DIMINAZENE
1. EXPLANATION
Diminazene aceturate, usually referred to simply as diminazene
(other names are 4,4'-(diazoamino) dibenzamidine diaceturate, berenil)
is used in tropical countries for the treatment of animal
trypanosomiasis and babesiosis, usually as an intramuscular injection
of 3-5 mg/kg. It is said to be effective in canine, ovine and bovine
babesiosis and, unlike some drugs, is less susceptible to relapse. It
may also possess antibacterial properties. This compound has not
previously been evaluated by the Joint FAO/WHO Expert Committee on
Food Additives.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
There are few data available on the distribution and excretion of
diminazene in laboratory animals and none on biotransformation. In the
rat, absorption appeared to be moderate after oral administration. For
example, after a dosage of 100 mg/kg bw, blood levels were about
0.25-2.25 µg/ml at 0-2 hours, 1.85 µg/ml at 7 hours and 0.5-0.6 µg/ml
at 28-31.5 hours, whereas after subcutaneous dosing levels were 26.35
µg/ml at 0-2 hours, 6 µg/ml at 7 hours and zero at 28-48 hours
(Raether et al., 1972).
In a relay study where rats were given liver from a calf dosed 7
days previously with 3.67 mg/kg diminazene (to give an oral dose to
the rats of 0.28-0.32 mg/kg), a large proportion of the dose was
eliminated in the urine (21-33%) and in the feces (37-48%). Only small
amounts were found in the bile (0.24-0.43%). It was estimated that
25-35% of the available dose was absorbed but the fractions present as
parent compound and/or metabolites were unknown (Kellner et al.,
1985).
When 3.5 mg/kg bw was given to rabbits by intramuscular
injection, biphasic pharmacokinetics with maximum blood levels at 15
minutes (1.3 µg/ml) and 3 hours (0.116 µg/ml) were noted. The highest
tissue levels at 7 days were in the liver (40 ppm), brain (2.5 ppm)
and kidney (3 ppm). Levels were low in other organs (0.4-2.0 ppm)
including muscle (2.1 ppm). By 7 days, 40-50% of the dose had been
excreted in the urine with 8-20% in the feces, the latter indicative
of biliary excretion (Gilbert & Newton, 1982; Gilbert, 1983).
In the monkey (Maccaca mulatta), peak plasma levels were
attained 25 minutes after an intramuscular dose of 20 mg/kg bw
diminazene and 6 hours after an oral dose of 40 mg/kg bw. The
elimination half-life was 15 hours for the oral dose and showed
monophasic kinetics. For the intramuscular dose, biphasic kinetics
were noted with half-lives of 1-2 hours and 18-19 hours (Raether et
al., 1974).
When 2 calves were dosed intramuscularly with 3.5 mg/kg bw
diminazene , maximum blood levels were reached after 15 and 45
minutes. Plasma clearance was biphasic with half-lives of 2 and 188
hours for each phase. By day 7, 47% of the dose had been excreted in
the urine and 7.1% in the feces. This was indicative of biliary
excretion in this species. Residue levels were low in edible tissues
when the calves were killed on days 7 or 20 after dosing, except for
the kidney, liver and myocardium (54.7, 75.5 and 6.6 ppm at day 7 and
12.1, 24.4 and 2.9 ppm, respectively, at day 20). Levels in skeletal
muscle were low at both days (0.5 and 0.3 ppm, respectively) (Kellner
et al., 1985). The results were similar to those noted in another
study where 3.5 mg/kg bw intramuscular diminazene produced peak plasma
levels at 0.5 hours and a maximum level of 4.5 µg/ml (Klatt & Hajdu,
1971; Klatt & Hajdu, 1976). Milk collected at 6 and 24 hours contained
0.2-0.5 µg/ml diminazene but the level fell to 0.1-0.2 µg/ml by 30
hours and was undetectable at 48 hours following dosing (limit of
detection 0.07 µg/ml). Two metabolites were detected in the urine of
calves; p-aminobenzamidine (22%) and p-aminobenzamidine (4%). The
remainder was the parent compound (74%) and approximately 80% of the
intramuscular dose was collected in the urine (Klatt & Hajdu, 1971).
In cattle, diminazene appeared to bind irreversibly to blood proteins
including hemoglobin (Alvi et al., 1985).
In goats given 3.5 mg/kg bw diminazene intramuscularly, systemic
availability was calculated to be 44-46% (Aliu et al., 1984). After
achieving a peak plasma level within one hour of administration,
plasma decay was triexponential. The plasma half-life (14-30h) was
longer than in sheep (10-13h) but shorter than in cattle (40-138h).
Following administration of diminazene (3.5 mg/kg bw) to sheep by the
intramuscular route, maximum plasma levels of 6.3-7.6 µg/ml were
attained in 20-45 minutes and plasma protein binding was high (65-85%)
and concentration-dependent. Based on the pharmacokinetic data in
sheep and goats, withdrawal periods of around 26 days were recommended
for tissue depletion although these times did not appear to take
account of retention by any specific issues.
2.2 Toxicological studies
2.2.1 Acute toxicity
There are few acute toxicity data available with diminazene in
the usual laboratory species. In the mouse (NMRI), in a preliminary
study for a micronucleus test (see section 2.2.5), an oral dose of
1500 mg/kg bw to 3 males and 3 females resulted in a single death
(female). Signs of toxicity included increased spontaneous activity,
tactile hyperesthesia and uncoordinated gait (Muller, 1988). The
LD50 by the subcutaneous route in the mouse was 258 mg/kg bw
(Berenil-Toxicology, 1988). Mice tolerated doses of 75 mg/kg bw but
unfortunately no other details including those of route and vehicle
were specified (Harant, 1979).
Brain damage has been reported in asses and dogs given the drug
(Aburu et al., 1984; Losos & Crockett, 1969). In the dog, spastic
paralysis, opisthotonos and nystagmus with involuntary running
movements were noted in dogs treated 24-72 hours previously with
diminazene. Intramuscular hemorrhage and diffuse intramuscular edema
were noted at the injection site (Losos & Crockett, 1969). Similar
signs accompanied by vomiting and death have been reported in dogs
given 30-35 mg/kg bw (Fussganger & Bauer, 1958) although the
recommended therapeutic dose is 3.5-8.0 mg/kg bw. In a small
experimental study, groups of 2 dogs of unspecified strain were given
single intramuscular doses of 10, 15, 20 or 60 mg/kg bw diminazene.
Animals given 20-60 mg/kg bw died 36-54 hours after administration;
clinical signs were the same as those reported for dogs treated
therapeutically with the drug. Extensive hemorrhagic malacia of the
brain stem was noted involving the mesencephalon and diencephalon
(Losos & Crockett, 1969).
Intramuscular injections of 8 mg/kg bw were well tolerated except
in the buffalo cow where "quivering" and restlessness followed dosing.
The animal recovered after an intravenous dose of dextrose (Verma et
al., 1970). Buffalo calves tolerated intramuscular doses of 20 mg/kg
bw diminazene. No acute effects occurred in cattle when given 6 times
the recommended dose (21 mg/kg bw) (Fairclough, 1963). Camels also
tolerated diminazene when given intramuscularly at the recommended
therapeutic dose of 3.5 mg/kg bw (Schillinger & Rottcher, 1986).
From a total of 154 donkeys usually given 0.5 mg/kg bw every 3
months to protect against trypanosome infection, a group of 31 became
infected with T. brucei and were treated with 7 mg/kg bw diminazene.
Around 48 hours later, 4 animals became weak and displayed staggering
and ataxia and by 96 hours, 29 donkeys had developed CNS effects; 6
died. The survivors recovered after 14-30 days. Post-mortem
examination revealed macroscopic and microscopic hemorrhages in the
cerebellum (Boyt et al., 1971).
Hepatoxicity in the dog has also been reported after single doses
of 3.5 mg/kg bw but pre-existing liver disease could not be excluded
as a contributing factor (Opping, 1969). Diminazene was also
hepatotoxic to camels; 10 or 40 mg/kg bw given to 3 dromedary camels
by the intramuscular route resulted in hyperaesthesia, salivation,
intermittent convulsions, frequent urination and defecation and
sweating. At necropsy, the lungs were congested and edematous while
the liver was congested and hemorrhagic with evidence of fatty change.
Congestion of the brain and urinary bladder was noted along with
hemorrhaging and congestive changes in the kidneys and heart (Homeida
et al., 1981).
2.2.2 Short-term studies
2.2.2.1 Rats
Groups of 20 male and 20 female Wistar rats were fed diets
containing 630, 1600 or 4000 ppm diminazene for 5 weeks after which
the levels were increased by 50% to 945, 2400 and 6000 ppm. After 3
months, 10 rats from each group were sacrificed and the remainder fed
the treated diets for a total of 9 months. Two other groups of 15 male
and 15 female rats were given gavage doses of 63 and 160 mg/kg bw/day
diminazene for 3 months when 5 rats of each sex were killed and the
remainder given the drug for a further 3 months. No signs of toxicity
were noted and no effects on food intake, body weights, hematology,
blood glucose or urine were observed. There were no macro- or
microscopic drug-related effects in any of the major organs (Baeder
et al., 1975).
2.2.2.2 Dogs
Diminazene was given to dogs for periods of up to 10 days. It was
reported that "ages varied from 6 months to 7 years and the breed from
purebred Alsatian to nondescript mongrels". When given 3.5 mg/kg bw on
two successive days by the intramuscular route, no signs of toxicity
were observed. A group of 3 males and 3 females were given 3.5 mg/kg
bw/day intramuscularly until signs of toxicity developed. Two dogs of
each sex showed signs of CNS toxicity on days 6-9 and all 4 were
killed on day 10; the remaining 2 dogs were unaffected. A further
group of 3 males and 3 females were treated in a similar manner with
10.5 mg/kg bw/day diminazene. All the dogs died on days 3-5. All the
affected animals showed cerebellar lesions characterized by
hemorrhages and areas of malacia. They were generally bilateral (Naude
et al., 1970).
In an oral study, groups of 3 male and 3 female beagle dogs were
given capsules containing 0, 20 or 60 mg/kg bw/day diminazene, 7 days
per week for 9 months. One dog of each sex died at the high dose. Body
weights were also reduced and general condition was poor in high dose
males. There were, however, no specific effects on hematology, urine,
serum analysis and blood glucose levels. In animals given 60 mg/kg
bw/day diminazene there were foci of softening in the brain stem and
cerebellum. There was also testicular atrophy and prostatic
dysfunction (Scholz & Brunk, 1969).
In a study where 10 dogs and 2 long-tailed monkeys were given an
unspecified dose of diminazene by an unspecified route for an
unspecified period of time, 7 dogs and both monkeys died within the
first week. Paralysis and lameness were noted prior to death.
Hemorrhagic necrosis in the CNS, mainly in the brain, was evident at
necropsy. The lesion was confined to the brain stem and cerebellum and
was thought to be due to necrosis of the capillaries and arteries
(Schmidt et al., 1977).
2.2.2.3 Oxen
An ox given intramuscular diminazene, 7 mg/kg bw/day for 15 days,
developed CNS-like signs of toxicity including ataxia and muscle
tremors, and the animal died on day 18. Necropsy revealed mild
swelling of the thalamic glia (Naude et al., 1970).
2.2.3 Long-term/carcinogenicity studies
No data are available.
2.2.4 Reproduction studies
No data are available.
2.2.5 Special studies on genotoxicity
There are few conventional studies available to assess the
genotoxic potential of diminazene, although there are a number of
publications suggesting a direct effect on DNA. It has been
demonstrated that diminazene is not a DNA intercalating agent (Waring,
1970 a-d; Bernard & Riou, 1980).
Treatment of Trypanosoma cruzi kinetoplast DNA with diminazine
resulted in an increased proportion of double-branched circular DNA
molecules. Replication of the DNA appeared to be blocked at specific
sites. Treatment resulted in a changed electron microscopic appearance
of the DNA which displayed a "lampbrush-like" appearance, possibly due
to the formation of replicating circular DNA molecules resulting in
specific blocking of the kinetoplast DNA replication (Brack et al.,
1972a and b). Berenil appears to bind to the trypanosome DNA including
DNA denatured by heat or other agents (Festy et al., 1970a and b).
The significance of these findings for the possible genotoxic effects
of diminazine on mammalian DNA is unknown.
Diminazene-induced respiration deficient petite mutations in
Saccharomyces cerevisiae (Mahler & Perlman, 1973; Perlman & Mahler
1973; Villa & Juliani, 1980). It is thought that diminazene binds to
yeast mitochondrial DNA (Perlman & Mahler 1973; Mahler 1973). Again,
whether these findings are relevant to mammals is unknown although
they appear to be involved in the trypanocidal activity of the drug
possibly in association with its inhibitory effects on diamine oxidase
and S-adenosylmethionine decarboxylase (Balana-Fouce et al., 1986).
In a micronucleus test, groups of 5 male and 5 female NMRI mice
were given gavage doses of 0 or 1500 mg/kg bw (highest tolerated doze)
of diminazene in water. Cyclophosphamide (50 mg/kg bw) served as a
positive control. Mice were killed 24, 48 and 72 hours after dosing.
There was no increased incidence of micronucleated polychromatic
erythrocytes in any of the test groups (1000 polychromatic erythrocyes
per mouse examined) but there was a marked increase in incidence in
animals given cyclophosphamide (Muller, 1988).
2.3 Observations in humans
Ninety-nine patients who had been treated 12-109 months earlier
with diminazene for early stage African trypanosomiasis were traced
and subjected to a medical examination. Each patient had received 3
doses of 5 mg/kg bw diminazene at one or two day intervals. Although
diminazene is known to produce a number of side effects in humans,
including pain in the soles of the feet, pyrexia, nausea, vomiting and
paralysis, no adverse effects were noted in the group (Abaru, et al.,
1984).
3. COMMENTS
No carcinogenicity studies on diminazene and only two
conventional mutagenicity studies were available. Trypanosome
kinetoplast studies suggested an interaction of diminazene with DNA
but the relevance to genotoxicity was unknown; the substance was not
an intercalating agent. Diminazene induced respiration-deficient
petite mutations in yeast but gave negative results in the mouse
micronucleus test. No teratology or other studies were available for
use in assessing the reproductive toxicity of the compound. Acute
toxicity studies in mice showed some evidence of effects on the
central nervous system at high doses. In dogs, cattle and donkeys,
intramuscular doses of 7-35 mg/kg bw/day diminazene produced clinical
effects on the nervous system, and necropsy revealed cerebellar
hemorrhages and edema. Hepatotoxic effects were reported in the dog at
a dose of 3.5 mg/kg bw/day although pre-existing liver disease could
not be ruled out. Intramuscular doses of 10-40 mg/kg bw/day were
hepatotoxic in camels.
Daily doses of 300-500 mg/kg bw diminazene for up to nine months
in the diet of rats produced no signs of toxicity. In dogs, repeated
intramuscular or oral administration produced signs of toxic effects
in the central nervous system, which were confirmed at necropsy by the
presence of cerebellar lesions. Feeding diminazene to dogs at 60 mg/kg
bw/day for nine months produced testicular atrophy; in this study, the
no-effect level was 20 mg/kg bw/day. There were no acceptable data in
humans on which to base any assessment.
The Committee was unable to establish an ADI because the results
of adequate studies of toxicity, including studies of carcinogenicity
(or genotoxicity), reproduction and teratogenicity, were not
available.
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