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Ivermectin distribution in the plasma and tissues of patients infected with Onchocerca volvulus

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To determine the distribution of ivermectin in plasma and tissues of onchocerciasis patients following a single oral dose of 150 micrograms kg-1. Medical Department at Soba University Hospital, Khartoum. Twenty five patients and fourteen healthy volunteers. Serial blood samples were obtained from both groups. Tissue samples were removed from various patients as full thickness skin punch biopsies or during nodulectomy. Ivermectin concentration was determined by radioimmunoassay. The plasma pharmacokinetic variables for patients were; maximum plasma concentration 52.0 ng ml-1; time to achieve maximum concentration, 5.2 h.; elimination half life, 35.0 h; and the area under the plasma concentration curve versus time, 2852 ng.h.ml-1. In healthy volunteers, the plasma ivermectin distribution was similar to that in patients, and both groups showed a tendency for a second rise in plasma concentration of the drug suggestive of enterohepatic recirculation. Ivermectin was detected in tissues obtained from patients. Fat showed the highest and most persistent levels, whilst values for skin, nodular tissues, and worms were comparable. Subcutaneous fascia contained the lowest concentrations. Infection with O. volvulus does not affect the pharmacokinetics of ivermectin, and filarial infected tissues and parasites themselves do take up the drug. There may be prolonged retention of ivermectin because of depot formation in fat tissue.
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Eur J Clin Pharmacol (1996) 50:407–410 © Springer-Verlag 1996
PHARMACOKINETICS AND DISPOSITION
O. Z. Baraka · B. M. Mahmoud · C. K. Marschke
T. G. Geary · M. M. A. Homeida· J. F. Williams
Ivermectin distribution in the plasma and tissues of patients
infected with
Onchocerca volvulus
Received: 16 August 1995/Accepted in revised form: 8 December 1995
Abstract Objective: To determine the distribution of
ivermectin in plasma and tissues of onchocerciasis
patients following a single oral dose of 150µgkg
[1
.
Setting: Medical Department at Soba University
Hospital, Khartoum.
Patients: Twenty ve patients and fourteen healthy
volunteers.
Methods:Serial blood samples were obtained from
both groups. Tissue samples were removed from vari-
ous patients as full thickness skin punch biopsies or
during nodulectomy. Ivermectin concentration was
determined by radioimmunoassay.
Results: The plasma pharmacokinetic variables for
patients were; maximum plasma concentration
52.0ngml¯
1
; time to achieve maximum concentration,
5.2h.; elimination half life, 35.0h; and the area under
the plasma concentration curve versus time,
2852 ng·h ml¯
1
. In healthy volunteers, the plasma iver-
mectin distribution was similar to that in patients, and
both groups showed a tendency for a second rise in
plasma concentration of the drug suggestive of entero-
hepatic recirculation. Ivermectin was detected in tis-
sues obtained from patients. Fat showed the highest
and most persistent levels, whilst values for skin, nodu-
lar tissues, and worms were comparable. Subcutaneous
fascia contained the lowest concentrations.
Conclusion:Infection with O. volvulus does not aect
the pharmacokinetics of ivermectin, and larial
infected tissues and parasites themselves do take up the
drug. There may be prolonged retention of ivermectin
because of depot formation in fat tissue.
Key words Ivermectin, Onchocerciasis; pharmaco-
kinetics, tissue concentration, enterohepatic
Introduction
Ivermectin is a macrocyclic lactone with potent
antiparasitic activity, widely used in veterinary medi-
cine (Campbell et al. 1983; Campbell 1985). Ivermectin
pharmacokinetics and tissue distribution, have been
extensively studied in animals (Chiu and Lu 1989).
Following successful clinical trials of ivermectin in
human onchocerciasis (Aziz et al. 1982; Taylor and
Greene 1989), it has become the drug of choice for
symptomatic treatment, with a potential for strategic
use in controlling the transmission of Onchocerca volvu-
lus infection (Greene 1992). However, despite its wide
popularity, knowledge of the pharmacokinetics of the
drug in man is rudimentary. There are only a few pub-
lished reports about oral bioavailability in healthy vol-
unteers (Edwards and Breckenridge 1988; Goa et al.
1991; Edwards et al. 1988), or in patients (Okonkwo
et al. 1993). Ivermectin concentration is determined by
HPLC (Downing 1989; Chiou et al. 1987). This method
although specic, generally requires large samples when
dealing with tissue samples, extensive instrumentation,
and sample preparation is time consuming.
The development of a radioimmunoassay (RIA) for
ivermectin by Marschke (1989), employing antibodies
with high avidity and specicity for the parent com-
pound, has made it possible to measure the drug in
very small volumes of plasma and tissue. The proce-
dure is sensitive down to 1 ng ml
[1
, the antibodies
show only 19–23% cross reactivity with avermectin,
and the amount required for 50% displacement of the
label (ED50) is 3 ng ml
[1
. This development has made
possible the study of ivermectin distribution in patients,
especially in organs with a high larial density, and the
O. Z. Baraka (*) ·M. A. Homeida
Department of Medicine, Faculty of Medicine, University of
Khartoum, Khartoum, P.O. Box 102, Sudan
B. M. Mahmoud
Department of Pharmacology, Faculty of Pharmacy, University
of Khartoum, Khartoum, Sudan
C. K. Marschke ·T. G. Geary
The UpJohn Company, Kalamazoo, MI, USA
J. F. Williams
Microbiology, Michigan State University, E. Lansing, MI, USA
results should shed light on some important issues in
drug/parasite/disease interactions.
In this study the distribution of ivermectin in
onchocerciasis patients was determined in samples of
plasma, skin, subcutaneous fascia, fat, onchocercal
nodules and worms, collected at various time points
after a single oral dose of 150µgkg
[1
in patients and
in volunteers.
Materials and methods
Subjects and sampling procedure
Twenty ve patients with onchocerciasis and microlaria-positive
skin snip biopsies participated in the study. Microlarial loads
ranged from 1.2 to 164 mfmg
[1
. All were admitted to hospital.
Fourteen healthy volunteers were recruited for the study of iver-
mectin plasma pharmacokinetics. Oral and written consent were
obtained from each subject. Ethical clearance for the study was
obtained from the University of Khartoum, Faculty of Medicine
Research Committee. All subjects were non smokers, did not con-
sume alcohol, and were not on medication. The females were non-
pregnant and not lactating. Each subject received a single oral dose
of ivermectin 150µgkg
[1
body weight after an overnight fast.
Subjects were allowed only water for 2h following treatment.
Heparinised venous blood samples 5ml were taken from an
indwelling cannula in the antecubital fossa at 0, 1, 2, 3, 4, 6, 8, 12,
24, and 48h post treatment no blood samples were obtained at 48
h from the volunteers. The plasma was immediately separated and
stored at [20
ο
C until the assay.
Tissue samples were obtained from the patients at various times
taking care not to subject any individual to more than one surgi-
cal intervention. Tissues were obtained either as full thickness 3 mm
skin punch biopsies or during surgical nodulectomy. Samples were
immediately frozen in liquid nitrogen and kept until assayed.
Analysis of plasma samples
Ivermectin concentrations in plasma samples were determined with-
out extraction by the RIA technique of Marschke (1989). One hun-
dred µl from each sample or standard (chromatographically puried
ivermectin), tritiated ivermectin (Amersham, 22.4 Ci/mmol
[1
,
6000 dpmper assay-tube), and antiserum (diluted 1/1000) were
added to 12 ×75 mm glass tube and mixed. The tube was kept at
4
ο
C for 16h. Stirred charcoal suspension, 0.7ml, (0.75% Sigma
activated C-4386) was added to all tubes except the Total Count
tube to which was added 0.7ml RIA buer. The tube contents were
mixed immediately and at 7.5 and 15min. Following centrifuga-
tion, the supernatant decanted into 10ml scintillation cocktail.
Tritium was determined with a liquid scintillation counter.
Ivermectin extraction from tissues
The tissue samples were dissected into their dierent components
to obtain skin, fascia, subcutaneous fat and nodules. Worm frag-
ments were obtained from onchocercal nodules by the collagenase
technique (Schultz-Key et al. 1980). Individual tissue samples were
weighed and assayed in duplicate. The tissue fragments were repeat-
edly homogenised in acetone using an electrical homogeniser with
vortex mixing. After centrifugation, the acetone layer was trans-
ferred to a new tube and evaporated. Acetonitrile was added to the
residue, which was repeatedly extracted into hexane. The acetoni-
trile layer was collected in 75 ×15 mm glass tubes and evaporated
to dryness. The residue was dissolved in 100µl of RIA buer. The
amount of ivermectin extracted from each tissue sample was then
determined by RIA.
To assess the ecacy of extraction, lean beef muscle and fat
samples were spiked with known amounts of ivermectin. About an
85% extraction rate was obtained.
Pharmacokinetic analysis
Pharmacokinetic parameters were calculated using GraphPAD
GPIP Inplot and the Medusa software package. The following were
determined; maximum plasma concentration (C
max
); the time to the
maximum concentration of the drug (t
max
); elimination half life (t
1/2
);
and the area under the plasma concentration curve time (AUC).
Statistical analysis
The unpaired t-test was applied. P <0.05 was considered signicant.
Results
Plasma ivermectin concentrations
No signicant dierences were observed between phar-
macokinetic parameters in patients with onchocerciasis
and healthy volunteers (Table 1). Ivermectin appeared
in plasma within 1h after the oral dose (5.7 to
38.8 ngml
[1
), and was detected for up to 48 h
(10.8–62.6 ngml
[1
). Some of the individual plasma
concentration proles (6 patients and 5 volunteers)
showed a second rise in plasma concentration follow-
ing an initial decrease. The secondary peak mostly
occurred between 6 and 12h after the dose. The curves
of the mean plasma concentrations of the patients and
the volunteers showed that the elimination phase was
similar and slow, and that it exhibits linear decay
(Fig.1).
Tissue ivermectin concentrations
Ivermectin concentrations in dierent tissues of
patients with onchocerciasis are shown in Table 2. The
drug was detected in all tissue sampled. Fat showed the
highest concentrations. Values for the skin, nodules,
and worms from the same patient were comparable.
The lowest concentrations were consistently seen in
subcutaneous fasciae.
408
Table 1 Ivermectin pharmacokinetics mean (SD) in 14 healthy vol-
unteers, and 14 patients infected with O. volvulus following oral
administration of 150µg·kg
C
max
t
max
t
1/2
AUC
(ngml
[1
)(h) (h) (µg·hml
[1
)
Healthy 54.4 (12.2) 4.9 (1.5) 36.6 (10.2) 3.18 (1.39)
subjects
Patients 52.0 (12.0) 5.2 (1.9) 35.0 (9.2) 2.850 (0.841)
Ivermectin administration to healthy volunteers did
not cause any post-treatment reactions. All the patien-
t’s skin snips obtained on D7 were negative for
microlariae.
Discussion
Our results show that infection with O. volvulus does
not aect the distribution of ivermectin in plasma of
patients compared to healthy volunteers. In both
groups there was a common tendency for a secondary
peak to appear, suggestive of enterohepatic circulation.
Accumulation of the parent drug in fat probably con-
tributes to prolonged retention of ivermectin in the
body.
The ivermectin plasma half-life in healthy volunteers
was reported to be 12, 22, and 28h (Fink and Porras
1989; Edwards 1987; Edwards and Breckenridge 1988),
and 56h in onchocercal patients (Okonkwo et al. 1993).
In comparison our results were 36.6, and 35h, respec-
tively. The AUC we obtained was higher than the
reported values of 885 (389) and 1545.3 (190.5)
ng·h ml
[1
, (Edwards 1987; Edwards and Breckenridge
1988; Okonkwo et al. 1993). The dierence in the sys-
temic availability of ivermectin can be attributed to sev-
eral factors. In previous reports it was not stated if
subjects had taken food during the 2hours immedi-
ately after dosing and we now know that food intake
can result in a signicant reduction in the amount of
ivermectin absorbed (submitted). Ivermectin consist of
a mixture of 80% dihydroavermectin B
1a
(H
2
B
1a
) and
20% dihydroavermectin B
1b
(H
2
B
1b
). The HPLC
method used measured only the H
2
B
1a
fraction of the
compound. Details of the specic recovery rates were
not given and values as low as 60% by extraction from
plasma have generally been accepted (Downing 1989).
The sensitivity of the RIA we used may have been supe-
rior, since the antibody is expected to react with the
whole compound and no extraction procedure was
applied during analysis of plasma samples. However,
we are aware of the fact that cross reactivity of the anti-
body with ivermectin metabolites could be an added
factor. The sensitivity of the RIA was demonstrated by
its ability to show secondary peaks similar to those
observed in a study of ivermectin disposition in four
healthy volunteers given the tritium-labelled drug (Fink
and Porras, 1989).
Ivermectin is mainly excreted in bile (Fink and
Porras 1989) and is undetectable in urine (Okonkwo
et al. 1993). The excretion of ivermectin in bile was
expected since it has a high molecular weight and is
very lipid soluble (Fisher and Morzik 1989). Hence,
enterohepatic circulation of the drug is to be expected.
Similar secondary peaks are frequently seen with com-
pounds that undergo hepatic recycling (Miller 1984;
Terhaag and Hermann 1986).
Ivermectin depletion appears very slow in most tis-
sues. The pattern of dierential distribution of iver-
mectin concentrations obtained in our human tissue
samples was comparable to that seen in animals (Chiu
and Lu 1989). The high concentration of ivermectin in
fat is a function of the lipid solubility of the drug, and
fat acts as a reservoir for ivermectin. This could explain
previous observations that ivermectin was detected in
human milk for 12 days following a single oral dose
(Chiou et al. 1987). Ivermectin concentrations in areas
of high larial density, the nodule, worm and skin were
comparable. This supports our previous observation
409
Fig. 1 Plasma Ivermectin concentrations [Mean (SEM)] in healthy
volunteers (n=14) and onchocerciasis patients (n=14) after
150µg/kg single oral dose
Table 2 Ivermectin concentration in tissues (ngg
[1
of tissue) of 10
patients with onchocerciasis, after a single oral dose of 150 µg kg
[1
NA sample not available; NB repetition of time points represents
analysis of tissues from dierent patients.
Time (h) Skin Fascia Fat Nodule Worm
4
a
90.9 NA 141 62.4 NA
670.5 31.8 NA 70.8 79.4
24
a
NA 26.2 NA 31.6 NA
30 71.7 38.2 NA 54.7 NA
48
a
66.6 NA NA 101.5 NA
72 NA 42.5 NA 56.4 NA
72 64.9 NA NA NA 44.2
72 41.4 18.5 NA 37.1 59.5
4 Days NA 58.9 117.6 NA NA
5 Days 15.6 NA 94.1 NA NA
a
Corresponding plasma values for these tissue samples were 46,
28.8 and 24 ngml
[1
, respectively
that, O. volvulus is accessible to blood-borne agents
(Mahmoud et al. 1991).
The sustained reduction of O. volvulus microlariae
in skin tissues has been explained by the eect of the
drug on the gravid uterus of the female worm (Albiez
et al. 1988). The sustainability of the eect may also
be attributable in part to prolonged retention of the
drug in the body. However, even allowing for prolonged
persistence of the drug, the relation between ivermectin
pharmacokinetics and its antiparasitic eect is still far
from being understood. Since ivermectin does not
directly aect target organisms (microlariae) invitro,
nor is there evidence of bioconversion invivo to metabo-
lites with direct activity (Soboslay et al. 1987). The like-
lihood is that suitable host responses act in concert
with ivermectin to bring about the antiparasitic effects
(Baraka et al. 1995). Dening these eector mecha-
nisms awaits further studies.
Acknowledgements We thank Dr. M. M. Ali for his assistance in
the parasitological aspect of this study. This work was supported
by MSU/NIH/SUDAN Medical Parasitology Grant No: A1-16312.
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