4
HCI
(2.7.5) Metamidium isomer (b) (ko-metamidium)
312 WILLIAMSON
The isomer (a) is purple, relatively water-insoluble and less active than the red isomer, (b), the structure of which was later confirmed by Berg (1960) who announced that it had been isolated in pure form in sufficient quantities for field trial under the name of iso-metamidium (M. & B. 4180A).
The original metamidium preparation con- tained the purple and red isomers in the proportions of 55% and 4570 respectively, and was less toxic than ethidium in mice (subcutaneous LD 50 (mg/kg): ethidium chloride, 70, purple isomer, 430, red isomer, 260, metamidium chloride hydrochloride, 230), and also more active against T. congol- ense. A dose of l/9 of the LD 50 of meta- midium protected mice from infection for up to 16 weeks, but neither ethidium nor berenil at doses of l/3 of the LD 50 showed any ap- preciable prophylactic activity under com- parable test conditions.
In African cattle (Fairclough, 1958) high subcutaneous doses (5 and 10 mg/kg) produced toxic symptoms locally and in the liver, similar to those caused by dimidium, but a dose of 1 mg/kg appeared completely non-toxic. Intramuscular injections are better tolerated (Smith and Brown, 1960; Stephen, 1960) but local reactions have been observed even at 0.5 mg/kg in some cases (Kirkby, 1960a).
Cures of T. vivax and T. congolense in- fections in both East and West Africa were achieved in some cases with doses down to 0.2 mg/kg (Marshall, 1958), but the prophy- laxis obtainable does not appear to be as great as was anticipated. In Kenya, in an area of low challenge, a dose of 3 mg/kg gave protection similar to that given by antrycide prosalt (Whiteside et al., 1960), and similar results were obtainable at the same dose in Northern Nigeria (Kirkby, 196Ob). In an area of high tsetse density, met- amidium at 4 mg/kg gave significantly longer protection (average 18 weeks) than prosalt (average 10 weeks) (Smith and Brown, 1960), and at a higher dose (5 mg/kg), Stephen (1960) has obtained protection against West African T. vivax infection for periods of over 200 days. Like ethidium and R.D. 2902, metamidium is not
curative for T. simiae in pigs (Stephen and Gray, 1961).
One marked advantage of metamidium is its activity against strains resistant to the other “cattle drugs,” antrycide, ethidium, prothidium, and berenil (Fairclough, 1958; Whiteside, 1958a, 1958b, 1960; Whiteside et al., 1960). Resistance to metamidium which may be expected to follow its use, as with other phenanthridinium compounds, has been experimentally examined, and a metamidium-resistant strain, although cross- resistant to antrycide, ethidium and prothid- ium, fortunately appears to retain its sus- ceptibility to berenil (Whiteside et al., 1960) (see section 3.3, Table VII).
The current position of iso-metamidium has been summarized as follows by Beveridge ( 1961) from Messrs. May and Baker Ltd., the manufacturers: ‘(On the basis of nearly four years laboratory and field investigation we shall be prepared to recommend the use of isometamidium as follows:
a) Curative: 0.5 mg/kg by deep intra- muscular injection.
b) Curative (drug-fast strains): l-2 mg/kg by deep intramuscular injection.
c) Prophylactic: 2 mg,/kg by deep intra- muscular injection.
‘(It has been demonstrated that trypano- somes can acquire a tolerance to metamidium in cattle under laboratory conditions and we expect this to apply to isometamidium. Berenil at 3.5-5 mg/kg will eliminate these infections. Whiteside has demonstrated the effectiveness of metamidium chloride in eliminating strains of T. congolense which have become resistant to homidium, pro- thidium and quinapyramine.
“Isometamidium, which is intrinsically more active than the isomeric mixture is expected to be at least as effective and possibly more SO. Local and systemic tolerance of cattle to injections of isometamidium methane sul- phonate is rather better than to metamidium chloride.”
A suramin salt of metamidium (M. & B. 4427) has been prepared, following an earlier demonstration of enhanced prophylaxis and reduced toxicity obtainable with other “cattle drugs” by this method (Williamson
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 313
and Desowitz, 1956; Williamson, 1957); its activity is described in section 2.9.
Although much synthetic work in this large drug group has been devoted to variation of the substituents on the phenanthridine nucleus, relatively little appears to have been done on the nuclear structure itself. Some trypanocidal activity has been shown in quaternary derivatives of 4,9-diazaphenan- threne (Davis, 1958b) and of the larger polycyclic phenanthridine analogue, azapyrene (Cooke and Moffatt, 1951). A similar series, based on 2,7-diaryl-1,6-diazapyrene (2.7.6) (Fairfull, Peak, Short and Watkins, 1952) was an attempt to double the trypanocidal phenanthridinium molecule, and a number of quaternary derivatives, including 4,9-diamino substituted compounds had some activity (Watkins, 1961).
(‘2.7.6)
Toxicity and Pharmacology of Phenanthridinium Derivatives
The use of dimidium (2.7.1) in African cattle caused a high incidence of delayed toxicity (Findlay, 1950; Davey, 1957). About 6 weeks after treatment, symptoms resembling photosensitization frequently ap- peared, characterized by acute dermatitis, lachrymation, nasal discharge, emaciation and jaundice, and accompanied by damage to the liver parenchyma. The drug causes a liver lesion (periportal fatty infiltration) (Thorold, Plowright and Burdin 1952a; Plowright, Burdin and Thorold, 1952; Burdin and Plowright, 1952a, 1952b; Plowright and Burdin, 1952) which appears to be of central importance, but external factors such as climate, nutritional status, and incidence of photodynamic substances in the diet may also be significant. In herbivora with liver damage, photosensitization can arise from the presence of phylloerythrin derived from
chlorophyll, and this reaction may be coupled with the dimidium liver lesion. Burdin and Plowright (1952a, 1952b) suggested that the methyl-substituted quaternary nitrogen possibly interfered with transmethylation reactions in the liver; this theory was sup- ported by the absence of liver toxicity in the ethyl-substituted analogue, ethidium (2.7.2) but is contraverted, as Goodwin and Chandler (1952) have pointed out, by the similar absence of liver lesions even after very high doses of the comparable methyl-substituted analogue, phenidium (Burdin and Plowright, 195213).
The compounds which have succeeded dimidium (ethidium, prothidium and met- amidium), are also capable of producing liver damage, but unlike dimidium, do so only at doses considerably higher than the thera- peutic level (Ford, Wilmshurst, and Karib, 1954; Wilson, 1954; Unsworth, 1954a; Robson, 1958a, b; Fairclough, 1958), and delayed toxicity and photosensitization symptoms have not so far followed their use in the field. Local tissue damage at the injection site can be severe with high doses of all active phenanthridinium drugs so far prepared, especially if given subcutaneously, and an investigation of the structural factors responsible for this histotoxic effect in the phenanthridinium and related series would be of considerable interest and importance.
Fluorescence in ultraviolet light is typical of active phenanthridinium drugs, and this property has been used to demonstrate the distribution of drug in the organs and tissues of anaesthetized experimental animals lapa- rotomized, injected intravenously and then observed under ultraviolet light. Goodwin, Goss and Lock (1950) used this method in rats, rabbits and cats to show the rapid appearance in the bile, liver and kidney cortex of 3-amino-9-p-carbethoxyamino- phenyl-lo-methylphenanthridinium ethane sulphonate, a derivative active against T. cruzi. The predominance of biliary excretion was established by analysis, and similar techniques applied by Taylor (1960b) with prothidium in rats, rabbits and cattle have shown that the bile is a major excretory route for this drug also. Both drugs were concentrated in the liver and kidneys and
314 WILLIAMSON
urinary excretion was negligible. The im- mediate distribution of ethidium after intra- venous injection in the rabbit appears to differ from that of prothidium (Williamson and Stephen: see Nash, 1958), in that although the kidney cortex fluoresces with both drugs, the thyroid, lung, and gall bladder rapidly become intensely fluorescent with the former drug only.
The prophylactic activity of prothidium after subcutaneous injection does not appear to depend on retention by plasma proteins, as with suramin, but to the formation of a local tissue depot at the injection site, rein- forced by strong binding of the drug in liver tissue (Taylor, 1960b’).
2.8. CARBOXYLATED AROMATIC ARSENICALS AND ANTIMONIALS
Butarsen
This compound ( y- (p-arsenosophenyl) - butyric acid, 70A) (Doak, Steinman and Eagle, 1940; Eagle, 1945) (2.8.1) was found to have exceptional trypanocidal activity, unlike most other acid-substituted phenyl- arsenoxides, and has been tested extensively in human sleeping sickness (Findlay, 1950).
O=As
(2.8.1) Butarsen
Despite its activity against tryparsamide- resistant trypanosomes, it was found to be effective only in the early stages of infection and has not come into general field use. Lapeyssonnie (1950) has, however, claimed that a combination of tryparsamide and butarsen can provide effective cure even in advanced cases.
Spirotrypan
An active acid-substituted arsenobenzene of unusual structure has been introduced by
Fussganger (Jahn and Haussler, 1951). This compound, Spirotrypan (Hoechst 10557)) (fi- [ 5 - { 3 - [bis - (2,3 - dihydroxypropyl) amino] - 4 - hydroxyphenyl - arseno} - 2 - benzoxazolylmercapto] propionic acid sodium salt) (2.8.2) is active against both trypano- somes and spirochaetes. Its development and properties have been reviewed in detail by Hilmer (1958).
The benzoxazolyl moiety maintains a formal resemblance to the m-amino-p-hy- droxy substituents in arsphenamine and its arsenoxide equivalent, oxophenarsine. Like arsenophenylglycine and other acid-substi- tuted arsenicals, it is active against tryp- anosomes with acquired resistance to neutral aromatic arsenicals (section 3.3, Table VI), or with a natural resistance, like T. lewisi or T. cruzi, although only the accessible blood forms of the latter are affected. Jahn and Haussler (1951) studied its absorption, ex- cretion and toxicity in the horse with a view to possible use against T. equinum infections in South America, but in sleeping sickness in man, Hilmer (1958) concludes that it is in- sufficiently promising to displace the existing effective drugs. This is confirmed by Neu- jean and Evens (1958), and its inactivity in T. vivax infections of cattle has been reported by Thienpont ( 1954).
Captostibone
A new acid-substituted phenylstibonate, captostibone, has been described by Schnit- zer, Kelly, Soo-Hoo, Grunberg and Unger ( 195 1) . This compound (sodium 2-carboxy- methylmercaptophenylstibonate, R.O. 2-l 160) (2.8.3) is active in experimental T. equi- perdum, T. brucei, T. rhodesiense and T. gambiense infections (Schnitzer et al., 1951; Packchanian, 1957) and also against “arsenic- resistant” trypanosomes, but although its toxicity for mice is low (LD 50 approximately 300 mg/kg), its activity is not high and is manifest only by repeated injection. Other
(2.8.2) Spirotrypan
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 315
S .CH,.COONa
u /OH !“\a OH
(2 J3.3) Captostibone
compounds of this general type with tryp- anocidal activity have been patented by Steiger and Keller (1952).
2.9. SULFONATED NAPHTHYLAMINE DERIVATIVES
Suramin
The principal member of this group is suramin (hexa-sodium 3,3’-ureido-his [ 8- (3- benzamido-p-toluido)-1,3$naphthalene sul- fonate] ) (synonyms: germanin, Bayer 205, moranyl, Fourneau 309, belganyl, naphuride, naganol, antrypol) ( 2.9.1) . Substances of related structure, such as Trypan Red, Try- pan Blue, and Pontamine Sky Blue SBX, have some trypanocidal activity but suramin is the only representative which is clinically effective. Its therapeutic and prophylactic activity in human sleeping sickness in Africa have been established by almost forty years of widespread use (Findlay, 1950; section 2.1) though its use is confined to early stages of the infection, usually in combination with tryparsamide.
The structure is highly specific, in that
removal of the two methyl groups, for ex- ample, virtually abolishes activity. Recent attempts to synthesize active analogues by replacing the naphthalene trisulfonate portion with heterocyclic nuclei have been unsuccess- ful (Adams, Ashley and Bader, 1956).
The possibility that suramin treatment may be responsible for cases of adrenal in- sufficiency has been investigated experi- mentally by Frisch and Gardner (1958). Careful histological examination failed to reveal any evidence of adrenal damage in suramin-treated rats.
Suramin complexes. The potential use of suramin has been extended by the discovery that the insoluble complexes formed by mutual precipitation of the anionic suramin and a number of cationic trypanocides, show considerable retention of therapeutic activity, enhancement of prophylactic activity and decrease of toxicity, compared with the individual constituents alone (Guimaraes and Lourie, 195 1; Williamson and Desowitz, 1956). Similar phenomena with related pharmacologically active materials such as curare and acid dyes, and including an antrycide-suramin interaction (Ormerod and Paton, 1950) have been noted frequently (Williamson, 1957; Mora, Young and Shear, 1959; Sangiorgi, 1959).
The initial observations on the inhibition of some pharmacological actions of pentam- idine by suramin as a result of salt forma- tion were considered to constitute “a unique situation in therapeutics where two com-
(2.9.1) Suramin
316 WILLIAMSON
pounds tend to annul one another’s pharma- cological or toxic effects while acting in concert (if not synergistically) as chemo- therapeutic agents . . .” (Guimaraes and Lourie, 1951). The effect was investigated clinically by Beaudiment and Zozol ( 1952) who showed that a prior or simultaneous intravenous dose of 0.5 g suramin enabled doses of pentamidine up to 8 mg/kg to be given safely. Subsequently a pentamidine- suramin salt (4.891 RP) was prepared com- mercially (SociCtk des usines chimiques RhBne-Poulenc, 1955a) which showed greater prophylactic activity in experimental infec- tions than either of its constituents (Cosar, Ducrot, Gailliot and Baget, 1954; Schneider and MontCzin, 1954). Despite its consider- able promise, this compound was poorly tolerated locally on intramuscular injection and was subsequently abandoned (Demarchi, 1958), although simultaneous injection of suramin intravenously and pentamidine intramuscularly was continued as a means of reducing any toxic side-effects of pentamidine (Demarchi, 1958; Neujean and Evens, 1958). Simultaneous treatment with suramin and pentamidine is claimed to have protected some 1500 people for 12-15 months in a French West African area, where 6-monthly pentamidine injections were difficult to en- force (Masseguin, Causse, and Ricosse, 1955), and in this case some enhancement of the prophylactic effect of pentamidine seems to have been achieved.
Insoluble complexes are also formed by combination of suramin with the cationic “cattle drugs” antrycide, dimidium, ethidium, prothidium, metamidium, cinnoline 528, Tozocide, and berenil (Williamson and Desowitz, 1956; Austin et al., 1957; Wragg et aZ., 1958). Maximal co-precipitation has a stoichiometric basis governed by the number of available ionized groups in the combining molecules (Williamson and Desowitz, 1956; Williamson, 1957); one molecule of suramin with six anionic groups combines maximally with six molecules of dimidium or ethidium, each with one cationic group, or with three molecules of antrycide, cinnoline 5 28, berenil, prothidium or metamidium, each with two cationic groups.
Complex formation in this way considerably
reduced the toxicity of the cationic drug component for rats by a factor varying from approximately 3 X for berenil to 33 X for RD 2902 (Williamson, 1957). Similar reduction of toxicity, especially with antry- tide, was observed in the first prophylactic trial in cattle (Williamson and Desowitz, 1956; Desowitz, 1957).
The induction or enhancement of prophy- lactic activity in small animals by insoluble suramin complex formation, was considered to arise from “depot” formation at the site of injection, and considerable prophylactic activity was in fact observed in African cattle exposed to regular and severe tsetse- borne T. vivax challenge. After a single dose, minimal periods of protection of 5% months for antrycide-suramin complex at 40 mg/kg, 13 months for ethidium-suramin complex at 10 mg/kg and 9 months for prothidium- suramin complex at 10 mg/kg were obtained in small numbers of experimental cattle (Desowitz, 1957). (Quoted doses of suramin complexes refer to the amount of cationic drug present and not to the total amount of complex.) High cost precluded further devel- opment of the antrycide-suramin complex in cattle, but its marked prophylactic and therapeutic activity against the important T. simiae infection in pigs (Watson and Williamson, 1958; Stephen and Gray, 1960a) continues to receive attention (see section 2.6). Larrat (1958) indicates that “le traitement associC antrycide-moranyl” has also given excellent results in T. brucei and T. vivax infections of horses and donkeys.
The outstanding activity of the ethidium- suramin complex shown in the preliminary field trial has subsequently been bedevilled by the severe injection-site reaction which develops after subcutaneous injection (Ste- phen, 1958; Stephen and Williamson, 1958; Williamson, 1958; Williamson and Stephen, 1961) . Swelling, ulceration and sloughing, with consequent loss of the drug depot, oc- curred in a high proportion of animals and a variety of attempts to eliminate or decrease the reaction have not so far been successful. Trials in East Africa (Marshall, 1958; Robson, 19586; Smith, 1959) have shown that lengthy protection can result in the few animals which do not lose their depot; in
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 317
the remainder, the prophylaxis is, in general, equivalent to that of prothidium.
Injection site reaction has also attended the use of the metamidium-suramin complex (M. & B. 4427) (Wragg et al., 1958) and attempts to control this by altering the particle-size of the suraminate (Smith and Brown, 1960) have shown that the severity may be lessened by increasing the particle- size to approximately 10 to 100 CL; a suspen- sion of particles less than 3 n was more toxic and somewhat more active prophylacti- cally. Williamson (1957) estimated the particle-size of freshly prepared suramin complex as about 0.5 CL, and later experi- ments with reconstituted freeze-dried ethidium complex in which particle-size was obviously increased, showed that there was an ap- preciable reduction of local toxicity, as- sociated also with a reduction in prophylactic activity (Stephen and Williamson, 1958).
Metamidium suraminate at 10 mg/kg has given protection periods in cattle varying from about 3 months against experimental tsetse-borne T. vivax and T. congolense in- fection in West Africa (Stephen, 1960), to about 5 months in an East African area densely infested with tsetse (Smith and Brown, 1960) ; experiments in Kenya at 5.5 mg/kg (Whiteside et al., 1960) also indicate that the prophylaxis obtained is superior to that of antrycide prosalt.
2.10. NITROFURANS
An entirely new structural type of tryp- anocide, based on the furan nucleus (2.10.1)
# I \ 0
(2.10.1)
was developed from the discovery by Dodd and Stillman (1944) that some simple furan derivatives had bacteriostatic action. S-Nitro- substituted derivatives were particularly active, and the 5nitro-2-furaldehyde semi- carbazone (Furacin, nitrofurazone) ( 2.10.2) was later found to be curative for experi- mental T. equiperdum infections in mice (Dodd, 1946; Giarman, 1951).
0
II
L-l / \ fNHz 02N 0
5’ (2.10.2) Furacin
A number of unsaturated lactones in- cluding furan derivatives, such as furfuryl alcohol, furfural and furoic acid, have tryp- anocidal activity which is abolished by saturation; tetrahydrofuran derivatives, for example, are inactive (Ziering and Buck, 1946; Rubin, 1948 ; Giarman, 195 1). Diamidinofuran analogues of some trypano- tidal aromatic diamidines have also been prepared (Newth and Wiggins, 1947), but only the 5,5’-diamidino-al3-2,2’-difurylethane showed trypanocidal activity; the correspond- ing diamidino-difurfuryl ether was inactive.
An analogue of furacin, furadroxyl (5 nitro-2-furaldehyde 2-( 2-hydroxyethyl)-semi- carbazone (2.10.3) was shown to produce a high survival rate in rats and mice infected with T. equiperdum, T. gambiense and T. rhodesiense (Cole, Frick, Hodges and Dux- bury, 1953); it was inactive against T. cruzi.
HC ’ ‘OH
(2.10.3) Furadroxyl
This compound, like furacin, is active on oral administration but because of its low therapeutic index, Cole et al. (1953) did not recommend its use in man.
Furacin
This drug is only slightly soluble in water and when given in the form of a suspension appears equally active by oral, subcutaneous
318 WILLIAMSON
or intraperitoneal routes (Giarman, 195 1; Packchanian, 1955). Packchanian (1955), in an extensive study of the action of furacin on T. gambiense infections in mice, showed that 100% cure was effected by oral doses of 50 mg/kg spread over 3 weeks to a total of 450-600 mg/kg.
Further experiments in guinea pigs in- fected with T. gambiense and T. rhodesiense (Evens, Niemegeers, and Packchanian, 1957a), and in mice with a recently isolated T. rhodesiense infection (Baker, 1959)) showed that a single intramuscular dose of 100 to 1.50 mg/kg could cure 50 to 100% of T. gambiense infections depending on the strain, but that T. rhodesiense infections were more refractory to both single and divided dose treatment.
The continued incidence of sleeping sick- ness cases which relapse to suramin, tryp- arsamide, pentamidine and even to the new Mel B treatment, prompted a clinical trial of furacin on this type of otherwise hopeless case (Evens, Niemegeers, and Packchanian, 19573). (Furacin might be expected to be active against a trypanosome strain resistant to these drugs because it is chemically un- related to them, but this important point does not seem to have been checked experi- mentally before the trial was begun.) In the event, furacin, which was found to be capable of entering the cerebrospinal fluid of dogs after an oral dose, was given orally three times daily in 2.1 to 12.5 mg/kg doses for 7 to 36 days in 32 human cases of T. gambiense infection of varying severity. Furacin alone cured 2 out of 3 early cases, and with pentamidine, cured 2 out of 3 children with central nervous system involvement. Nine out of 20 hopeless drug-resistant ad- vanced cases responded favorably to a “cocktail” treatment of furacin given in con- junction with suramin, pentamidine and Mel B.
A pilot trial of furacin on 9 similar cases of advanced T. rhodesiense sleeping sickness which had relapsed to all other forms of treatment, including Mel B, also showed en- couraging results (Apted, 1960). Oral furacin treatment (170-583 mg/kg divided into 4 doses over a period of 5 to 7 days), either alone or in conjunction with Mel B, ap-
parently cured 4 cases who would otherwise have died, and considerably prolonged the lives of the remainder. Similar good but variable results have been reported by Fierlafyn (1960) in 82 advanced cases of sleeping sickness, 64 of which had relapsed to other drugs; 12 of the latter group had also relapsed to Mel B.
Toxicity. Both Apted (1960) and Fierlafyn (1960) noted that furacin caused a periph- eral neuritis, and the latter also showed that the blood pyruvate level was raised during treatment; aneurin was therefore given to counteract this effect.
Work on isolated enzyme systems had previously suggested that furacin may inter- fere with pyruvate oxidation (Paul, Bryson, and Harrington, 1956) (see section 3.2), and Robertson (1961) has noted further ex- amples in man of a beri-be&type of poly- neuropathy and disordered pyruvate metab- olism following furacin treatment.
Nitrofurans such as furadroxyl (Prior and Ferguson, 1950) and furacin (Nissim, 1957) also have a specific cytotoxic effect on testic- ular tissue in animals; furacin, because it causes degeneration of seminiferous tubules, has in fact been used to treat disseminated seminoma of the testis in man (Wildermuth, 195.5). This potential danger is overshadowed however, by recent observations which show that furacin is capable of inducing an acute haemolytic anaemia in individuals with the heritable glucose-6-phosphate dehydrogenase deficiency trait (Gilles and Taylor, 1961; Robertson, 1961). The incidence of this trait, which appears to affect erythrocyte integrity, is especially high (15 to 20%) in African negroes (Sonnet and Michaux, 1960), and in such deficient individuals, a number of im- portant drugs such as 8-aminoquinoline antimalarials, aniline derivatives and p- aminosalicylic acid, in addition to furacin, may precipitate haemolysis.
2.11. ANTIBIOTICS
Although a number of established anti- biotics have some suppressive action on T. cruzi (Packchanian, 1953 ; Goldman, 1960)) none of the commoner types, such as peni- cillin, streptomycin, chloramphenicol, ter- ramycin, and chlortetracycline, is active
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 319
against African trypanosomes (Pires and De Almeida, 195 1; Hewitt, Wallace, Gumble, Gill, and Williams, 1954; Miihlpfordt, De Almeida, Girale, and Andrade Lima, 1954; Tani and Takano, 1958; Neujean and Evens, 1958; Sebastiani, 1959). The general insus- ceptibility of protozoa, unlike bacteria, to antibiotic action, may indicate that they are closer phylogenetically to the metazoa (Hutner, Nathan, Aaronson, Baker and Scher, 1958) ; certainly those antibiotics which have antiprotozoal activity are usually also quite toxic for the mammalian host (see below).
At least five antibiotics have been reported as active against African trypanosomes. Four of these, including amphomycin, puromycin and nucleocidin, were obtained from Strepto- myces spp. and the fifth, I.C.I. No. 13959, was isolated from a strain of Paecilomyces. Puromycin and nucleocidin have undergone trial in Africa, the former in man and cattle and the latter so far only in cattle. Cosar, Ninet, Pinnert-Sindico and Preud’homme (1952) described a crystalline product with trypanocidal activity isolated from a Strep- tomyces. The structure, other than ele- mentary composition, was not reported, but the compound was especially active against T. congolense in mice, but apparently at doses not far removed from the maximum tolerated.
Amphomycin (Heinemann, Kaplan, Muir and Hooper, 1953), a polypeptide-like anti- biotic isolated from Streptomyces canus, a new species, cured experimental T. rho- desiense and T. gambiense infections in mice treated intraperitoneally with doses near the maximum tolerated (100 to 150 mg/kg for 4 to 5 consecutive days) (Packchanian, 1956; TrincHo, Da Graca, Da Fonseca and Nogueira, 1958) ; it was ineffective when given orally.
Another polypeptide-like antibiotic, I.C.I. No. 13959, is highly active against T. congol- ense in mice, but again only at doses near the maximum tolerated (Davey, 1961). Chemi- cally, it is of some interest as the isolation from it of the amino acids, a-aminoisobutyric acid and P-hydroxyleucine, represents the first occasion on which these acids have been isolated from natural sources.
Puromycin This substance was originally labeled
Achromycin by Lederle Laboratories Divi- sion, American Cyanamid Co. (Porter, Hewitt, Hesseltine, Krupka, Lowery, Wallace, Bohonos and Williams, 1952). Subsequently the trade name was changed to Puromycin and then to Stylomycin, the label Achro- mycin having been assigned to a Lederle brand of tetracycline.
Originally isolated from Streptomyces albo-niger (Porter et al., 1952), puromycin (6 - dimethylamino - 9 - (3’ - p - methoxy - L-
phenylalanylamino - 3’ - deoxy - p - D - ribo- furanosyl) purine) (Waller, Fryth, Hutch- ings and Williams, 1953; Baker, Schaub, Joseph and Williams, 1954) (2.11.1) can
(2.11.1) Puromycin
also be degraded by loss of the phenylalanyl portion to a form known as puromycin aminonucleoside (6 - dimethylamino - 9 - (3’- amino - 3’ - deoxy - p - D - ribofuranosyl pu- rine) (2.11.2). The striking resemblance of puromycin to the terminal adenylic acid- amino acid “tail” postulated for “transfer” ribonucleic acid is commented on elsewhere (section 3.2)) but it may be noted that the trypanocidal action in vivo and in vitro can be selectively antagonized by adenine (Hewitt, Gumble, Wallace and Williams, 1954; Agosin and von Brand, 1954).
Porter et al. (1952) showed that puromycin was curative for T. equiperdum in mice either orally or parenterally, and that approximate
320 WILLIAMSON
HS\N,PS
(2.11.2) Puromycin aminonucleoside
acute LD 50 figures for mice were 350 mg/kg, 525 mg/kg and 675 mg/kg for the intravenous, intraperitoneal and oral routes respectively. In further work (Hewitt, Wallace, Gumble, Gill and Williams, 1953), a total intraperitoneal dosage of 70 to 140 mg/kg divided into seven doses was found to cure all mice infected with T. equiperdum; twice this dose range was necessary to cure infected rabbits. T. equinum, T. equiperdum and T. evansi infections of mice are more susceptible to puromycin than T. gambiense and T. rhodesiense infections (Tobie, 1954); T. congolense is particularly insusceptible. In a comparative study of 52 puromycin ana- logues (Hewitt, Gumble, Wallace and Wil- liams, 1955) none was found more active against T. equiperdum than puromycin aminonucleoside. The trypanocidal activity in the series varied in a manner parallel to antitumor activity, and interference with nucleic acid or nucleoprotein synthesis was suggested to be a locus of drug action, in view of the reversal of this action by a number of purine derivatives (Hewitt et al., 1954). Tobie and Highman (1956) concluded in agreement with the earlier results of Hewitt et al. (1954) on T. equiperdum in- fections, that the aminonucleoside was more actively trypanocidal than puromycin itself. Further studies by Goldman, Marsico and Angier (1956) on a variety of puromycin aminonucleoside analogues substituted in the
6-position of the purine moiety, showed that the 6-diethylamino- and 6-dipropylamino- analogues were 16 to 32 times more active against T. equiperdum in mice than puromy- tin ; no comparative toxicity data were recorded.
Other experiments on T. gambiense and T. rhodesiense infections (Trincao, Nogueira and France, 1955a, 19553; Trincao and Nogueira, 1957) confirmed the effectiveness of puromycin when given in divided doses from the early stage of the infection; one or two large doses either before or soon after infection were ineffective. T. rhodesiense in mice had been noted as somewhat less sus- ceptible than T. gambiense (Tobie and High- man, 1956), and Baker (1957), with a freshly isolated strain in rats, was able to cure only a small proportion with puromycin; for this reason he suggested that it was un- likely to be effective clinically.
Clinical trials have however been made against T. gambiense sleeping sickness. In Portuguese Guinea, fifteen cases were treated orally with 1 to 2.25 g daily over 7 to 10 days (Trincao, France, Nogueira, Pinto and Miihlpfordt, 1955; Trincao, Pinto, Franc0 and Nogueira, 1956). Ten of these remained free of infection for a period of 2.5 months, but four out of five relapses were in the nervous stage of the disease. Other clinical tests have also given effective results only in the early stages of infection (Heuls, Orio, Ceccaldi and Merveille, 1958) or have not been considered successful (Neujean and Evens, 1958). Puromycin thus appears un- likely to supplant existing drugs for the treatment of African sleeping sickness, but it is likely to be of considerable experimental value in studies on the mode of trypanocidal drug action (see section 3.2). The activity of its aminonucleoside, in conjunction with primaquine, in experimental T. cruzi infec- tions (Moraes, Faria and Fernandes, 1960) may be of considerable practical value in South American trypanosomiasis.
Nucleocidin
This antibiotic, isolated from Streptomyces calvus (Thomas et al., 1957; American Cyanamid Co., 1959) unlike puromycin, is more active against T. congolevz.se than
CHEMOTHERAPY OF AFRICAN TRYPANOSOMIASIS 321
against T. gambiense in rats and mice (Tobie, 1957). Three daily subcutaneous doses of 0.05 mg/kg were 95% curative for T. c,ongol- ense but only 73% curative for T. gambiense.
Its structure has only partially been elucidated (Waller, Patrick, Fulmor and Meyer, 1957) but it resembles puromycin in being a 9-substituted adenine glycoside. The carbohydrate portion appears to be bound by ester formation to sulphamic acid (2.11.3).
(2 .11.3) Nucleocidin (partial structure)
Following the demonstration of high ac- tivity in experimental T. congolense infec- tions, a therapeutic trial in African cattle has been reported (Stephen and Gray, 1960b). A single intramuscular dose of 0.025 mg/kg effectively cleared the blood of a tsetse-borne mixed T. vivax and T. congobnse infection for periods of 18 to 33 days, but was not curative.
None of these trypanocidal antibiotics ap- pears to have any prophylactic activity, but they may conceivably be of value in some form of combined therapy with other established trypanocides.
2.12. MISCELLANEOUS COMPOUNDS
Trypanocidal activity of varying degree continues to be demonstrated in a number of compounds, not readily classifiable under the preceding chemical groups. The activity has been of experimental interest primarily, but in some cases it may be related to struc- tural features similar to those in established trypanocides; in one case, p-aminosalicylic acid, a drug widely used for tuberculosis treatment, has shown hitherto unsuspected trypanocidal activity (Pick, 1950).
A heparin-like sulfonated galacturonic acid which is active but not curative in T. brucei infections of mice (Taubmann and Winkler,
1951), resembles suramin and the related sulfonated dyes Trypan Red and Trypan Blue in being both trypanocidal and anti- coagulant. Two compounds, 6-amidino-2- p-amidinophenylbenzthiazole ( 2.12.1) and the corresponding benziminazole analogue
NH (2.12.1)
(Bower, Stephens and Wibberley, 1950) have marked trypanocidal activity against T. rhodesiense in mice. The latter compound, which is at least six times more active than the former, has a subcutaneous CD 50 of 4 mg/kg and an LD 50 of 160 mg/kg; its superior activity is ascribed to the increased possibilities of tautomerism associated with the iminazole system. Benziminazoles can in fact be regarded as cyclic amidines (Hunter and Marriott, 1941) and there is an obvious affinity between the active derivative prepared by Bower et aZ. (1950) and the aromatic diamidines (section 2.S) and even with the phenanthridinium series (cf. 2.7.1). A number of related properties such as a high degree of internal conjugation, cationic res- onance, possession of hydrogen-bonding sub- stituents, molecular planarity and extended molecular area, are shared by several active trypanocides in the “cationic” group (sections 2.3-2.6), and probably contribute to the ease and strength of adsorption of these drugs at cellular receptor sites.
The high trypanocidal activity of aliphatic diguanidines, diisothioureas and diamidines emphasizes functional, possibly “bio-isosteric,” similarities in the terminal groups, and Volini, Stubbs and Ercoli (1956) have shown that in a series of long-chain aliphatic monoisothiouronium chlorides
NH . HCl
RS/ . . . \
NH2
322 WILLIAMSON
the coconut-oil derivative (R = LOCO) had some curative activity in T. equiperdum in- fections of mice. The functional similarity in trypanocidal action of the guanidine- and thiourea-type groupings is illustrated more effectively in the series of l-substituted dithiobiuret derivatives reported by Woolfe (1953) and Fairfull and Peak (1955). These compounds were markedly active only against T. congolense and the two most effective products, l-methyl- and l-n-propyl- l-phenyldithiobiuret (2.12.2), although poorly water-soluble, were of low toxicity (LD 50, 0.1 and 0.5 g/kg respectively, subcutaneously in mice) and curative at low doses (CD 50, 50 mg/kg and 125 mg/kg respectively divided into 5 daily doses).
s ii II
\
2 xl I
HN-N-N ’ H I
(2.12.2)
Despite these high therapeutic ratios, the insolubility of the drugs and the necessity for repeated dosage seemed to preclude their further development for use in African cattle.
A highly original approach to the chemo- therapy of T. congolense is provided by Goble and Boyd (1959) who tested a series of tetrapyrrole derivatives in mice on the sup- position that they might act as antagonists of hematoporphyrin-type metabolites known or assumed to be essential for a number of Trypanosomidae. Of a variety of derivatives, only magnesium or copper chlorophyllins and haematoporphyrin were active; prolonged intravenous administration of these at dose levels of 600 to 1000 mg/kg was curative, and their activity could be reversed com- pletely or partially by haemin, but not by protoporphyrin. The compounds had no action on T. cruzi infections.
This interesting result suggests that T. congolense has a specific requirement for an iron porphyrin, although cytochrome pig- ments do not appear to be present (Ryley,
1956; Fulton and Spooner, 1959). Further complications in analysis of the drug sus- ceptibility of T. congolense are illustrated by the activity of certain surface-active polyoxyethylene ethers which are active against murine tuberculosis and leprosy (Goble, Boyd and Fulton, 1960). In these compounds, a p-lert-octyl-phenol unit (2.12.3) can be arranged either linearly, or condensed into a cyclic structure composed of 4 units. Only the linear type appears to have trypanocidal activity (but not against T. CYUZ~), although both types are active against
(2.12.3) Structural unit of polyoxyethylene ethers
Leishmania infections. This association of polymeric structure with leishmanicidal ac- tivity may be reflected also in the pentavalent organic antimonials which have a marked tendency to polymerize through antimony- oxygen linkages. T. congolense in mice could be cured by large intravenous doses (5 g/kg) of a linear derivative (WR-1339) and some activity was also evident in vitro. The com- pounds were chosen for test, because it was thought that they might act on trypano- somes by interference with the reticuloendo- thelial system, but the results have shown that other factors are also involved.
Although none of the compounds described in this section has proved of practical use, the importance of studies on the trypano- tidal activity of unconventional chemical structures is considerable, not only for an understanding of the chemical basis of tryp- anocidal action, but also, as suggested for the antibiotics in the preceding section, as possible synergic agents in combined therapy.
