The ocular deposition of chloroquine
Howard Bernstein, Nathan Zvaifler, Martin Rubin, and
Sister Agnes Mary Mansour
per kilogram of chloroquine dihydrocluonde, the drug is retained in the uoeai tract of tin
pigmented rabbit eye in high concentration for at least 1 month. In pigmented rats receiving
daily oral chloroquine phosphate for 6 months, the iris contained 80 times as much drug as
c, ' h 1 o r o q u i n e (7-chloro-4- (4'-diethyl-
amino-l'-methylbutylamino) quinoline) is
an antimalarial drug which has been used
since 1950 in the treatment of rheumatoid
arthritis, discoid and systemic lupus ery-
thematosus, and dermal light sensitivity
eruptions. A retinopathy produced by the
long-term administration of this drug was
first recognized by Hobbs1 in 1959. Subse-
quently, over 50 cases of this toxicity have
been reported. The clinical and electro-
physiologic characteristics of the retinop-
athy have been described in detail.2 The
From the Ophthalmology Branch, National Insti-
tute of Neurological Diseases and Blindness,
National Institutes of Health, United States
Public Health Service, Department of Health,
Education and Welfare, Bethesda Md., and
from the Departments of Medicine and Bio-
chemistry, Georgetown University School of
Medicine, Washington, D. C.
This study was supported in part by United States
Public Health Service Grant 2A-5042 and by
the Arthritis and Rheumatism Foundation of
Metropolitan Washington, D. C.
384
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depressed to absent electroretinogram in
advanced cases indicates damage to the
rods and cones. However, the basic mecha-
nism by which chloroquine produces this
retinopathy is still unknown.
As part of a general program of investi-
gation into the basic pharmacology and
biochemistry of chloroquine, we have
studied the uptake and tissue distribution
of the drug in the ocular and visceral tissues
of experimental animals. Because of an
observation by Potts12 at the 1962 Ophthal-
mic Biochemistry Meeting on chloroquine
adsorption to melanin granules, particular
attention was given to the pigmented ani-
mal. This report is concerned with the
striking affinity of chloroquine for the
melanin-containing tissues of the eye—the
iris, choroid, and pigment epithelium, and
the possible relationship of this unusual
biochemical characteristic to the develop-
ment of the retinopathy.
"Aqueous chloroquine dihydrochloride, SO ing
centimeter, was supplied through the courte
throp Laboratories, New York, N. Y.

Materials and Methods
A single intravenous injection of aqueous chloro-
quine dihydrochloride,0 5 ing. per kilogram of
body weight, was given to pigmented (chin-
chilla) and pure-bred albino rabbits. At intervals
ranging from 15 minutes to 28 clays following
administration of the drug, the rabbits were killed
with an overdose of pentobarbital, and the eyes
were enucleated. Blood samples and tissues were
obtained for analysis. Albino and pigmented
rabbits were also given 5 mg. per kilogram of
chloroquine dihydrochloride by intramuscular in-
jection, and were killed after 48 hours and 63 days.
To evaluate drug levels after long-term adminis-
tration, chloroquine phosphate was added to the
drinking water (0.6 mg. per milliliter) of both
pigmented (Sprague-Dawley) and albino rats for
a minimum of 6 months. The average ingestion
was 125 to 250 mg. per week. The rats were
killed with ether anesthesia while on the medica-
tion. All tissues were processed in a similar
manner to those of the rabbit.
The dissection technique for the rabbit eye
was as follows: The aqueous was aspirated with
a 27 gauge needle on a 1 ml. syringe. With a
razor blade, a circumferential cut was made an-
terior to the equator, and the posterior segment
separated. Vitreous was collected by aspiration
into a 1 ml. syringe. The retina was brushed to-
gether with the reverse end of a forceps and cut
free at the nerve head. The choroid was separated
from sclera by blunt and fine dissection. With this
procedure, the pigment epithelium remained with
the choroid. The sclera of the pigmented rabbit
was scraped as free as possible from residual
pigment. The lens was separated from its zonular
attachments and the iris cut off from the ciliary
body. A segment of cornea was then dissected
free from the adjacent sclera. All tissues were
immediately weighed on a torsion balance to the
nearest 0.01 mg.
The dissection procedure for the rat, essentially
similar to that described above, was performed
under magnification and on moist filter paper to
prevent desiccation of the tissues. No aqueous or
vitreous samples were taken. Wet tissue weight
was obtained on an electrobalance with a sensiti-
vity of 0.0001 mg.
Chloroquine and its major metabolite, the N-
dcsethylated derivative, 7-chloro-4-(4'-ethylamino-
l'-methylbutylamino) quinoline (Fig. 1), were
determined after tissue homogenization in ethanol
for all tissues except hair, iris, and choroid. These
tissues were homogenized in 0.1N hydrochloric
acid. Aliquots of the ethanol or acid extracts were
then transferred to methylene chloride. Fractional
extraction of the drug from the methylene chloride
was performed with aqueous buffer solution, pll
7.85, after washing with a pH 9.5 buffer. Chloro-
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Ocular deposition of chloroquine 385
quine and metabolite concentrations were then
determined by fluorescence with an Aminco-
Bowman spectrophotoHuorometer at 400 m/i, fol-
lowing activation at 335 mn in alcoholic alkali.
Details of the method have been described.'1
Chloroquine and metabolite at concentration
levels of 0.05 gamma per gram of sample could
be measured with an accuracy of ± 10 per cent.
The ocular tissue levels are expressed as the
average value of the two eyes of each animal,
and other tissue levels by the average of at least
two samples. The study in the rabbit of the drug
distribution over a period of 28 clays following
a single intravenous injection represents one animal
at each time interval. This study was repeated,
and the tissue drug levels were found to be quite
similar in pattern and magnitude.
Results
The most striking observation in this
study is the preferential storage of chloro-
quine in the iris and choroid of the pig-
mented experimental animal. In rabbits
killed at 48 hours following a single intra-
venous dose of 5 mg. per kilogram of
chloroquine dihydrochloride (Table I), the
drug is found in high concentrations in the
pigmented iris and choroid, and is absent in
the nonpigmented uveal ocular tissues of
the pigmented rabbit and in all the ocular
tissues of the albino rabbit. The concen-
tration of chloroquine in the pigmented
choroid is four to five times greater than
that found in any other systemic tissue.
Data obtained from the intramuscular
administration of the same dose of chloro-
HH-C—c-c-c-<
i i i i \c
H-C-H H H H L
CHLOROQUINE
METABOLITE
H H H .CJHJ
HN-C C-C-C-N'
I i i I x
H-C-H H H H
H
Fig. 1.
 3S6 Bernstein el cd. 'Investigative Ophthalmology
August 1963

quine dihydrochloride (Table II) show lowing intravenous administration, the

again, at 4S hours, the presence of chloro- choroid level is 54 gamma and the iris con-
quine in the choroid of the pigmented tains 35 gamma. This difference probably
animal and its absence in the choroid of represents a slower release of chloroquine
the albino animal. After intramuscular in- from the site of injection when administered
jection, the chloroquine level in the intramuscularly.
choroid is only 14.6 gamma, and no drug The time course of chloroquine uptake
is found in the iris. At the same time, fol- and excretion in the pigmented rabbit
following a single intravenous dose, 5 mg.
per kilogram of body weight, can be seen
Table I. Chloroquine levels at 48 hours in Fig. 2. Peak drug levels in the plasma
following a single intravenous dose are present at 15 minutes, decrease grad-
ually over the next 4 to 6 hours, and are no
Gamma/gram wet tissue weight
longer detectable after 6 hours.
Pigmented Albino Deposition of the drug in the liver gradu-
(2 eyes) (2 eyes)
Chloro-
ally increases until a maximum level, 20
Chloro-
quine quine gamma, is reached at about 20 hours. The
Chloro- metabo- Chloro- metabo- chloroquine concentration then gradually
Tissue quine lite quine lite decreases until the drug is no longer de-
Iris 35 0 0 0 tected in the liver between the fourteenth
Choroid 54 0 0 0
and twenty-eighth days.
Cornea 0 0 0 0
Aqueous 0 0 0 0 Chloroquine uptake in the pigmented
Lens 0 0 0 0 choroid and iris follows a different pattern.
Vitreous 0 0 0 0
Retina 0 0 0 0
The drug first appears in these tissues
Sclera 0 0 0 0 between 6 and 12 hours after administra-
Liver 10.8 10.6 8.0 11.8 tion, at a time when it can no longer be
Kidney 14.0 8.0 7.0 9.0
detected in plasma. There is, then, a very
Spleen 2.8 4.0 4.4 6.2
Lung 6.8 4.4 4.0 1.6 rapid buildup to maximum levels occurring
Heart 7.0 1.6 4.6 3.2 within 24 hours for the choroid and 4S

Table II. Chloroquine levels at 48 hours and 63 days following a single

intramuscular dose
Camma/gmm wet tissue weight
48 liours 63 days
Pigmented Albino Pigmented Albino
(2 eyes) (2 eyes) (2 eyes) (2 eyes)
Chloro- Chloroquine Chloro- Chloroquine Chloro- Chloroquine Chloro- Chloroquine
Tissue quine metabolite quine metabolite quine metabolite quine metabolite
Iris 0 0 0 0 42.0 0 0 0
Choroid 14.6 0 0 0 30.0 0 0 0
Aqueous 0 0 0 0 0 0 0 0
Lens 0 0 0 0 0 0 0 0
Vitreous 0 0 0 0 0 0 0 (I
Retina 0 0 0 0 0 0 0 0
Sclera 0 0 0 0 0 0 0 0
Liver 3.6 3.6 2.2 3.2 0 0 0 0
Kidney 18.7 7.2 8.9 3.0 0 0 0 0
Spleen 6.1 2.8 1.3 1.4 0 0 0 0
Lung 10.1 4.2 2.9 1.8 0 0 0 0
Heart 2.3 0.84 1.6 1.8 0 0 0 0

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 Volume Ocular deposition of chloroquine 387
Numhe

hours for the iris. Drug levels at this time Dcu-Sma/kaBWlM

for the choroid are 68 gamma and for the ,'\ I Plasma?,
iris 35 gamma. Chloroquine levels decrease
at 7 days, and then return to their original
peak levels by 28 days. At this time, there
is no longer any detectable drug in the liver
or any of the other organs studied.
The presence of chloroquine in the
pigmented uveal tract for such a remarkably
long period of time is further emphasized
by the substantial concentration (31 gamma
per gram) of the drug found in the choroid
of a pigmented rabbit 63 days after a Fig. 2. Chloroquine uptake in the pigmented
single intramuscular injection (Table II). rabbit.
At this time, the drug is no longer detect-
able in any other tissue examined.
The high concentration of the drug in
the pigmented uveal tract is confirmed in
a second species, the rat, following ad-
ministration of chloroquine phosphate in
drinking water for at least 6 months (Table
III). The concentration of the drug in the
iris of the pigmented rat, 20,910 gamma per
gram, is over SO times the concentration
found in the liver. Again, chloroquine is
not detected in the albino iris or choroid.
Chloroquine is also present, but in a
much lower concentration, in the retina
and solera of the pigmented and albino Fig. 3. Chloroquine uptake in the pigmented
rat. The higher level in the pigmented rat versus albino rabbit.
probably represents pigment contamination
from the adjacent choroid which was ob-
served at the time of dissection, especially Chloroquine levels in the systemic tissues
of the sclera. However, since chloroquine appear to be of the same magnitude in the
is found in the albino retina, it would pigmented and albino animals, both in the
appear that, on long-term administration, single dose and chronic administration ex-
the retina has a significant drug uptake of periments (Fig. 3). One exception, how-
its own, comparable in magnitude to that ever, is the drug level in the hair of the
of the liver. rat (Table III). A significantly higher level
A measure of the capacity of the pig- is found in hair of the pigmented rat, 780
mented uvea to store chloroquine is sug- gamma per gram, as compared to the
gested by a comparison of the drug level albino rat, 140 gamma per gram.
in the iris of the rabbit after a single dose, The major metabolite of chloroquine, its
35 gamma, and the iris of the rat after long- N-desethylated derivative (Fig. 1), can
term administration, 20,910 gamma per be detected in the liver of the rabbit as
gram. The latter shows a six hundred fold early as 30 minutes following an intra-
increase, whereas comparable levels in the venous injection. In the liver, the metabo-
liver, 14.0 gamma and 240 gamma, show lite levels follow a very similar time course
less than a twentyfold increase on long- to those of chloroquine itself. However,
term administration of the drug. the metabolite cannot be detected in the

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 38S Bernstein et ul. Investigative
August. 1063

Table III. Chloroquine levels in rats drug cannot be detected in the liver
following long-term oral administration and other systemic tissues, there is as much
chloroquine present in the pigmented
Gamma/gram wet tissue wviglit
ocular tissues as was present after 24 hours.
Pigmented Albino These two basic pharmacologic charac-
(6 months) (14 months)
(4 eyes) (4 eyes) teristics, (1) extensive tissue accumulation
Chloro- Chloro- and (2) prolonged retention in the tissues
quine quine after drug administration has ceased, have
Chloro- metab- Chloro- metab- been observed to a lesser degree in earlier
Tissue quine olite quine olite
studies of the physiologic disposition of
Iris 20,910 3,250 0
0
0
0
chloroquine.4'3 The relatively high systemic
Choroid 7,820 1,190
Cornea 62 0 0 0 tissue levels compared to blood levels were
Lens 0 0 0 0 demonstrated by Berliner,4 who adminis-
Vitreous 0 0 0 0 tered chloroquine phosphate, 25 mg. per
Retina 312 75.3 143.3 39.3
Sclera 360 74.5 14.0 0 kilogram daily, to rats for 10 days with
Brain 15 10 20.0 10 resulting tissue/plasma concentration ratios
Liver 240 130 140 80 of 670 for kidney, 640 for lung, and 420 for
Hair 780 160 140 20
liver.
The marked tissue binding of chloroquine
results in a slow disappearance of the drug
pigmented iris or choroid until the twenty- from the body when its administration is
eighth clay. At that time, chloroquine discontinued. Urinary excretion of chloro-
metabolite is found in the choroid only at quine and its metabolite has been found
a concentration of 19.5 gamma per gram. to continue at low levels for at least 10
The metabolite is also present in both the days following the ingestion of a single
pigmented iris and choroid of the rats after dose of from 0.1 to 1.0 Gm. of the drug."' r
long-term administration of the drug In patients who have been on long-term
(Table III). chloroquine therapy, we have found traces
It should be noted that in the single dose of chloroquine in the blood and urine
experiment, the dosage of 5 nig. per kilo- several years after they have discontinued
gram of body weight is equivalent to 250 to the drug.8
500 mg. of oral chloroquine phosphate, It has been suggested that the retention
which is the average daily close in humans. of chloroquine in systemic tissues is the
It is also comparable to the single weekly result of binding of the drug to nucleopro-
close of chloroquine base given as prophy- tein and nucleic acids."'10 However, as
laxis for malaria. demonstrated in the kinetic study of the
drug redistribution in the tissues following
Discussion a single dose in the pigmented rabbit (Fig.
The iris and choroid of the pigmented 2), the uptake and disposition of the drug
animal have been shown to store chloro- in pigmented uveal tissues differ from those
quine in a much higher concentration than found in systemic tissues in at least five
other systemic tissues. The difference has ways. There is (1) a delay in uptake of
ranged from a fourfold one 48 hours after chloroquine from 6 to 12 hours from time
a single dose in rabbits to eightyfold after of injection, (2) a very much higher tissue
long-term oral administration of the drug drug concentration reached, (3) a redistri-
to rats. bution of chloroquine levels in the eye
Chloroquine is also retained in the pig- which occurs between the second and
mented iris and choroid for a much longer fourteenth days, (4) a retention of the
period than by other tissues. One month drug by the pigmented tissues for a much
following a single intravenous dose, when longer period of time, and (5) the N-

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 Volume 2 Ocular deposition of chloroquinc 389
Number 4

Fig. 4. Macula in chloroquine retinopatliy illustrating the unusual perifoveal highlights and
patchy depigmentation commonly seen.

Fig. 5. Macula in chloroquine retinopathy illustrating a marked loss of pigment in the macular

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 390 Bernstein et al.
Fig. 6. Histologic section from a case of chloroquine retinopathy. Note the loss of rods and
cones with migration of pigment granules into the inner nuclear layer. (xl43.)
desethylated metabolite is not found in the
pigmented uvea as it is in other systemic
tissues until at least 1 month after drug
administration. It is felt that, because of
these differences, the nature of chloroquine
binding to pigmented tissues will be sub-
stantially different from that now asso-
ciated with the binding of the drug to
nonpigmented tissues.
The affinity of chloroquine for the pig-
mented iris and choroid and its absence
in comparable albino tissues suggest that
melanin pigment may play a principal role
in this unusual type of binding. The only
known cellular defect in the albino is a
decrease in the normal content of the
enzyme tyrosinase, which results in a de-
ficiency of melanin in the melanocytes.11
Evidence for the implication of melanin
in drug binding has recently been demon-
strated by Potts12 in his comprehensive
study on uveal pigment fixation of the
various phenothiazine derivatives. Several
phenothiazine derivatives (prochlorpera-
zine and chlorpromazine) were shown to be
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tive Ojihttwhuology
AuKttst 1963
taken up in the iris and choroid of the
pigmented experimental animal in a very
similar manner to that described in the
above experiments for chloroquine. The
combination of these phenothiazine com-
pounds with suspensions of isolated melanin
granules was demonstrated in vitro, and
it was concluded that the melanin granules
were the site of the in vivo storage of
phenothiazines.
Relationship of chloroquine localization
to the development of retinopathy
The high levels of chloroquine in the
melanin granules of the choroid and pig-
ment epithelium would strongly suggest
that the initial pathologic alterations lead-
ing to the development of chloroquine
retinopathy originate in either one or both
of these areas. Potts12 has shown that the
two phenothiazine derivatives, NP-207 and
thioridazine, which have produced retinop-
athies, are also heavily concentrated in
the pigmented uveal tract of the eye.
The appearance of the fundus in chloro-

quine retinopathy is that of an unusual
pigmentary disturbance. In the perimacular
area there is often seen an oval patch of
depigmentation, the so-called "bulls-eye"
appearance (Fig. 4). A complete disappear-
ance of macula pigment has also been
observed (Fig. 5). In the peripheral ret-
ina there is usually a prominent choroidal
pattern with a fine gray or brown stippling
effect. There are no gross pigment deposits
as seen in retinitis pigmentosa.
Histopathologic examination of a case of
chloroquine retinopathy, which will be re-
ported in greater detail,13 shows a seg-
mental disappearance of rods and cones
with migration of the melanin pigment
from the pigment epithelium to the inner
nuclear layer (Fig. 6). The epithelium in
areas is completely depigmented, but other-
wise appears anatomically normal. In por-
tions of the retina where the rods are
present, the pigment epithelium is undis-
turbed and the pigment has the normal
fine granular appearance.
The rather characteristic fundus appear-
ance, the histopathologic picture, and the
high levels of chloroquine found in the
melanin-containing tissues of the eye, would
suggest strongly that the retinopathy is a
result, at least initially, of a disturbance of
melanin in the choroid and pigment epithe-
lium.
One of the characteristics of chloroquine
retinopathy is frequently a progressive loss
of visual acuity following discontinuation
of the drug. This may be related to the
prolonged retention of chloroquine in high
concentrations in the pigmented uvea, as
shown by its presence for at least 28 days
after a single intravenous dose in the
rabbit.
Methods for the rapid decrease of these
tissues stores of chloroquine would repre-
sent a rational approach to the treatment
of this condition. Administration of am-
monium chloride and BAL (2, 3, dimer-
captopropanol) has been shown to increase
chloroquine excretion.13 Whether this in-
creased excretion depletes chloroquine
from both uveal and systemic tissues or
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Ocular deposition of chloroquine 391
just the latter alone has still to be deter-
mined.
As originally shown by Potts, the prefer-
ential binding of certain ringed compounds
to pigmented uveal tissues or melanin pig-
ment is a new pharmacologic concept of
utmost importance to ophthalmology. Al-
though only certain of these compounds
have produced retinal damage (chloro-
quine, NP-207, and thioridazine), any drug
which is concentrated in the uveal tract
and pigment epithelium may be potentially
retinotoxic, especially in long-term use. As
new drugs are developed, it would
certainly appear advisable to determine
their concentration in the pigmented ocular
tissues. If elevated levels are found, the
ophthalmologist should be alerted to the
possibility of a drug-induced retinal toxicity.
These findings call attention to the role
of melanin pigment and the pigment epithe-
lium in the visual processes and in the
maintenance of the normal integrity of
the rods and cones. To quote Glockin and
Potts,14 "It has been well established from
clinical and experimental observations that
normal vision is dependent upon the
metabolic integrity of the pigment cell
epithelium and upon its physical contact
with the visual cells of the retina, but little
is known of the actual biochemical proces-
ses involved." It is anticipated that further
study of the biochemical effects of com-
pounds such as chloroquine may provide a
few of the answers to this problem.
We would like to gratefully acknowledge tlie
technical assistance of Mrs. Almuth Ewing in
this study.
REFERENCES
1. Hobbs, H. E., Sorsby, A., and Freedman,
A.: Retinopathy following chloroquine ther-
apy, Lancet 2: 478, 1959.
2. Okun, E., Gouras, P., Bernstein, H., and von
Sallmann, L.: Chloroquine retinopathy: A
report of eight cases with ERG and dark
adaptation findings, A. M. A. Arch. Ophth.
69: 59, 1963.
3. Rubin, M., Sister Agnes Mary Mansour, and
Zvaifler, N.: Fluorescence analysis of chloro-
quine (in preparation).
4. Berliner, R. W., Earle, E. P., Jr., Taggert,
 392 Bernstein el. al.
J. V., Zubrod, C. C , Welch, W. J., Cannon,
N. J., Bauman, E., Scudder, S. T., and
Shannon, J. A.: Studies of the chemotherapy
of the human malarias. VI. The physiological
disposition, antimalarial activity and toxicity
of several derivatives of 4-aminoquinoline,
J. Clin. Invest. 27: (Pt. 2) 98, 1948.
5. McChesney, E. W., and McAuIiff, J. P.:
Laboratory studies of the 4-aminoquinoline
antimalarials: I. Some biochemical character-
istics of chloroquine, hydroxychloroquine, and
SN-7718, Antibiotics & Chemother. 11: 800,
1961.
6. Fuhrmann, C , and Koenig, K.: Unter-
suchungen iiber die Resorptions und Aus-
scheidu ngsverhaltnisse von Resortren, Re-
sochin und Yatren, Ztschr. Tropenmed. u.
Parasitol. 4: 155, 1953.
7. Kuroda, K.: Detection and distribution of
chloroquine metabolites in human tissues, J.
Pharmacol. & Exper. Therap. 137: 148, 1962.
8. Rubin, M., Bernstein, H., and Zvaifler, N.:
Downloaded from iovs.arvojournals.org on 05/01/2020
vstigatioeOiilalwluwhtsu
August 1UC3
Studies on the pharmacology of chloroquine
with recommendations for the treatment of
chloroquine retinopathy, A. M. A. Arch.
Ophth. (In press.)
9. Parker, F., and Irvin, ].: The interaction of
chloroquine with nucleic acids and nucleo-
proteins, J. Biol. Chem. 199: 897, 1952.
10. Kurnick, M., and Radcliffe, I.: Reaction be-
tween DNA and quinacrine and other anti-
. malarials, J. Lab. & Clin. Med. 54: 669, 1962.
11. Fitzpatrick, T., Seiji, M., and McGugan, A.:
Melanin pigmentation, New England J. Med.
265: 374, 1961.
12. Potts, A. M.: Uveal pigment and pheno-
thiazine compounds, Tr. Am. Ophth. Soc. 60:
517, 1962.
13. Bernstein, N., and Ginsberg, J.: The ocular
pathology of chloroquine retinopathy, A. M.
A. Arch. Ophth. (In press.)
14. Glockin, V., and Potts, A.: The metabolism
of retinal pigment cell epithelium, INVEST.
OPHTH. 1: 111, 1962.