The safety, tolerability and pharmacokinetics of levamisole alone, levamisole plus ivermectin, and levamisole plus albendazole, and their efficacy against Onchocerca volvulus


Awadzi, K.; Edwards, G.; Opoku, N.O.; Ardrey, A.E.; Favager, S.; Addy, E.T.; Attah, S.K.; Yamuah, L.K.; Quartey, B.T.

Annals of Tropical Medicine and Parasitology 98(6): 595-614

2004


Two randomized, double-blind, placebo-controlled trials, in which levamisole (2.5 mg/kg) was given alone or co-administered with ivermectin (200 µg/kg) or albendazole (400 mg), were conducted. In Trial 1, safety and drug— drug interaction were explored in 42 healthy male volunteers. During Trial 2, the safety of the same treatment regimens and their efficacy against the adult worms and microfilariae of Onchocerca volvulus were investigated in 66 infected subjects of both sexes. Safety was determined from the results of detailed clinical and laboratory examinations before treatment, during hospitalization and on day 30. The pharmacokinetic parameters for levamisole alone and the combinations were determined in Trial 1 and then compared with historical data for ivermectin and albendazole, given as single agents, to determine if drug—drug interaction had occurred. The level of efficacy against the adult worms was determined by the examination of histology sections of nodules excised 6 months posttreatment and from the changes seen in the levels of microfilaridermia within a year of treatment. Microfilaricidal efficacy was estimated from the reductions in the levels of microfilaridermia between day 0 (1 day pre-treatment) and day 30. Although the regimens were generally well tolerated, there were unexpected adverse effects in both healthy volunteers and infected subjects. Clinically significant drug—drug interactions resulted in an increase in the bio-availability of ivermectin but a reduction in that of albendazole when these drugs were co-administered with levamisole. Levamisole given alone or with albendazole had little effect on 0. volvulus. The combination of levamisole with ivermectin was neither macrofilaricidal nor more effective against the microfilariae and the adult worms than ivermectin alone. The pathogenesis of the adverse events and the drug—drug interactions are discussed.

Annals
of
Tropical
Medicine
&
Parasitology,
Vol.
98,
No.
6,
595-614
(2004)
The
safety,
tolerability
and
pharmacokinetics
of
levamisole
alone,
levamisole
plus
ivermectin,
and
levamisole
plus
albendazole,
and
their
efficacy
against
Onchocerca
volvulus
K.
AWADZI*,
G.
EDWARDSt
4
,
N.
0.
OPOKU*,
A.
E.
ARDREYt,
S.
FAVAGERt,
E.
T.
ADDY*,
S.
K.
ATTAH*,
L.
K.
YAMUAH§
and
B.
T.
QUARTEY*
*Onchocerciasis
Chemotherapy
Research
Centre
(OCRC),
Hohoe
Hospital,
P.O.
Box
144,
Hohoe,
Ghana
*Department
of
Pharmacology
and
Therapeutics,
The
University
of
Liverpool,
Ashton
Street
Medical
School,
Liverpool
L69
3GE,
U.K
*Molecular
and
Biochemical
Parasitology
Research
Group,
Liverpool
School
of
Tropical
Medicine,
Pembroke
Place,
Liverpool
L3
SQA,
U.K
§Armauer
Hansen
Research
Institute
(AHRI),
P.O.
Box
1005,
Addis
Ababa,
Ethiopia
Received
30
March
2004,
Revised
20
May
2004,
Accepted
24
May
2004
Two
randomized,
double-blind,
placebo
-controlled
trials,
in
which
levamisole
(2.5
mg/kg)
was
given
alone
or
co
-administered
with
ivermectin
(200
µg/kg)
or
albendazole
(400
mg),
were
conducted.
In
Trial
1,
safety
and
drug
drug
interaction
were
explored
in
42
healthy
male
volunteers.
During
Trial
2,
the
safety
of
the
same
treatment
regimens
and
their
efficacy
against
the
adult
worms
and
microfilariae
of
Onchocerca
volvulus
were
investigated
in
66
infected
subjects
of
both
sexes.
Safety
was
determined
from
the
results
of
detailed
clinical
and
laboratory
examina-
tions
before
treatment,
during
hospitalization
and
on
day
30.
The
pharmacokinetic
parameters
for
levamisole
alone
and
the
combinations
were
determined
in
Trial
1
and
then
compared
with
historical
data
for
ivermectin
and
albendazole,
given
as
single
agents,
to
determine
if
drug
—drug
interaction
had
occurred.
The
level
of
efficacy
against
the
adult
worms
was
determined
by
the
examination
of
histology
sections
of
nodules
excised
6
months
post-
treatment
and
from
the
changes
seen
in
the
levels
of
microfilaridermia
within
a
year
of
treatment.
Microfilaricidal
efficacy
was
estimated
from
the
reductions
in
the
levels
of
microfilaridermia
between
day
0
(1
day
pre-treatment)
and
day
30.
Although
the
regimens
were
generally
well
tolerated,
there
were
unexpected
adverse
effects
in
both
healthy
volunteers
and
infected
subjects.
Clinically
significant
drug
—drug
interactions
resulted
in
an
increase
in
the
bio-availability
of
ivermectin
but
a
reduction
in
that
of
albendazole
when
these
drugs
were
co
-administered
with
levamisole.
Levamisole
given
alone
or
with
albendazole
had
little
effect
on
0.
volvulus.
The
combination
of
levamisole
with
ivermectin
was
neither
macrofilaricidal
nor
more
effective
against
the
microfilariae
and
the
adult
worms
than
ivermectin
alone.
The
pathogenesis
of
the
adverse
events
and
the
drug
—drug
interactions
are
discussed.
Levamisole
hydrochloride
is
a
broad-
spectrum
anthelmintic
and
an
immuno-
stimulant
that
restores
cell
-mediated
immune
mechanisms
that
have
become
depressed
in
peripheral
T
-lymphocytes
(Renoux,
1980).
Awadzi
et
al.
(1982)
found
that
levamisole
had
little
effect
on
the
Reprint
requests
to:
K.
Awadzi.
E-mail:
awadzi@ghana.com;
fax:
+233
21
668871.
microfilariae
(mff)
or
the
adult
worms
of
Onchocerca
volvulus
when
given,
at
2.5
mg/
kg,
on
two
occasions
during
the
fi
rst
week
and
then
weekly
for
3
weeks,
to
patients
infected
with
the
parasites.
When
the
same
dose
was
administered
on
two
occasions
in
the
week
preceding
a
21
-day
treatment
with
mebendazole,
however,
levamisole
was
seen
to
augment
the
modest
effects
of
the
mebendazole
against
the
mff
and
the
©
2004
The
Liverpool
School
of
Tropical
Medicine
DOI:
10.1179/000349804225021370
596
AWADZI
ETAL.
intra-uterine
embryos
of
0.
volvulus
(Awadzi
et
al.,
1982).
Although
the
mechanism
was
unclear,
this
benefit
was
attributed
to
a
`priming'
of
the
immune
system
by
pre-
treatment
with
levamisole,
and
not
to
any
alteration
in
the
pharmacokinetics
of
the
mebendazole
(Awadzi
et
al.,
1982)
.
Alb
en-
dazole,
another
benzimidazole
carbamate
derivative,
has
little
activity
against
the
mff
of
0.
volvulus
but
has
been
found
to
disrupt
severely
all
the
intra-uterine
stages
in
the
adult,
female
worms
(Awadzi
et
al.,
1991).
Moreover,
as
albendazole
is
much
more
potent
than
mebendazole,
significant
activity
against
0.
volvulus
is
achieved
with
a
single
dose,
and
prolonged
treatment,
like
that
required
with
mebendazole,
is
unnecessary
(Awadzi
et
al.,
1994).
Should
the
activity
of
albendazole
against
adult
0.
volvulus
be
augmented,
like
that
of
mebendazole,
by
levamisole,
then
treatment
with
levamisole
followed
by
albendazole
may
possibly
lead
to
a
more
prolonged
embryotoxic
effect
than
seen
with
levamisole/mebendazole,
or
even
a
permanent
sterilization
of
the
adult,
female
worms.
Ivermectin,
the
only
drug
currently
recommended
for
the
treatment
of
oncho-
cerciasis,
neither
kills
nor
sterilizes
the
adult
worms
of
0.
volvulus
when
given
in
single
doses
of
150-800
lug/kg
bodyweight
(Awadzi
et
al.,
1999).
Given
repeatedly,
however,
ivermectin
causes
a
variable
level
of
mortality
among
the
adult,
female
worms
(Chavasse
et
al.,
1992;
Duke
et
al.,
1992;
Klager
et
al.,
1996;
Gardon
et
al.,
2002).
If
a
single
dose
of
ivermectin
has
a
significant
but
hidden
macrofilaricidal
potential
that
could
be
revealed
by
the
immunomodulating
pro-
perties
of
levamisole,
the
co
-administration
of
the
two
drugs
should
radically
improve
the
control
of
onchocerciasis.
Combinations
of
levamisole
with
alben-
dazole
or
ivermectin
have
not
previously
been
used
in
humans.
The
aims
of
the
present
study
were
to
determine
the
safety
and
tolerability
of
levamisole,
when
used
alone
or
in
combination
with
albendazole
or
ivermectin,
in
healthy
volunteers,
to
explore
the
pharmacokinetic
interactions
of
the
two
drug
combinations
in
the
same
volunteers,
and
then
to
determine
the
safety
and
efficacy
of
the
same
three
treatment
regimens
in
patients
infected
with
0.
volvulus.
SUBJECTS
AND
METHODS
Ethical
Aspects
In
each
of
the
communities
in
which
the
subjects
of
the
present
study
resided,
the
aims
of
each
trial,
the
procedures
involved
and
any
potential
risks
and
benefits
were
explained
in
detail
to
all
the
participants,
the
village
co-ordinators
who
are
supported
by
the
Onchocerciasis
Chemotherapy
Research
Centre
(OCRC)
at
Hohoe,
Ghana,
and
the
community
leaders,
prior
to
any
investiga-
tive
procedures
and
admissions
to
hospital.
Each
subject's
consent
to
participate
in
the
study,
given
verbally,
was
witnessed
and
documented
on
a
form
by
the
local
OCRC
village
co-ordinator
and
by
an
impartial
witness
from
the
community.
The
form
was
then
countersigned
and
dated
by
one
of
the
investigators.
The
study
protocol
was
approved
by
the
Ethics
Committee
of
the
Ministry
of
Health,
Ghana,
and
by
the
Secretariat
Committee
for
Research
Involving
Human
Subjects
(SCRIHS)
of
the
World
Health
Organization.
The
trials
took
place
at
the
OCRC
between
February
1998
and
June
2000.
They
were
conducted
in
accordance
with
good
clinical
practices
and
the
Declaration
of
Helsinki
(1996
revision).
Subjects
All
108
participants
in
the
two
trials
that
were
conducted
came
from
onchocerciasis-
endemic
communities
in
the
basin
of
the
River
Tordzi,
in
the
forested
region
of
south—eastern
Ghana.
These
communities
lie
in
a
focus
that
has
not
been
under
vector
control
and
is
not
part
of
any
ivermectin-
treatment
programme.
The
subjects
were
aged
18-60
years
and
in
good
general
health,
and
each
weighed
at
least
40
kg
and
had
no
significant,
concurrent,
clinical,
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
597
haematological
or
biochemical
abnormali-
ties
(see
details
below).
TRIAL
I
In
Trial
1,
42
male
subjects
who
appeared
free
from
the
filarial
parasites
prevalent
in
the
area
(i.e.
0.
volvulus,
Mansonella
streptocerca
and
Wuchereria
bancroftz)
were
cautiously
exposed
to
the
treatment
regi-
mens
that
had
never
before
been
given
to
humans.
Only
six
men
were
initially
enrolled,
with
two
assigned
to
each
treat-
ment
regimen.
After
close
observation
of
this
first
group
revealed
no
serious
untoward
effects,
a
second
group
of
nine
subjects,
followed
by
third
of
12
subjects,
and
finally
a
fourth
group
of
15
subjects
were
investi-
gated,
giving
a
final
total
of
14
subjects
in
each
of
the
three
treatment
arms.
The
time
intervals
between
the
treatments
of
succes-
sive
groups
of
subjects
were
12,
14
and
21
days.
TRIAL
2
For
Trial
2,
66
onchocercal
nodule
carriers
of
both
sexes
were
enrolled,
with
five
groups
(of
12-15
subjects
each)
tested
one
after
the
other
and
22
subjects
eventually
assigned
to
each
treatment
regimen.
None
of
the
women
investigated
was
breastfeeding
and
all
gave
negative
results
in
pregnancy
tests.
Although
the
initial
group
consisted
of
lightly
infected
subjects
(with
<
10
mff/mg
skin
-snip),
more
heavily
infected
individuals
were
enrolled
in
the
subsequent
groups.
Study
Design
and
Blinding
Each
trial
was
randomized,
double-blind
and
placebo
-controlled.
Randomization
to
treatment
regimens,
blinding,
and
the
moni-
toring
for
drug
safety,
were
conducted
in
a
very
similar
manner
in
the
two
trials;
the
treatment
regimens
were
identical.
In
each
trial,
subjects
satisfying
the
protocol
require-
ments
were
given
code
numbers
and
ran-
domized
into
three
treatment
arms,
using
three
columns
of
random
integers
generated
by
the
MINITAB
computer
programme
(MINITAB
Inc.,
State
College,
PA).
The
drugs
were
packaged
individually
into
enve-
lopes
and
labelled
with
the
participant's
name,
code
number
and
date
of
admini-
stration.
The
code
number
and
treatment
schedule
for
each
participant
were
recorded
on
a
piece
of
paper
which
was
then
deposited
in
a
sealed,
opaque
envelope.
The
sealed
envelopes
were
kept
in
a
locked
cabinet
for
the
duration
of
the
trials.
The
investigators
who
monitored
the
trials
were
different
to
those
who
arranged
the
randomization.
All
subjects
received
the
same
number
of
tablets;
their
nature
was
unknown
to
the
participants
and
to
those
who
administered
them.
Individuals
who
were
unaware
of
the
treatment
regimens
performed
the
labora-
tory
tests
and
drug
analyses.
All
assessors
were
un-blinded
only
after
all
the
data
col-
lected
over
the
trial
period
had
been
verified,
validated
and
locked
by
a
clinical
monitor,
who
also
closely
examined
the
procedures
during
Trial
2.
Primary
and
Secondary
Endpoints
The
primary
endpoint
in
Trial
1
was
the
safety
of
the
novel
drug
combinations
(levamisole—ivermectin
and
levamisole—
albendazole).
The
secondary
endpoint
was
an
assessment
of
drug
—drug
interaction.
The
primary
endpoints
in
Trial
2
were
the
efficacy
against
the
adult
worms
and
the
tolerability
of
the
combinations
in
onchocer-
ciasis
patients.
The
secondary
endpoint
was
the
efficacy
against
the
mff
in
the
skin
and
eyes.
Treatments
Merck
(Merck
&
Co.,
Inc.,
Whitehouse
Station,
NJ)
provided
6
-mg
tablets
of
ivermectin
and
matching
placebo
tablets,
GlaxoSmithKline
(Brentford,
U.K.)
pro-
vided
200
-mg
tablets
of
albendazole
and
matching
placebo
tablets,
and
Janssen
Pharmaceutica
(Beerse,
Belgium)
provided
50
-mg
tablets
of
levamisole.
All
participants
received
levamisole
(at
approximately
598
AWADZI
ETAL.
2.5
mg/kg).
In
addition,
the
subjects
in
the
levamisole-only
(LO)
treatment
arm
received
two
placebo
tablets
to
match
the
ivermectin
and
two
placebo
tablets
to
match
the
albendazole,
those
in
the
levamisole—
ivermectin
(LI)
treatment
arm
received
two
ivermectin
tablets
(12
mg)
and
two
placebo
tablets
to
match
the
albendazole,
and
those
in
the
levamisole—albendazole
(LA)
treat-
ment
arm
received
two
albendazole
tablets
(400
mg)
and
two
placebo
tablets
to
match
the
ivermectin.
The
tablets
were
given
after
an
overnight
fast,
under
the
direct
observa-
tion
of
a
medical
officer
and
two
nurses,
and
from
07.00
hours
(`hour
0')
on
day
1.
The
date
and
time
of
administration
and
the
number
of
the
tablets
given
were
recorded
in
the
notes
for
each
participant.
In
Trial
1,
a
standard
breakfast
was
taken
2
h
after
drug
administration
whereas
in
Trial
2
the
tablets
were
given
with
a
standard
breakfast,
in
order
to
enhance
the
absorption
of
albendazole
(Awadzi
et
al.,
1994)
.
Monitoring
Safety
Variables
Each
subject
was
admitted
3-5
days
before
the
administration
of
the
treatment
regimens
and
hospitalized
for
8
days
afterwards.
Adverse
events
(AE)
were
determined
from
changes
in
symptoms
and
vital
signs,
and
the
results
of
physical
and
ocular
examinations,
electrocardiogrammes
(ECG),
and
labora-
tory
tests
(haematological,
biochemical
and
urine).
Each
ocular
examination
included
the
taking
of
an
opthalmic
history,
visual
acuity
tests
using
an
illiterate
-E
chart,
visual
-field
measurement
using
a
standard-
ized
Goldmann
perimeter,
an
external
ocular
examination,
and
tests
for
ocular
motility.
The
anterior
segment
of
each
eye
was
examined
using
a
Haag
-Streit
900
slit
-lamp
(Haag
-Streit,
Konitz,
Switzerland)
after
head
-down
positioning
for
at
least
5
min.
Microfilariae
in
the
anterior
chamber,
living
and
dead
corneal
mff,
and
onchocercal
punctate
opacities
were
counted
and
their
positions
noted.
The
ocular
fundi
were
examined
by
direct
and
indirect
ophthal-
moscopy
after
pupillary
dilatation.
A
TRC-
50VT
retinal
camera
(Topcon
America,
Paramus,
NJ)
was
used
for
fundus
photo-
graphy
(limited
to
the
initial
examination)
and
fl
uorescein
angiography
(using
20%
fl
uorescein
sodium).
The
haematological
variables
recorded
were
haemoglobin
con-
centrations,
haematocrits,
total
and
dif-
ferential
leucocyte
counts,
platelet
and
reticulocyte
counts,
erythrocyte
sedimenta-
tion
rates,
presence
or
absence
of
glucose-6-
phosphate-dehydrogenase
(G6PDH)
defi-
ciency,
and
the
results
of
haemoglobin
electrophoresis
(HbE).
Biochemical
tests
included
total
and
direct
bilirubin,
aspartate
aminotransferase
(ASAT),
alanine
amino-
transferase
(ALAT),
alkaline
phosphatase,
glutamyl
transferase
(GGT),
lactate
dehy-
drogenase
(LDH),
albumin,
urea,
creati-
nine,
sodium,
potassium,
bicarbonate
and
chloride.
Urine
was
examined
for
pH,
specific
gravity,
protein,
glucose,
ketones,
bilirubin,
urobilinogen,
blood,
nitrites
and
leucocytes,
using
a
reagent
strip,
and
a
cen-
trifuged
deposit
was
examined
for
cells,
casts
and
parasite
ova.
Symptoms
were
documented
whenever
they
occurred
and
during
formal
enquiries
at
06.00,
14.00
and
21.00
hours
each
day
throughout
the
admission
period.
Vital
signs
were
recorded
at
the
same
time
and
a
detailed
physical
examination
was
done
at
least
once
daily.
Ocular
examinations
were
repeated
on
day
8.
ECG
were
repeated
approximately
4
h
after
dosing
and
on
days
2
and
3.
The
haematological
(except
G6PDH
and
HbE),
biochemical
and
urine
tests
were
repeated
on
days
4
and
8.
In
Trial
1,
the
subjects
were
visited
in
their
villages
on
day
18
or
20,
and
all
AE
that
had
occurred
since
discharge
were
then
recorded.
On
day
30,
all
the
participants
were
asked
to
report
to
the
OCRC,
when
they
were
given
detailed
systemic,
clinical
and
ocular
examinations
and
the
haematological,
biochemical
and
urine
tests
were
repeated.
Each
type
of
AE
observed
was
listed
on
a
standard
form
for
each
subject,
together
with
its
date
of
onset,
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
599
duration,
severity,
number
of
episodes
and
causality.
The
AE
were
graded
and
causality
attributed
using
modifications
of
the
criteria
published
by
Awadzi
(1980)
and
Hero
et
al.
(1992).
The
data
collected
up
to
day
30,
when
Trial
1
was
terminated,
determined
the
safety
of
the
test
compounds.
Drug
-concentration
Measurements
and
Definition
of
Pharmacokinetic
Parameters
Samples
(10
ml)
of
heparinized
blood
were
collected
from
each
subject,
pre-treatment
and
1,
2,
3,
4,
6,
8,
12,
24,
36,
48
and
72
h
post
-treatment,
through
an
indwelling
but-
terfly
needle
kept
patent
with
isotonic
saline.
Each
sample
was
centrifuged
(2000
x
g
for
15
min)
within
30
min
of
its
collection,
so
that
the
plasma
could
be
recovered,
split
between
two,
1.8
-ml
cryotubes,
and
then
frozen
at
—70°C.
One
of
each
pair
of
tubes
was
shipped
on
dry
ice
to
the
Department
of
Pharmacology
and
Therapeutics
at
Liver-
pool
University
(Liverpool,
U.K.)
but
the
other
was
kept
at
Hohoe,
as
a
standby,
until
all
the
drug
analyses
were
complete.
In
Liverpool,
each
plasma
sample
was
extracted
with
chlorobutane
before
the
con-
centrations
of
levamisole
were
determined
by
reverse
-phase
HPLC
with
ultra
-violet
detection
(at
225
nm),
using
quinidine
as
an
internal
standard,
and
a
Hypersil
®
BDS
C18
column
(Thermo
Electron,
Waltham,
MA;
4-ium
particle
size;
15
cm
long,
with
an
inter-
nal
diameter
of
4.6
mm).
The
mobile
phase
consisted
of
CH
3
CN
(15%)
and
water
(85%)
containing
KH
2
PO
4
(0.05
M)
and
1
-
pentane
sulphonic
acid
sodium
salt
(5
mm;
pH
3.0),
fl
owing
at
1.0
ml/min.
Under
these
conditions,
the
coefficient
of
variation
was
<
15%
and
the
limit
of
quantification
was
20
ng/ml.
Plasma
concentrations
of
albendazole
sulphoxide
(Hoaksey
et
al.,
1991;
Rawden
et
al.,
2000)
and
ivermectin
(Edwards
et
al.,
1988)
were
also
determined
by
reversed
-
phase
HPLC,
using
previously
validated
methods.
The
area
under
the
curve
(AUC)
of
plasma
concentration
v.
time,
to
the
last
experimentally
determined
concentration,
was
determined
by
the
linear
trapezoidal
rule.
The
maximum
plasma
concentrations
(C
max
),
and
the
times
at
which
they
were
achieved
(T
max
)
were
noted
directly.
Monitoring
Efficacy
variables
The
primary
efficacy
variable
was
the
death
of
the
adult
worms
(macrofilaricidal
effect)
or
their
permanent
sterilization.
The
viabil-
ity
and
reproductive
activity
of
the
adult
worms
were
determined
by
the
examination
of
histology
sections
prepared
from
oncho-
cercal
nodules
removed
on
day
180.
At
this
time,
all
located
nodules
were
aseptically
excised,
under
local
anaesthesia
with
2%
xylocaine,
and
processed
as
described
previ-
ously
(Awadzi
et
al.,
1997).
The
features
examined
are
listed
in
Table
4.
Additional
information
was
obtained
from
the
repo-
pulation
of
the
skin
by
mff,
assessed
on
days
180,
270
and
365.
The
secondary
efficacy
measurements
were
the
percentage
reduc-
tions
seen
in
the
levels
of
microfilaridermia
(mff/mg
skin
-snip)
between
day
0
and
day
30
(microfilaricidal
effect).
In
each
subject,
skin
-snips
were
taken
from
four
sites
(both
iliac
crests
and
both
calves)
with
a
Walser
-
type
corneo—scleral
punch.
Punches
were
sterilized
between
subjects
in
an
autoclave.
Each
snip
was
weighed
immediately,
on
a
sensitive
balance,
and
then
incubated
for
approximately
24
h
in
isotonic
saline
in
a
well
of
a
fl
at-bottomed
microtitre
plate.
The
mff
that
had
emerged
were
then
counted
using
an
inverted
microscope.
The
density
of
mff
at
each
site
was
expressed
as
the
number
of
mff/mg
skin.
The
'count'
for
each
indi-
vidual
was
the
sum
of
the
densities
at
the
four
sites
(mff/4
mg
of
skin).
Data
Management
and
Analysis
When
the
trial
codes
were
broken,
all
the
data
collected
for
individual
subjects
were
arranged
by
treatment
regimen.
The
AE
were
listed
by
body
system
or
category
and
then
their
incidences
and
severities
were
tabulated.
All
three
treatment
arms
were
initially
compared,
to
identify
variables
with
600
AWADZI
ET
AL.
significant
inter
-arm
differences,
which
were
then
explored
in
paired
comparisons,
using
x2
test
or
Fisher's
exact
tests
and
version
6
of
the
Epi
Info
software
package
(Centers
for
Disease
Control
and
Prevention,
Atlanta,
GA).
The
drug-
and
non
-drug
-related
AE
were
compared
separately.
The
pharmacoki-
netic
parameters
(AUC,
C.
and
T
max
)
were
used
to
determine
if
a
drug
—drug
interaction
had
occurred.
Briefly,
the
geometric
means,
geometric
mean
ratios
(value
of
the
para-
meter
for
the
drug
when
used
in
a
com-
bination:corresponding
value
for
the
drug
used
alone),
and
the
90%
confidence
inter-
vals
(90%
CI)
about
the
geometric
mean
ratios
(GMR)
were
determined,
using
the
computer
programme
Confidence
Interval
Analysis
(CIA;
British
Medical
Journal,
London,
U.K.).
The
pharmacokinetic
para-
meters
defined
previously,
for
ivermectin
used
alone
and
albendazole
used
alone
(Awadzi
et
al.,
2003),
were
used
as
historical
controls.
It
was
concluded
that
a
clinically
significant
interaction
had
occurred
when-
ever
the
90%CI
for
a
systemic
exposure
ratio
fell
entirely
outside
the
equivalence
range
of
0.8-1.25
(Anon.,
1999).
The
percentages
of
adult
worms
and
nodules
with
each
of
the
various
features
listed
in
Table
4
were
compared
across
all
three
treatment
arms,
with
subsequent
paired
comparisons
being
limited
to
the
areas
of
difference
so
identified.
The
densities
of
mff
in
the
skin
were
logarithmically
transformed
prior
to
analy-
sis.
Analysis
of
variance
(ANOVA)
was
used
to
compare
the
three
treatment
arms,
the
unpaired
t
-test
to
compare
geometric
means
at
various
time
-points
for
the
LO
and
LA
treatment
arms,
and
the
paired
t
-test
to
compare
adjacent
counts
in
the
LI
treatment
arm,
in
order
to
define
the
pattern
of
response
(i.e.
the
microfilarial
kinetics).
The
ocular
counts
of
mff
were
generally
low
and
highly
variable
within
and
between
treatment
arms;
analysis
of
the
effect
of
treatment
on
these
counts
was
thus
limited
to
a
description
of
the
pattern
of
response
within
each
treatment
arm.
For
all
compari-
sons,
the
null
hypothesis
was
rejected
at
P<
0.05.
RESULTS
Background
Characteristics
The
42
healthy
volunteers
of
Trial
1
were
all
adult
males
aged
18-56
years
(mean
=34
years).
All
but
six
weighed
.-
50
kg.
Two
had
choroido-retinal
lesions
of
non-onchocercal
origin.
The
baseline
characteristics
of
the
66
subjects
in
Trial
2
are
summarized
in
Table
1.
Overall,
the
subjects
in
the
three
treatment
arms
in
Trial
2
were
similar
with
respect
to
age,
gender
distribution,
physical
characteristics
and
the
paucity
of
onchocer-
cal
lesions,
of
which
the
main
manifestations
were
subcutaneous
nodules.
The
percentage
of
subjects
in
the
LA
treatment
arm
who
had
mff
in
the
anterior
chamber
of
the
eye
(54.5%)
was,
however,
significantly
higher
than
the
18.2%
in
the
LI
treatment
arm
(x
2
=
6.29;
P=0.012)
and
higher,
but
not
significantly
so,
than
the
36.4%
in
the
LO
treatment
arm
(x
2
=
1.47;
P=
0.226).
In
Trial
2,
there
were
no
significant
gender
differences
in
the
numbers
of
nodule
sites
or
in
the
intensities
of
infection
with
0.
volvulus.
Nineteen
(54.2%)
of
the
35
male
subjects
and
22
(71.0%)
of
the
31
females
each
had
two
or
more
nodule
sites
(x
2
=
1.94;
P=0.16).
The
geometric
mean
values
and
95%
confidence
intervals
(95%
CI)
for
the
initial
levels
of
microfilari-
dermia
were
86.4
(66.1-112.8)
for
the
male
subjects
and
74.4
(52.4-105.6)
for
the
female
subjects
(95%
CI
for
the
difference
=
0.126-0.253;
T=
0.67;
degrees
of
free-
dom
=
57;
P=
0.51).
On
average,
the
women
were
5
years
older,
5
kg
lighter
and
10
cm
shorter
than
the
men.
Adverse
Events
HEALTHY
VOLUNTEERS
Adverse
events
were
pooled
for
the
42
per-
sons
included
in
Trial
1,
as
the
frequencies
appeared
unrelated
to
the
treatment
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
601
TABLE
1.
Baseline
characteristics
of
the
66
infected
subjects
of
Trial
2,
who
were
treated
with
levamisole
only
(LO),
levamisole
plus
ivermectin
(LI)
or
levamisole
plus
albendazole
(LA)
Variable
Treatment
LO
LI
LA
No.
of
subjects
22 22
22
%
of
subjects
male
50.0
45.5
63.6
Mean
age
and
(range)
(years)
38
(18-58)
40
(23-60)
43
(19-60)
Mean
weight
and
(range)
(kg)
50.1
(40.0-65.5)
55.1
(42.0-75.0)
53.4
(43.0-65.0)
Mean
height
and
(range)
(cm)
161.6
(152.0-180.0)
162.3
(147.5-180.5)
162.1
(142.5-177.5)
NO.
AND
(%)
OF
SUBJECTS
WITH:
One
or
two
nodule
sites
20
(90.9)
15
(68.2)
18
(81.8)
More
than
two
nodule
sites
2
(9.1)
7
(31.8)
4
(18.2)
Normal
visual
function
19
(86.4)
20
(90.9)
18
(81.8)
Microfilariae
in
the
anterior
chamber
8
(36.4)
4
(18.2)
12
(54.5)
Ocular
pathology
2
(9.1)
0
(0)
4
(18.2)
Geometric
mean
count
of
microfilariae
in
the
skin
and
(95%
confidence
interval)
(microfilariae/4
mg
skin)*
82.1
(56.9-118.3)
71.9
(45.1-114.2)
88.5
(66.6-117.5)
*Each
'count'
was
the
sum
of
densities,
in
microfilariae/mg,
in
snips
from
both
iliac
crests
and
both
calves.
regimen.
The
most
common
drug
-related
events
were
general
weakness
and
headache,
each
reported
by
nine
subjects
(21.4%),
fol-
lowed
by
itching
in
six
subjects
(14.3%),
rash
in
five
(11.9%),
and
joint
pains
in
four
(9.5%).
With
the
exception
of
grade
-2
itch-
ing
in
one
subject
in
the
LA
treatment
arm,
and
grade
-2
headache
and
fever
resulting
from
malaria
in
another,
all
the
AE
recorded
in
Trial
1
were
of
grade
-1
severity.
Itching
began
relatively
early
in
two
sub-
jects:
at
8.25
h
in
one
subject
from
the
LO
treatment
arm,
and
at
12.25
h
in
one
subject
in
the
LI
arm.
In
one
other
subject
from
the
LI
treatment
arm,
one
from
the
LO,
and
two
from
the
LA,
itching
was
not
noted
until
at
least
23
h
post
-treatment.
Itching
lasted
for
8-25
h
except
in
one
subject
in
the
LA
treat-
ment
arm,
who
had
mild
itching
throughout
most
of
the
admission
period.
A
papular
rash
developed
over
the
trunk
in
four
subjects
who
developed
itching.
One
subject
without
itching
developed
papules
on
the
right
arm
on
day
3.
Swelling
of
the
foot
or
leg,
begin-
ning
on
day
1
or
day
3
and
lasting
for
9
h,
occurred
in
two
subjects
(one
LA
and
one
LI).
During
Trial
1,
no
important
changes
in
the
ECG
or
in
visual
function
were
observed
and
there
were
no
abnormalities
of
clinical
significance
in
any
of
the
laboratory
tests.
SUBJECTS
INFECTED
WITH
0.
volvulus
The
most
common
drug
-related
events
occurring
in
the
66
infected
subjects
of
Trial
2
were
itching
and
rash,
occurring
in
36
(54.5%)
and
31
(47.0%)
of
the
subjects,
respectively.
Other
AE,
occurring
with
moderate
frequency,
were
headache
in
20
subjects
(30.3%),
arthralgia
in
11
(16.7%),
and
other
body
pains
in
11
(16.7%).
None
of
the
AE
was
serious
and
most
were
of
grade
-1
severity.
The
commonest
grade
-2
AE
were
itching
(17
subjects),
followed
by
headache
(six),
rash
(three)
and
dizziness
(three).
Lymphadenitis,
myalgia,
fever
and
anorexia
of
grade
2
were
each
observed
in
one
subject.
Two
subjects
in
the
LI
treatment
arm
had
grade
-3
itching.
Lymphadenitis
(five
subjects)
and
swelling
of
the
face
or
limb
(five
subjects)
were
limited
to
the
subjects
treated
with
ivermectin.
The
other
drug
-
related
AE
that
each
occurred
in
at
least
five
of
the
66
subjects
are
tabulated,
and
the
fre-
quencies
of
all
such
events
among
the
three
treatment
arms
are
compared,
in
Table
2.
This
shows
that
the
three
treatment
arms
602
AWADZI
ET
AL.
TABLE
2.
Comparison
of
the
frequencies
of
the
more
common
drug
-related
adverse
events
(each
of
which
occurred
in
at
least
five
subjects)
among
the
66
subjects
infected
with
Onchocerca
volvulus
who
were
treated
with
levamisole
only
(LO),
levamisole
plus
ivermectin
(LI)
or
levamisole
plus
albendazole
(LA)
Treatment
LO
LI LI
No.
of
subjects
investigated
NO.
OF
SUBJECTS
DEVELOPING:
22 22 22
Pruritus
8
18
10
0.01
Rash
10
12
9
0.653
Headache
7
8
5
0.605
Arthralgia
1
7
3
0.047
Other
body
aches
2
9
0
<
0.0001
Ocular
pain/discomfort
2
4
0
0.111
Myalgia
1
3
1
0.421
Fever
1
7
0
0.002
Dizziness
2
3
0
0.22
Sinus
bradycardia
3
2
1
0.577
Elevated
aminotransferases
3
2
4
0.680
were
dissimilar
in
the
frequencies
of
pruri-
tus,
arthralgia,
other
body
aches,
and
fever.
Further
examination
revealed
that
all
of
these
AE
were
more
frequent
in
the
LI
treat-
ment
arm
than
in
the
LO
or
LA,
the
latter
two
treatment
arms
being
similar
in
the
frequencies
of
all
the
AE
listed.
In
view
of
the
unexpectedly
high
incidence
of
itching
and
rash
recorded
in
Trial
2,
the
time
of
onset,
severity
and
duration
of
the
itching
and
the
type
and
anatomical
distribu-
tion
of
the
rash
were
compared
between
the
treatment
arms.
The
itching
began
'early'
(i.e.
within
10
h
of
treatment)
in
all
eight
subjects
reporting
this
AE
in
the
LO
treat-
ment
arm,
in
nine
of
the
18
subjects
who
reported
it
in
the
LI
treatment
arm,
and
in
eight
of
the
10
subjects
reporting
it
in
the
LA
arm.
Nine
subjects
given
LI
and
two
given
LA
began
itching
'late'
(i.e.
at
least
22
h
post
-treatment).
The
three
treatment
arms
differed
significantly
in
terms
of
when
itching
began
(x
2
=7.25;
P=0.027).
The
main
factor
was
the
much
earlier
onset
of
this
AE
among
the
subjects
given
levamisole
alone,
as
compared
with
the
LI
(x
2
=
4.11;
P=
0.043)
or
LA
treatment
arms.
Itching
was,
however,
more
severe
with
LI
than
LO
or
LA,
being
scored
grade
-2
or
-3
for
only
three
subjects
given
LO
and
only
five
of
those
given
LA
but
13
of
those
given
LI
(x
2
=
11.73;
P=
0.003).
The
onset
of
rash,
which
was
mainly
papular,
parallelled
the
onset
of
itching.
The
rash
appeared
in
<
10
h
in
nine
of
the
10
LO
subjects
who
developed
this
AE,
five
of
the
12
LI,
and
seven
of
the
nine
LA.
The
rash
seen
on
the
LO
subjects
involved
mainly
the
posterior
part
of
the
upper
trunk
whereas
that
on
the
LI
and
LA
subjects
was
evenly
distributed
between
the
upper
limbs
and
the
upper
trunk.
The
three
treatment
arms
did
not
differ
significantly,
however,
in
the
frequency
of
rash
or
the
extent
of
the
rash.
As
in
Trial
1,
no
important
changes
in
the
ECG
or
in
visual
function
were
observed
during
Trial
2,
and
there
were
no
abnormali-
ties
of
clinical
significance
in
any
of
the
labo-
ratory
tests.
Many
AE
that
did
not
appear
to
be
drug
-related
(46,
33
and
32
in
the
LO,
LI
and
LA
treatment
arms,
respectively;
P>
0.05)
were
recorded
during
the
30
-day
safety
assessment.
Concomitant
Medication
The
main
therapeutic
intervention
was
the
administration
of
acetaminophen
for
headaches
(four
subjects),
gland
pain
(one
subject),
myalgia
(one
subject)
or
diffuse
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
603
aches
and
pains
(one
subject).
Two
patients
who
had
malaria
also
received
chloroquine.
Three
patients
received
tincture
of
bella-
donna
and
one
magnesium
trisilicate,
for
abdominal
pain.
Chlorpheniramine
was
given
to
12
subjects
during
the
first
30
days,
for
the
prevention
and
treatment
of
reactions
to
intravenous
fl
uorescein
sodium.
These
reactions
manifested
as
itching,
urticaria,
nausea,
vomiting,
abdominal
pain
and
palpi-
tation.
The
treatment
arms
did
not
differ
in
their
requirement
for
additional
medication.
Pharmacokinetic
Parameters
in
Healthy
Volunteers
Plots
of
mean
plasma
concentrations
v.
time
are
shown
for
ivermectin,
in
the
pres-
ence
and
absence
of
levamisole,
in
Figure
1.
Similar
plots
for
albendazole
sulphoxide
and
levamisole
are
shown
in
Figure
2,
and
45
40
U
35
a.)
to
30
o
25
I
i)
20
U
8
15
czt
cd
E
czt
N
10
0
those
for
levamisole,
levamisole
plus
alben-
dazole,
and
levamisole
plus
ivermectin
appear
as
Figure
3.
(Error
bars
have
been
omitted
to
avoid
overcrowding
of
the
figures.)
Levamisole
and
ivermectin
were
both
rapidly
absorbed,
as
indicated
by
the
short
times
taken
to
reach
their
peak
plasma
concentrations.
Albendazole
was
not
detected
in
any
plasma
sample,
reflecting
this
drug's
rapid
conversion
to
albendazole
sulphoxide.
In
nearly
all
subjects,
the
con-
centrations
of
levamisole,
ivermectin
and
albendazole
sulphoxide
fell
below
the
lower
limits
of
their
quantification
by
72
h
post-
treatment.
The
number
of
subjects
in
each
treatment
regimen,
and
the
pharmacokinetic
variables
for
levamisole
(when
given
alone
or
with
albendazole
or
ivermectin),
albendazole
sulphoxide
when
albendazole
is
given
with
levamisole,
and
ivermectin
when
given
with
levamisole,
together
with
those
for
i•
0
10
20
30
40
Time
(h)
50
60
70
80
FIG.
1.
The
mean
plasma
concentrations
of
ivermectin
in
11
subjects
receiving
ivermectin
plus
levamisole
(•;
present
study)
and
13
receiving
ivermectin
alone
(0;
Awadzi
et
aL,
1993).
604
AWADZI
ET
AL.
140
a)
0
cz1
120
-zs
100-
80
-
60-
Q
o
40
-
sEL,
czt
20
-
10
20
30
40
50
60
70
80
Time
(h)
FIG.
2.
The
mean
plasma
concentrations
of
albendazole
sulphoxide
in
14
subjects
receiving
albendazole
plus
levamisole
(•;
present
study)
and
14
receiving
albendazole
alone
(0;
Awadzi
et
aL,
1993).
ivermectin
used
alone
and
for
albendazole
sulphoxide
when
albendazole
is
used
alone
[obtained
from
historical
data
(Awadzi
et
al.,
2003)],
are
listed
in
Table
3.
The
geometric
mean
ratio
(GMR),
and
the
related
90%
CI
for
the
AUC,
C
max
,
and
T
m
,
are
summarized
for
each
paired
drug
combi-
nation,
together
with
the
equivalence
range,
in
Figure
4.
Levamisole
produced
a
clinically
significant
increase
in
the
AUC
and
C
max
of
ivermectin.
The
GMR
was
>
2
in
each
case,
whereas
the
90%Cl
were
all
outside
the
`no
effect'
range
(of
0.8-1.25);
there
was
a
modest
reduction
in
the
T
m
.
When
com-
bined
with
albendazole,
levamisole
signifi-
cantly
reduced
the
AUC,
C
m
,
and
T
max
of
albendazole
sulphoxide.
The
GMR
for
the
AUC
and
C
max
were
<0.5,
with
the
90%Cl
well
outside
the
'no
effect'
range.
With
the
T
m
,
the
upper
limit
of
the
90%
CI
(0.875)
barely
crossed
the
lower
limit
of
the
no
effect
range
(0.800).
Ivermectin
had
no
influence
on
the
PK
parameters
of
levamisole,
whereas
albendazole
sulphoxide
produced
a
modest
reduction
in
the
AUC
(but
not
in
the other
parameters)
of
levamisole.
Thus,
the
only
clinically
significant
drug
—drug
interactions
were
levamisole-attributable
increases
in
the
AUC
and
C
max
of
ivermectin,
and
decreases
in
the
AUC
and
C
max
of
albendazole
sulphoxide.
Efficacy
Against
the
Adult
Worms
Overall,
278
onchocercomata
were
excised
from
64
of
the
subjects
of
Trial
2
(the
nod-
ules
from
the
other
two
subjects
contained
no
worm
material).
Table
4
summarizes
the
effects
of
the
three
drug
regimens
on
the
viability,
reproductive
activity
and
micro
-
filarial
output
of
the
adult
worms
(as
repre-
sented
by
the
presence
of
mff
in
nodular
tissue).
The
treatment
arms
were
similar
in
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
605
450
400
-
350
-
ct
to
300
o
250
+
al
200
150
-
100-I
a)
50
........
0
0
10
20
30
40
50
60
70
80
Time
(h)
FIG.
3.
Mean
plasma
concentrations
of
levamisole
in
14
subjects
receiving
levamisole
alone
(0),
14
receiving
levamisole
and
albendazole
(•)
and
14
receiving
levamisole
and
ivermectin
(•).
terms
of
the
low
percentages
of
the
female
worms
considered
dead
or
moribund,
or
dead
and
calcified,
and
of
the
low
percent-
ages
of
male
worms
found
dead,
indicating
the
lack
of
a
macrofilaricidal
effect.
The
major
differences
were
in
the
effects
on
reproductive
activity
and
microfilarial
out-
put
(P<0.0001).
In
the
LI
treatment
arm,
there
was
a
suppression
of
embryo
produc-
tion,
with
only
40%
of
the
adult
female
worms
showing
active
embryogrammes,
as
compared
with
approximately
66%
in
the
LO
and
LA
treatment
arms.
Moreover,
those
female
worms
producing
embryos
in
the
LI
treatment
arm
contained
predomi-
nantly
degenerate
forms,
with
the
result
that
<25%
of
the
onchocercomata
from
this
treatment
arm
contained
mff.
This
treat-
ment
arm
differed
significantly
from
each
of
the
two
other
treatment
arms
in
all
these
respects,
whereas
LO
and
LA
produced
similar
effects
on
the
adult
female
worms.
There
was
no
apparent
effect
on
spermatogenesis
in
any
treatment
arm.
Efficacy
Against
the
Microfilariae
The
initial
levels
of
microfilaridermia
and
the
changes
observed
in
the
year
post-
treatment
are
presented
in
Table
5.
The
initial
densities
were
similar
in
all
three
treat-
ment
arms
(ANOVA;
F=0.32;
P=
0.724).
In
the
LO
and
LA
arms,
the
levels
of
micro-
filaridermia
observed
post
-treatment
were
always
similar
to
the
pre-treatment
values
(P>
0.05)
and
the
counts
remained
similar
in
the
two
treatment
arms
at
each
time
-
point.
In
contrast,
massive
reductions
in
the
level
of
microfilaridermia
occurred
rapidly
in
the
LI
treatment
arm,
and
it
seems
more
appropriate
to
describe
the
pattern
of
response
within
this
treatment
arm
than
to
'7V
la
IZUV
1X1V
TABLE
3.
The
values
of
three
pharmacokinetic
parameters
determined
for
apparently
healthy
volunteers
treated
with
ivermectin
(alone
or
with
levamisole),
albendazole
(alone
or
with
levamisole)
or
levamisole
alone
Parameter
Ivermectin,
when
given:
Albendazole
sulphoxide,
when
albendazole
was
given:
Levamisole,
when
given:
Alone*
With
levamisole
Alone*
With
levamisole
Alone
With
ivermectin
With
albendazole
No.
of
subjects
13t
11*
14 14 14 14 14
ARITHMETIC
MEAN
VALUE
AND
(S.D)
FOR:
AUC0-72h
(ng.h/m1)
527
(309)
1056
(453)
3581 (1432)
913
(514)
3298
(1742)
3696
(2263)
2189
(1211)
C
max
(ng/ml)
23
(12)
57
(53)
153
(52)
73
(38)
482
(223)
534
(280)
449
(244)
Median
and
(range)
for
T
ax
(h)
2
(3-12)
4
(2-6)
3
(1-6)
4
(3-24)
2
(1-4)
2.5
(1-6)
3
(1-4)
*The
'historical'
data
of
Awadzi
et
al.
(2003).
tIvermectin
was
not
detected
in
one
subject.
*Ivermectin
not
detected
in
three
subjects.
AUC,
Area
under
the
plot
of
plasma
concentration
v.
time;
C„,
ax
,
maximum
plasma
concentrations;
Tax,
time
to
C„,ax.
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
607
pr
A
1VM+LEV
ALB+LEV
LEV+IVM
LE
V+ALB
FIG.
4.
Geometric
mean
ratios
(GMR)
for
the
pharmacokinetic
parameters
AUC
(
C
max
(0)
and
T
ax
(•)
for
ivermectin
when
given
with
levamisole:alone
(IVM
+
LEV),
albendazole
when
given
with
levamisole:alone
(ALB
+
LEV),
levamisole
when
given
with
ivermectin:alone
(LEV
+
IVM),
and
levamisole
when
given
with
albendazole:alone
(LEV
+ALB).
The
horizontal
lines
indicate
the
equivalence
range
(0.8-1.25).
Whenever
the
vertical
lines,
which
indicate
90%
confidence
intervals,
lie
entirely
outside
of
the
equivalence
range,
a
clinically
significant
drug
-drug
interaction
is
indicated.
TABLE
4.
The
effect
of
treatment
with
levamisole
only
(LO),
levamisole
plus
ivermectin
(LI)
or
levamisole
plus
albendazole
(LA)
on
the
viability
and
reproductive
activity
of
adult
Onchocerca
volvulus
collected
from
nodules
180
days
post
-treatment
Variable
Treatment
LO
LI
LA
No.
of
onchocercomata
excised
84
112
82
NO.
AND
(%)
OF
FEMALE
WORMS
Recovered
106
127
101
Dead
or
moribund
18
(17.0)
23
(18.1)
9
(8.9)
0.12
Dead
and
calcified*
8
(7.5)
10
(7.9)
2
(2.0)
0.126
Live
88
(83.0)
104
(81.9)
92
(91.1)
NO.
AND
(%)
OF
LIVE
FEMALE
WORMS
Producing
embryos
58
(65.9)
42
(40.4)
64
(69.6)
<0.0001
With
degenerate
embryos/
22
(37.9)
35
(83.3)
28
(43.8)
<0.0001
Not
producing
embryos
30
(34.1)
62
(59.6)
28
(30.4)
With
relict/degenerate
embryos*
5
(16.7)
15
(24.2)
2
(7.1)
0.148
NO.
AND
(%)
OF
MALE
WORMS
Recovered
47
57
52
Dead
or
moribund
0
(0)
1
(1.8)
2
(3.8)
Live
47
(100.0)
56
(98.2)
50
(96.2)
No.
and
(%)
of
live
male
worms
with
normal
spermatogenesis
46
(97.9)
53
(94.6)
48
(96.00)
0.702
No.
and
(%)
of
nodules
with
microfilariae
in
the
capsule
46
(54.8)
26
(23.2)
54
(65.8)
<0.0001
*The
percentages
shown
are
of
the
total
population
of
adult
female
worms.
/The
percentages
shown
are
of
the
live
females
producing
embryos.
*The
percentages
shown
are
of
the
live
females
not
producing
embryos.
co
'7V
la
IZUV
1X1V
TABLE
5.
Changes
in
the
levels
of
microfilaridennia
seen
in
the
year
following
treatment
with
levamisole
only,
levamisole
plus
ivermectin
or
levamisole
plus
albendazole,
showing
the
percentage
reductions
compared
with
the
day
-0
values
Time
Treatment
arm
Levamisole
only
(N=
22)
Levamisole-ivermectin
(N=
22)
Levamisole-albendazole
(N=
22)
GMC
and
(CI)
Reduction
(microfilariae/4
mg
skin)
(%)
GMC
and
(CI)
(microfilariae/4
mg
skin)
Reduction
(%)
GMC
and
(CI)
(microfilariae/4
mg
skin)
Reduction
(%)
Day
0
82.1
(56.90-118.30)
71.9
(45.10-114.23)
88.5
(66.60-117.50)
Day
4
75.0
(51.21-109.73)
8.6
5.0
(2.99-8.07)
93.0
63.3
(44.67-89.46)
28.5
Day
8
56.0
(34.64-90.24)
31.8
1.2
(0.62-2.00)
98.3
76.4
(51.19-113.72)
13.7
Day
30
60.7
(37.19-98.64)
26.1
0.05
(-0.02-0.14)
99.9
86.2
(60.74-122.25)
2.6
Day
180
75.6
(49.69-114.69)
7.9
1.4
(0.63-2.42)
98.1
69.9
(48.64-100.13)
21.0
Day
270
71.6
(45.30-112.81)
12.8
3.7
(1.80-6.79)
94.9
72.7
(49.82-105.94)
17.9
Day
365
60.0
(36.71-97.58)
26.9
4.0
(1.89-7.46)
94.4
67.3
(46.58-97.09)
24.0
GMC,
The
geometric
mean
of
the
sum
of
densities
(in
microfilariae/mg)
found
in
the
snips
from
both
iliac
crests
and
both
calves;
CI,
95%
confidence
interval.
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
609
compare
the
LI
values
with
those
in
the
other
two
treatment
arms.
In
the
LI
treat-
ment
arm,
the
counts
of
mff
were
already
much
reduced
by
day
4
and
were
even
lower
on
days
8
and
30.
Between
day
30
and
day
270
they
appeared
to
increase
slightly
but
then
they
appeared
to
remain
unchanged
between
day
270
and
the
final
check
on
day
365
(Table
5).
About
30%
of
all
the
subjects
of
Trial
2
each
had
mff
in
the
anterior
chamber
of
at
least
one
eye;
the
counts
were
highly
variable
and
the
initial
values
differed
between
treat-
ment
arms.
The
means
and
(ranges)
of
the
counts
for
both
eyes
were
1.1
(one
to
eight)
for
the
LO
treatment
arm,
3.6
(one
to
68)
for
the
LI
and
7.7
(one
to
95)
for
the
LA.
A
meaningful
assessment
of
the
effect
of
each
treatment
was
thus
limited
to
a
description
of
the
pattern
of
response
within
each
treat-
ment
arm.
There
was
no
significant
reduc-
tion
in
the
ocular
parasite
counts
in
the
LO
or
LA
treatment
arms
over
the
period
of
observation.
The
day
-180
counts
in
the
LI
treatment
arm
were,
however,
significantly
lower
than
the
pre-treatment
counts,
although
two
of
the
four
LI
subjects
who
had
ocular
mff
pre-treatment
still
had
one
or
eight
mff
in
their
eyes
on
day
365
(data
not
shown).
DISCUSSION
In
both
trials,
the
levamisole
alone
or
in
combination
with
ivermectin
or
albendazole
was
well
tolerated,
although
there
was
an
unexpectedly
high
incidence
of
itching
and
rash.
The
co
-administration
of
levamisole
with
ivermectin
or
albendazole
resulted
in
clinically
significant
drug
—drug
interactions.
Treatment
with
levamisole
alone
or
levamisole—albendazole
led
to
AE
of
similar
frequency
and
severity
and,
in
the
subjects
with
onchocerciasis,
had
minimal
effects
on
the
mff
and
adult
worms.
The
combination
of
levamisole
with
ivermectin
was
not
macrofilaricidal,
and
did
not
augment
the
potent
effect
against
skin
and
intra-uterine
mff
that
has
been
observed
with
ivermectin
alone
(Awadzi
et
al
.
,
1995a,
2003).
The
combinations
of
levamisole
with
albendazole
or
ivermectin
have
never
been
considered
previously
for
the
treatment
of
filarial
infections.
Thus,
in
any
development
of
such
drug
regimens
for
human
use,
their
safety
and
pharmacokinetic
interactions
must
first
be
explored
in
healthy
volunteers.
Only
if
the
results
of
these
first
trials
are
encouraging
should
the
efficacy
and
toler-
ability
of
the
regimens,
in
subjects
infected
with
the
target
parasite,
be
investigated.
Ini-
tially
in
Trial
1,
only
two
healthy
volunteers
were
exposed
to
each
of
the
treatments
(LO,
LA
and
LI),
the
size
of
each
successive
group
investigated
increasing
by
three
subjects
to
a
maximum
of
five
subjects/regimen
in
the
final
group.
The
subjects
were
closely
observed
in
hospital,
were
visited
in
their
villages
after
discharge,
and
were
seen
in
hospital
at
trial
termination
on
day
30.
In
Trial
2,
only
subjects
with
light
infections
with
0.
volvulus
were
initially
treated,
before
the
responses
to
treatment
of
heavily
infected
individuals
were
explored.
The
drug
-related
AE
recorded
in
the
healthy
volunteers
investigated
in
Trial
1
and
the
relatively
high
frequencies
of
the
AE
in
the
LO
and
LA
treatment
arms
of
Trial
2
were
unexpected.
The
explanation
for
these
find-
ings
is
best
found
by
considering
some
of
the
features
of
untreated
onchocerciasis,
and
the
pathogenesis
of
the
various
AE
that
occur
during
the
treatment
of
the
disease.
The
key
elements
are
summarized
in
Figure
5.
The
tissue
damage
produced
by
the
death
of
mff
is
responsible
for
much
of
the
pathology
in
onchocerciasis.
A
competent
immune
system
determines
the
human
host's
ability
to
kill
mff
and
defines
the
clini-
cal
spectrum
of
a
disease
that
includes:
(1)
endemic
normals
or
the
putatively
immune
individuals
who
have
lived
in
endemic
areas
for
many
years,
have
been
exposed
to
infection,
but
who
are
seemingly
free
of
infection;
(2)
individuals
with
microfilari-
dermias
associated
with
generalized
or
no
skin
manifestations;
and
(3)
individuals
with
610
AWADZI
ET
AL.
Microfilariae
Detectable
in
skin,
eye
nodular
tissue,
and/or
vector
Immune
system
Competent
V
Natural
disease
(spontaneous
death
of
microfilariae)
Endemic
controls
Sowda
Trial
1.
Induction
of
AE
(healthy
volunteers)
Drug
treatment
direct
effect
on
microfilariae
immunostimulation
by
levamisole
(acute
death
of
microfilariae)
Occult
Incompetent
—110
Generalized
onchocerciasis
Trial
2.
Modification
of
time
-frame
of
AE
(infected
subjects)
Mazzotti
reaction
1-
Mazzotti
test.
Priming
with
levamisole
Trial
1.
Induction
of
AE
(healthy
volunteers)
FIG.
5.
The
interaction
of
the
microfilariae
and
the
immune
system
in
the
genesis
of
the
pathology
of
onchocercia-
sis,
and
the
influence
of
levamisole
on
the
Mazzotti
reaction
in
subjects
(healthy
volunteers
and
infected
individuals)
treated
with
levamisole
only,
levamisole
plus
ivermectin,
or
levamisole
plus
albendazole.
AE,
Adverse
events.
hyper
-reactive
onchodermatitis
(or
Sowda)
with
severe,
asymmetric
skin
lesions
and
light
microfilaridermias
(Ottesen,
1995;
Ali
et
al.,
2002).
A
cellular
(lymphocyte)
immune
hypo
-responsiveness
to
onchocer-
cal
antigens
is
characteristic
of
those
with
generalized
onchocerciasis;
endemic
nor-
mals
have
higher
cellular
immune
responses
but
lower
specific
antibody
responsiveness,
whereas
those
with
the
hyper
-reactive
form
have
the
highest
titres
of
specific
antibodies
(WHO,
1995;
Ali
et
al.,
2003).
In
the
present
study,
the
subjects
of
Trial
1
were
equivalent
to
endemic
normals,
whereas
those
of
Trial
2
represented
subjects
with
generalized
onchocerciasis.
The
death
of
mff
also
evokes
a
series
of
events
(symptoms,
signs
and
laboratory
events)
known
collectively
as
the
Mazzotti
reaction.
In
areas
where
onchocerciasis
is
endemic,
subjects
undergo
a
chronic
reac-
tion
even
in
the
absence
of
any
intervention.
This
is
the
result
of
the
death
of
mff
at
the
end
of
their
normal
life
-span,
or
of
the
immune
hyper
-reactivity
that
is
characteris-
tic
of
the
localized
form
of
the
disease.
The
mff
that
contribute
to
the
Mazzotti
reaction
may
be
readily
demonstrable
in
skin
-snips
or
the
eyes
and
nodular
tissue
of
the
human
residents
and
in
the
Simulium
vector,
or
may
be
occult,
being
undetectable
even
by
sensitive
parasitological
methods.
Their
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
611
presence
in
humans
can
be
inferred,
how-
ever,
from
the
observation
of
a
Mazzotti
reaction
in
response
to
diethylcarbamazine
treatment
(WHO,
1987).
The
AE
that
follow
the
treatment
of
0.
volvulus
infections
result
from
the
intrinsic
(pharmacological)
properties
of
the
drug,
the
death
of
the
mff
(Mazzotti
reaction),
progressive
disability
from
pre-existing
lesions,
coincidental
illness
occurring
during
or
after
the
drug
administration,
or
unknown
factors.
In
the
present
study,
the
main
factors
that
are
relevant
are
the
intrinsic
drug
effects,
the
Mazzotti
reaction,
and
the
unknown.
The
series
of
drug
-related
AE
described
in
Trials
1
and
2,
taken
as
a
whole,
are
consistent
with
those
that
occur
in
the
Mazzotti
reaction
(WHO,
1987).
As
they
are
not
characteristic
of
the
AE
associated
with
any
of
the
three
drugs
when
they
are
administered
to
healthy
volunteers,
they
are
unlikely
to
be
intrinsic
drug
effects.
The
marked
difference
in
the
frequency
and
severity
of
the
AE,
between
the
presumably
uninfected
volunteers
of
Trial
1
and
the
subjects
with
microfilaridermias
investigated
in
Trial
2,
support
a
relationship
with
the
intensity
of
infection
with
0.
volvulus.
Levamisole
acts
as
an
immunostimulant
and
restores
cell
-mediated
immune
mecha-
nisms
that
have
become
depressed
in
periph-
eral
T
-lymphocytes,
although
the
drug
has
only
marginal
effects
in
the
immunocompe-
tent
(Renoux,
1980).
The
itching
and
rash
seen
in
six
of
the
42
healthy
volunteers
(endemic
controls)
in
Trial
1
probably
resulted
from
the
enhancement
of
the
immune
response
by
levamisole,
such
that
these
six
individuals
were
capable
of
killing
the
mff
that
were
present
in
them.
Although
no
mff
had
been
found
in
skin
-snips
taken
from
seven
sites
(right
outer
canthus,
both
scapulae,
both
iliac
crests
and
both
calves)
on
each
of
the
volunteers
of
Trial
1,
the
presence
of
occult
mff
cannot
be
excluded.
Thus,
the
interaction
of
an
enhanced
immune
response
with
the
occult
mff
present
in
a
few
subjects
resulted
in
a
Mazzotti
reaction.
The
drug
-related
AE
in
Trial
2
were
probably
the
result
of
the
putative
effect
of
levamisole
on
the
immune
system,
the
presence
of
(demonstrable)
mff
in
the
skin,
and
the
potent
effect
of
iver-
mectin
on
the
mff.
The
major
AE
in
this
trial
were
itching
and
rash.
For
the
data
analysis,
the
onset
of
itching
was
divided
into
early
(
-.,
10
h
post
-treatment)
and
late
(
>
10
h).
Early
itching
occurred
in
all
eight
subjects
in
the
LO
treatment
arm
who
reported
pruri-
tus,
nearly
all
(eight
out
of
10)
such
subjects
in
the
LA
treatment
arm
and
in
half
(nine
out
of
18)
of
those
in
the
LI
treatment
arm.
Since
early
itching
is
uncharacteristic
of
ivermectin
treatment,
and
occurred
at
higher
frequencies
after
LO
or
LA
than
after
LI,
it
may
represent
the
priming
effects
of
levamisole
on
the
immune
system.
Late
itch-
ing
that
occurred
from
22
h
post
-treatment
was
virtually
limited
to
the
subjects
treated
with
ivermectin,
and
is
consistent
with
the
expected
onset
of
itching
caused
by
ivermectin
alone
(Awadzi
et
al.,
1990a).
As
well
as
the
more
frequent
and
more
severe
itching,
ivermectin
treatment
was
associated
with
arthralgia,
other
body
aches,
lymphad-
enitis,
fever
and
swellings
(Table
2)
AE
that
appear
to
result
from
the
direct
effect
of
ivermectin
on
the
mff.
Thus,
in
Trial
2,
the
subjects
given
LI
experienced
a
`biphasic'
Mazzotti
reaction,
with
early
and
late
phases
attributable
to
the
effects
of
levamisole
and
ivermectin,
respectively.
Since
levamisole
and
albendazole
have
little
effect
on
the
mff,
the
AE
in
the
LO
and
LA
treatment
arms
would
be
expected
to
result
predominantly
from
the
putative,
immunostimulatory
effects
of
levamisole
(as
occurred
in
Trial
2).
The
proposed
mechanisms
of
the
Mazzotti-type
reactions
seen
in
Trial
1
and
of
the
alteration
in
the
time
frame
of
the
AE
to
ivermectin
treatment
in
Trial
2
cannot
yet
be
supported
by
any
immunological
findings.
Although
similar
phenomena
were
not
observed
in
previous
studies
with
levamisole,
in
a
different
population
(Awadzi
et
al.,
1982,
1990b;
Taylor
et
al.,
1985),
the
mechanisms
proposed
above
appear
to
be
612
AWADZI
ET
AL.
the
only
plausible
explanation
for
the
events
that
were
observed
in
the
present
study.
The
successful
implementation
of
com-
bination
chemotherapy
demands
that
no
serious
AE
occur,
including
any
that
are
attributable
to
drug
—drug
interactions.
Phar-
macokinetic
studies
elucidate
those
drug
drug
interactions
that
may
contribute
to
an
improved
response
to
multiple
-drug
therapy.
The
present
assessment
of
drug
drug
interactions
drew
on
data
from
a
previous
investigation
in
which
albendazole
and
ivermectin
had
been
administered
as
single
agents
(Awadzi
et
al.,
2003).
The
use
of
these
'historical'
data
seems
justified
on
several
grounds.
Firstly,
the
studies
providing
the
historical
data
were,
like
the
present
study,
randomized,
double-blind
and
placebo
-controlled.
Secondly,
the
older
data
were
obtained
from
a
group
of
patients
whose
demographic
characteristics
were
essentially
identical
to
those
of
the
present
subjects.
Thirdly,
the
methods
used
by
Awadzi
et
al.
(2003),
for
sample
collection,
handling,
storage
at
the
trial
site
and
ship-
ment,
were
the
same
as
those
used
in
the
present
study.
Finally,
in
the
earlier
study,
the
relevant
plasma
drug
concentrations
were
determined
by
the
same
laboratory
personnel,
using
the
same
analytical
procedures,
as
in
the
present
study.
Taken
together,
the
present
results
provide
no
evidence
indicating
that
systemic
exposure
to
levamisole
is
altered
substan-
tially
by
concomitant
administration
of
either
albendazole
or
ivermectin.
Compari-
son
of
the
pharmacokinetic
parameters
for
albendazole
sulphoxide
and
ivermectin,
as
determined
in
the
present
study,
with
those
obtained
previously
(Awadzi
et
al.,
2003)
indicates
that
co
-administered
levamisole
elicits
a
decrease
in
the
'area
under
the
curve'
of
albendazole
sulphoxide
and
an
increase
in
the
same
parameter
for
iver-
mectin.
The
reasons
for
these
effects
and
their
clinical
consequences
are
not
immedi-
ately
apparent.
Albendazole
and
ivermectin
are
both
substrates
of
cytochrome
P450
(CYP)
3A4,
the
most
widely
distributed
human
P450,
which
is
expressed
both
in
the
liver
and
small
intestine.
There
is,
therefore,
the
potential
for
drug
—drug
interactions
at
each
of
these
sites.
The
information
provided
by
Rawden
(1999)
indicates
that
albendazole
may
be
converted
to
alben-
dazole
sulphoxide,
the
major
plasma
metabolite
in
man,
by
microsomes
prepared
from
human
intestinal
tissue.
Moreover,
ivermectin
(Kwei
et
al.,
1999),
but
not
albendazole
(Merino
et
al.,
2002),
is
a
substrate
for
p-glycoprotein.
This
transport
protein
is
expressed
in
a
variety
of
tissues,
and
interactions
at
the
cellular
level
could
conceivably
occur
through
direct
com-
petition
for
binding.
While
conversion
of
levamisole
to
a
4-hydroxy
metabolite
has
been
demonstrated
(Kouassi
et
al.,
1986),
the
anthelmintic
properties
of
this
metabo-
lite
and
its
contribution
to
the
pharmacolo-
gical
profile
of
levamisole
are
unknown.
Furthermore,
there
is
no
information
avail-
able
regarding
the
site
of
the
metabolism,
the
enzymes
involved,
or
the
probability
that
levamisole
is
a
substrate
for
p-glycoprotein.
Such
information
is
needed
if
the
LA
and
LI
interactions
are
to
be
explained
and
their
clinical
importance
is
to
be
established.
Levamisole
had
little
effect
on
the
efficacy
of
ivermectin
or
albendazole
against
0.
volvulus.
The
increase
in
the
bio-availability
of
ivermectin
seen
when
levamisole
was
co
-administered
was
unlikely
to
alter
the
efficacy
of
the
ivermectin,
since
doses
much
higher
than
the
standard
dose
used
in
Trials
1
and
2
are
no
more
effective
against
the
mff
and
adult
worms
(Awadzi
et
al.,
1995b,
1999;
Gardon
et
al.,
2002).
The
reduction
in
the
bio-availability
of
albendazole
(measured
as
the
sulphoxide)
seen
when
levamisole
was
co
-administered
was
probably
not
a
factor
in
the
observed
lack
of
efficacy
of
the
alben-
dazole,
since
a
similar
dose
of
albendazole
(400
mg)
also
showed
little
activity
when
given
as
a
single
agent
(Awadzi
et
al.,
2003).
These
findings,
taken
together
with
the
results
of
previous
studies
with
albendazole
in
which
higher
doses
were
used
(Awadzi
et
al.,
1991,
1994),
indicate
that
the
LEVAMISOLE
IN
THE
TREATMENT
OF
ONCHOCERCIASIS
613
minimum
dose
of
albendazole
for
the
treat-
ment
of
onchocerciasis
should
be
800
mg.
In
the
earlier
study
of
mebendazole
(Awadzi
et
al.,
1982),
two
doses of
levamisole
were
given
48
h
apart
and
the
mebendazole
was
started
at
least
24
h
after
the
second
dose.
At
the
time
the
mebendazole
treatment
was
initiated,
the
plasma
concentrations
of
leva-
misole
would
have
been
too
low
to
affect
the
kinetics
of
mebendazole.
The
augmented
efficacy
of
mebendazole
observed
was
there-
fore
attributed
to
the
immunomodulating
properties
of
levamisole,
and
formed
the
basis
of
the
current
studies.
It
is
uncertain
whether
the
use
of
the
same
dose
regimen
of
levamisole
with
ivermectin
or
albendazole
would
yield
more
favorable
results.
The
combination
of
levamisole
with
iver-
mectin
or
albendazole
induced
unexpected
adverse
events,
altered
the
time
-frame
of
reactions
to
ivermectin,
and
resulted
in
significant
drug
—drug
interactions.
These
findings
emphasise
the
need
to
study
drug
combinations
cautiously,
as
in
the
present
studies,
prior
to
their
use
in
large
groups
of
subjects.
This
requirement
is
independent
of
whether
or
not
the
drugs
are
already
in
use
for
other
indications.
ACKNOWLEDGEMENTS.
The
authors
thank
GlaxoSmithKline,
Merck
&
Co.,
and
Janssen
Pharmaceutica
for
supplying
the
drugs
and
placebo
tablets
used.
This
inves-
tigation
received
financial
support
from
the
African
Programme
for
Onchocerciasis
Control,
the
Onchocerciasis
Control
Pro-
gramme
in
West
Africa
and
the
UNDP/
World
Bank/WHO
Special
Programme
for
Research
and
Training
in
Tropical
Diseases.
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