Site of ocular hydrolysis of a prodrug,

dipivefrin, and a comparison of its ocular

metabolism with that of the parent
compound, epinephrine
Janet A. Anderson, Wendy L. Davis, and Chau-Po Wei

Dipivefrin is a prodrug of epinephrine which is hydrolijzed to epinephrine after absorption into

the eye. The major site of this hydrolysis is shown to be the cornea. Some dipivefrin is absorbed
unchanged into ocular tissues, but most appears as epinephrine and its metabolites within 15
min after topical application. Metanephrine is the major metabolite of epinephrine found in
ocular tissues. It is found as soon as 15 min after application of either dipivefrin or epinephrine
and appears in all the tissues tested. The data indicate that the epinephrine which is liberated
by hydrolysis of topically applied dipivefrin is metabolized similarly to topically applied epi-
nephrine. The monoamine oxidase metabolites of epinephrine appear 1 to 3 hr after treatment
and are found mainly in the aqueous humor. After ocular application of either compound there
appear to be uptake and storage of the exogenous epinephrine in the iris plus ciliary body.
There is also some storage of unmetabolized epinephrine in the cornea.

Key words: prodrug, epinephrine, dipivefrin, dipivalyl epinephrine,

biotransfbrmation, ocular metabolism, esterases, catechol-o-methyl transferase,
monoamine oxidase

D ipivalyl epinephrine, or dipivefrin, is a

pivalic acid diester of epinephrine which ef-
fectively reduces intraocular pressure in hu-
mans and rabbits at concentrations 5% to
10% of the commonly used epinephrine con-
centrations.1* 2 This is probably due to the
lipophilic nature of the compound which al-
lows it to be more readily absorbed into the
eye than the parent compound, epineph-
From the Department of Ophthalmology, California Col-
lege of Medicine, University of California, Irvine.
Supported in part by Allergan Pharmaceuticals, Inc.,
Irvine, Calif.
Presented in part before the Third International Con-
gress of Eye Research, Osaka, Japan, May 1978.
Submitted for publication Dec. 1, 1978.
Reprint requests: Janet Anderson, Ph.D., Department
of Ophthalmology, California College of Medicine,
University of California, Irvine, Calif. 92717.
0146-0404/80/070817+07$00.70/0 © 1980 Assoc. for Res. in Vis. and Ophthal., Inc.
rine.3 Ten times as much radioactive material
is found in rabbit eyes 30 min after topical
application of 14C-dipivefrin as after topical
application of 14C-epinephrine.3 In human
eyes, 17 times as much drug penetrated the
cornea after dipivefrin treatment as after epi-
nephrine treatment.4
Dipivefrin is hydrolyzed to epinephrine in
ocular tissues in vitro5 and appears as epi-
nephrine in the aqueous humor after topical
application both in humans and rabbits.3" 4
We were interested in determining the site
of hydrolysis of the prodrug and in comparing
the ocular metabolism of the liberated epi-
nephrine to that of topically applied epi-
nephrine.
Materials and methods
The animals used in these studies were female
white New Zealand rabbits weighing from 2.0 to
817

818 Anderson, Davis, and Wei
Table I. Percent of radioactive material
extracted (mean ± S.D.)
Tissue 7- uC-Epinephrine 7- l4C-Dipivefrin
Cornea 81.67 ± 5.09 87.88 ± 4.15
(n = 12) (n = 13)
Iris plus 90.86 ± 5.62 91.75 ± 7.07
ciliary body (n = 12) (n = 11)
Aqueous humor 94.6 ± 5.8 94.6 ± 5.5
(n = 4)
3.2 kg (Curd's Caviary, La Puente, Calif.). Un-
labeled dipivefrin and 7-14C-dipivefrin (HCl salt,
1.0 mCi/mmol, MW 388) were obtained from
INTERx, Lawrence, Kans. 7-14C-epinephrine
(bitartartrate salt, 33 mCi/mmol, MW 333) was
obtained from Amersham Corp., Arlington
Heights, 111. Unlabeled epinephrine bitartrate was
obtained from 3M Laboratories, Ltd., Lough-
borough, England, Metabolites were from Sigma
Chemical Co., St. Louis, Mo., except for dihy-
droxymandelic acid and methoxyhydroxymandelic
acid from Calbiochem, La Jolla, Calif. All other
chemicals used were reagent grade.
Drugs were applied to the eyes in 50 /JL\ drops
from a micropipette. The eyedrop was placed
above the cornea and allowed to spread over the
surface of the eye. Then the eye was gently held
closed for 30 sec so that no blinking occurred.
Mass spectrometry. Dipivefrin hydrolyzes to
two compounds, one of which has been identi-
fied by two different thin-layer chromatographic
methods,'• 4 by high-pressure liquid chromatogra-
phy, 5 and by ultraviolet spectral analysis (unpub-
lished data) as epinephrine. The other compound
migrates in our thin-layer chromatographic sys-
tem 3 with an Rf of 0.73 and, judging from the
times of its appearance and disappearance, is an
intermediate in the hydrolysis of dipivefrin to epi-
nephrine. Both compounds are found after enzy-
matically catalyzed hydrolysis of dipivefrin and
also after the slow hydrolysis of dipivefrin in solu-
tion that occurs with time. A sample of dipivefrin,
0.5% in 0.2M citrate buffer, pH 2.5, which had
been made up 3 years previously and stored in a
sealed ampule under N2, was chromatographed as
previously described.'1 The section of the chroma-
togram with an Rf value of 0.7 to 0.8 was scraped
off the glass plate and eluted with 0.01N HCl in
methanol, and the eluted material was dried
under N2. This material was then analyzed by
low-resolution chemical ionization (isobutane)
mass spectrometry on an AEI MS 9.
Corneal hydrolysis studies. Rabbits were in-
Invest. Ophthalmol. Vis. Sci.
July 1980
jected intramuscularly with 0.5 ml of morphine
sulfate (1 mg/ml) followed by 1 to 2 ml of 5% so-
dium pentobarbital injected intravenously until
the animal was sufficiently anesthetized. Each eye
was then treated with a 50 /xl drop of a 0.1% solu-
tion of 14C-dipivefrin in 0.01N acetate buffer, pH
4.0. After 2 min, the eyes were washed with sa-
line. From 4 to 18 min after treatment, the cor-
neas were removed from the anesthetized ani-
mals, washed with saline, and quickly placed in
cold 0.01N HCl in methanol. The time from
treatment to placement in acidic methanol was de-
termined. The corneal tissue was extracted two
times in 3 ml of acidic methanol in a ground-glass
homogenizer. The extraction solutions were com-
bined and centrifuged at 4° C for 30 min at
1000 X g. The extracted corneal tissue and pellet
were combined and solubilized in NCS (Amer-
sham) at 55° C overnight. The samples were
counted and corrected for quenching as previously
described. 3 The extraction fluid supernatant was
reduced in volume under a stream of N2 as was a
control sample of 14C-dipivefrin in 0.01N HC1-
methanol. The samples were chromatographed,
and distribution of radioactivity was determined as
previously described. 3 The percent of radioactive
material extracted was 88.4% ± 5 . 0 (mean ±
S.D.) of the total amount in the cornea. Rates of
corneal hydrolysis were also determined in a
group of unanesthetized animals. These animals
were sacrificed with a peripheral ear vein injection
of 35% sodium pentobarbitol immediately before
removal of the corneas.
When the role of the corneal epithelium in the
hydrolysis of dipivefrin was determined, the rab-
bits were not given a general anesthetic but were
given 1 drop of the local anesthetic, propara-
caine (Ophthetic; Allergan Pharmaceuticals, Ir-
vine, Calif.), in each eye. The surface of one cor-
nea was scraped with a No. 11 scalpel blade until it
became dull-appearing and smooth. At this time, a
5% solution of collodion in ether and alcohol was
applied to the eye, and the film of collodion was
pulled off in order to remove any remaining epi-
thelial cells. The surface of the eye was rinsed with
normal saline and blotted dry. After 2 min, a 50 /xl
drop of a 0.1% solution of 14C-dipivefrin in 0.01N
acetate buffer, pH 4.0, was placed on each eye.
Each eye was rinsed with normal saline 2 min after
drug application. The animal was sacrificed as de-
scribed above, and the corneas were rapidly re-
moved. After removal, the corneas were ex-
tracted, and the extract was chromatographed as
described above.
Volume 19
Number 7
Table II. Corneal hydrolysis of dipivefrin'1
Dipivefrin
Rate of disappearance 0.262 ± 0.043 min-'
Rate of appearance
Time when 50% of radioactive 6.08 min
material is in this form
"Twelve eyes from seven rabbits were tested. Mean values ± S.D. are given.
Table III. Rates of corneal hydrolysis in scraped and control eyes (min-1)
Corneal hydrolysis rates
Pretreatment of eye Dipivefrin
Epithelium removed (n = 6) 0.028 ± 0.024
Control (n = 6) 0.109 ± 0.029
Significance of difference p <0.05
between scraped and
control eyes*
* Determined by analysis of covariance.
t Assuming that the data from the control and scraped eyes had the same intercept values.
The rates of hydrolysis were calculated with the
formula for a first-order rate of loss. In order to
eliminate scatter in the hydrolysis rate calculations
due to variations in absorption between animals,
the calculations of corneal hydrolysis rates were
based on the percent of total radioactive material
in the cornea which was present as dipivefrin or its
hydrolysis products. In the calculation of the rate
of loss of dipivefrin alone or of dipivefrin plus
monopivalylepinephrine (MPE), the natural loga-
rithm of the percent of the total radioactive mate-
rial in the cornea appearing as these compounds
was plotted against time, and the slope of the re-
gression was determined. The rate of appearance
of hydrolyzed material was determined by cal-
culating the difference between the percent of ra-
dioactive material present as dipivefrin plus MPE
in the applied drop and the percent of radioactive
material found as epinephrine and metanephrine
in the cornea at various times after drug applica-
tion. The slope was calculated as described above.
With the use of this method of analyzing the
data, the assumptions are made that (1) all com-
pounds were quantitatively extracted from the
cornea and (2) there was no change in the total
concentration of radioactivity due to absorption
into or elimination from the cornea over the ex-
perimental period. Since we were able to extract
88% of the radioactive material from the cornea
and since both dipivefrin and epinephrine were ex-
tracted in large amounts (80% to 90% of the total
radioactivity) at different times, the first assump-
tion is probably correct. The second assumption
Ocular hydrolysis of a prodrug, dipivefrin 819
Dipivefrin + MPE Epinephrine + metanephrine
0.176 ± 0.028 min-
0.156 ± 0.024 mill" 1
8.63 min 8.64 min
Rates of appearance of
Dipivefrin + MPE epinephrine and metanephrine
0.032 ± 0.012 0.033 ± 0.012
0.098 ± 0.028 0.102 ± 0.029
p <0.05 p = 0.07
(p = 0.0031)
clearly is not true, but experimental conditions
were selected to reduce the concentration changes
due to absorption and elimination. The eyes were
washed copiously with saline 2 min after drug ap-
plication to remove unabsorbed drug. The period
of the experiment was short, so that loss of drug
due to elimination was not large. The rate of loss of
radioactive material from the cornea calculated
from previous data3 indicates that in 18 min, 12%
of the initial radioactivity would be lost by elimi-
nation. The actual variation in the total radio-
activity present in the corneas tested was not
large (mean ± S.D. = 4.56 ± 1.41 x 103 cpm),
and there was a very low correlation of loss of
radioactive material with time (r = 0.31).
Ocular metabolism studies. Rabbits were treat-
ed in each eye with a 50 fxl drop of either a 0.1%
solution of 14C-dipivefrin in acetate buffer, pH 4.0,
with a specific activity of 2.58 /x-Ci/ml or a 0.1%
solution of 14C-epinephrine in the same buffer
with a specific activity of from 25 to 40 fiCi/m\.
The animals were sacrificed as previously de-
scribed, and the aqueous humor, cornea, and iris
plus ciliary body were removed in that order. Tis-
sues were homogenized two times in 2 ml of 0.01N
HCl-methanol with a ground glass pestle in a
ground-glass tissue grinder kept in ice. The solu-
tions were combined, centrifuged for 30 min at
1000 x g at 5° C. The supernatants were filtered
on a glass fiber filter (Whatman GF/C), and the
filter was washed with sufficient 0.01N HCl-
methanol to bring the final volume to 5 ml. One
milliliter of filtrate was taken for radioactivity de-
 Invest. Ophthalmol. Vis. Sci.
820 Anderson, Davis, and Wei July 1980

60 m

-« cornea
aqueous humor
—» iris and ciliary body

Fig. 1. Thin-layer chromatographic distribution of radioactive material extracted from ocular

tissues at various times after treatment with 7-14C-epinephrine. The average values of several
experimentally determined values are given. Vertical bars represent S.E.M. Where no verti-
cal bar is shown, the S.E.M. is smaller than the symbol. The number of samples tested was
three except in the following cases: aqueous humor—15 min (n = 4), 30 min (n = 5), 60 min
(n = 5), 3 hr (n = 4); cornea—60 min (n = 4), 3 hr (n = 2).

termination. The remaining filtrate was reduced in
volume under a stream of N2, and the concen-
trated filtrate was chromatographed as described
previously/' Acidified methanol (0.025 ml) was
added to the aqueous humor samples, which were
then centrifuged. The pellets were washed with
0.5 ml of 0.01N HCl-methanol, the supernatants
were combined, and a 0.2 ml sample was taken for
counting. The remainder was reduced under N2
and chromatographed. Standard drug solutions
were chromatographed with every experiment.
No samples are reported if the total counts on the
chromatogram were less than 90 cpm above back-
ground.
The precipitates from each tissue homogenate
were solubilized in NCS at 55° C overnight, neu-
tralized with acetic acid, counted, and corrected
for quenching with the external standard channels
ratio. The average tissue recoveries are shown in
Table I. Occasionally there was low recovery of
radioactive material from a tissue. Data from such
tissues were discarded unless at least 75% of the
total radioactivity was extracted.
Results
Mass spectrometry. Material with an RF of
0.73 in our chromatographic system was iso-
lated from a partially hydrolyzed sample of
dipivefrin and analyzed for structure. Chemi-
cal ionization mass spectrometry and gas
chromatography data strongly indicated that
the unknown hydrolysate of dipivefrin is a
monopivalate ester of epinephrine (MPE).
The analytical data do not show which one of
the two possible esters is present or whether
both are present in the sample analyzed.
Corneal hydrolysis of dipivefrin. Corneas
taken from anesthetized rabbits after topical
application of 14C-dipivefrin were extracted
and chromatographed. Dipivefrin migrated
with an Rf of 0.91 in this system. The rate of
disappearance of dipivefrin in these extracts
with time is shown in Table II. The percent
of radioactive material migrating with an Rf
value similar to that of MPE increased ini-
tially and then began to disappear. The per-
cent of radioactive material appearing in the
epinephrine and metanephrine portions of
the chromatogram increased with time. The
times at which 50% of radioactive material
could be expected to appear in the various
forms are given in Table II. These times are
later than the half-times calculated from the
hydrolysis rates, probably because of con-
Volume 19
Number 7 Ocular hydrolysis of a prodrug, dipivefrin 821

60m

—. cornea
aqueous humor
iris and ciliary body

Fig. 2. Thin-layer chromatographic distribution of radioactive material extracted from ocular

tissues at various times after treatment with 7-14C-dipivefrin. The average values of several
experimentally determined values are given. Vertical bars represent S.E.M. Where no verti-
cal bar is shown, the S.E.M. is smaller than the symbol. The number of samples tested was
three except in the following cases: aqueous humor—15 min (n = 2), 30 min (n = 5), 60 min
(n = 6); cornea—15 min (n = 5); iris plus ciliary body—15 min (n = 2).

tinued absorption of the unhydrolyzed di-
pivefrin at the early time points.
Corneal extracts from the five unanes-
thetized animals gave results similar to those
reported above. In these animals, dipivefrin
was hydrolyzed at a rate of 0.260 ± 0.056
min"1 (mean ± S.D.) and dipivefrin plus
MPE at a rate of 0.157 ± 0.015 min"1.
Role of the corneal epithelium in hydroly-
sis of dipivefrin. Eyes from which the epi-
thelium had been removed were treated with
14
C-dipivefrin, and the corneas were then
analyzed for the rates of hydrolysis of di-
pivefrin and MPE. One eye of each rabbit
did not have the epithelium removed and
was used as a control. The rates of hydroly-
sis and the appearance of epinephrine and
metanephrine are shown in Table III. The
differences between the control and de-
epithelized eyes were significant as deter-
mined by analysis of covariance. The rate of
hydrolysis decreased about threefold when
the corneal epithelium was removed. The
rates of hydrolysis in the control eyes were
lower than those observed in the previous
studies. The decreased rates may be due to
the presence of the local anesthetic propara-
caine which was not used in the previous
studies.
Ocular metabolism. Results of thin-layer
chromatography of the tissue extracts are
presented graphically in Figs. 1 and 2. Fif-
teen minutes after topical application of epi-
nephrine, most of the drug in the ocular tis-
sues was present as epinephrine. Thirty
minutes after epinephrine treatment, the
percent of radioactive material present as
epinephrine was still high, with a portion ap-
pearing as metanephrine. In the cornea,
there was also some more rapidly moving ma-
terial appearing where the metabolites di-
hydroxyphenylglycol and dihydroxymandelic
acid migrate. Sixty minutes after treatment,
the aqueous humor had an increased percent
in the metanephrine area plus significant
amounts in the sections where methoxyhy-
droxyphenylglycol (MHPG) and methoxyhy-
droxymandelic acid (VMA) migrate. After 3
hr, the cornea and iris plus ciliary body had
very little material that did not chromato-
graph like epinephrine. In the aqueous hu-
mor, the percent of radioactivity present as
 Invest. Ophthalmol. Vis. Sci.
822 Anderson, Davis, and Wei July 1980

Table IV. Percent of applied dose present as epinephrine in ocular tissues

Treatment
Dipivefrin Epinephrine Dipivefrin/
(% of dose as epinephrine) (% of dose as epinephrine) epinephrine
30 min:
Cornea 2.112 0.108 19.6
Iris/ciliary body 0.241 0.121 2.0
Aqueous 0.091 0.048 1.9
Total 2.444 0.277 8.8

60 min:
Cornea 1.336 0.090 14.8
Iris/ciliary body 0.363 0.119 3.0
Aqueous 0.178 0.019 9.4
Total 1.877 0.228 8.2

3 hr:
Cornea 0.124 0.045 2.8
Iris/ciliary body 0.094 0.085 1.1
Aqueous 0.032 0.007 4.6
Total 0.250 0.137 1.8

MHPG and/or VMA had increased greatly.
Fifteen minutes after application of 14C-
dipivefrin, most of the radioactive material
present in the cornea migrated like epineph-
rine or metanephrine. The aqueous humor
and iris plus ciliary body showed radioactivity
in the portions of the chromatogram where
dipivefrin and MPE migrate, but the major
portion of the radioactive material was found
in the slower moving areas where epineph-
rine and metanephrine migrate. Since more
than 86% of the total radioactive material in
the tissues tested was in the cornea at 15 min,
less than 10% of the radioactive material in
the ocular tissues tested was present as di-
pivefrin at this time. After 3 hr, most of the
radioactive material in the cornea and aque-
ous humor appeared as metanephrine. In the
aqueous humor, significant amounts of radio-
active material began to appear as MHPG
and VMA. The pattern in the iris plus ciliary
body was different, with most of the material
still chromatographing like epinephrine after
3hr.
In previously reported experiments,3 the
percent of drug absorbed into ocular tissues
after treatment with radioactively labeled
dipivefrin and epinephrine was measured.
When those data are combined with the data
available in this study on the metabolism of
these drugs in the eye, the percent of the
original dose present as the active drug, epi-
nephrine, in the various ocular tissues can be
calculated. The results of such calculations
are shown in Table IV.
Conclusions
Dipivefrin is hydrolyzed to epinephrine
shortly after entry into the eye. The cornea is
probably the major site of ocular hydrolysis,
since the rate of corneal hydrolysis is rapid
and very little dipivefrin appears in other
ocular tissues. The calculated rates of hydro-
lysis are probably underestimations, since
there was no correction for continued ab-
sorption into the cornea or elimination of ma-
terial from it. Since radioactive drug is ab-
sorbed as dipivefrin and eliminated mainly as
epinephrine from the cornea, changes in
drug concentrations due to these processes
would mask the effects of corneal hydrolysis.
Although there was a large reduction in the
rate of hydrolysis when the corneal epithe-
lium was removed, some hydrolytic activity
was still present in the stroma and/or en-
dothelium. Studies have shown that dipivef-
rin is rapidly hydrolyzed in human eyes also.
As in rabbit eyes, very little material appears
in the aqueous humor as dipivefrin after ocu-
lar application.4
Volume 19
Number 7 Ocular hydrolysis of a prodrug, dipivefrin 823
During the 3 hr following ocular applica-
tion of epinephrine, most of the radioactive
material found in the cornea and iris plus
ciliary body was present as epinephrine. The
expected metabolites were also present. En-
zymes which metabolize epinephrine have
been observed in ocular tissues.6' 7 Cate-
chol-o-methyl transferase activity has not
been previously demonstrated in the cornea,
but the appearance of metanephrine in this
tissue suggests that this enzyme is present in
the cornea.
During the first hour after treatment with
14
C-dipivefrin, the radioactive compounds
present in ocular tissues were similar to those
seen after 14C-epinephrine treatment. Three
hours after treatment with dipivefrin, the dis-
tribution of radioactive material was some-
what different from that in the epinephrine-
treated eye. This difference maybe related to
the fact that much more material is absorbed
into the eye after dipivefrin3 treatment. The
data suggest that the rate-limiting step in the
ocular metabolism of exogenous epinephrine
is the monoamine oxidase oxidation of meta-
nephrine.
Most of the radioactive material in the iris
plus ciliary body is present as epinephrine
after treatment with either epinephrine or
dipivefrin. These tissues have heavy concen-
trations of adrenergic fibers,8 and the seques-
tering of exogenous epinephrine is probably
related to the adrenergic innervation of the
tissues. Kramer and Potts9 have shown that
following carotid artery infusion of 3H-nor-
epinephrine, the major portion (>60%) of the
radioactive material in the iris and ciliary
body was present as the unmetabolized cate-
cholamine norepinephrine. These data and
ours suggest that exogenous catecholamines
are taken up and retained in the adrenergic
neurons in the iris and ciliary body in an up-
take process similar to uptake 1 described by
Iverson.10
There may also be uptake of exogenous
epinephrine into the adrenergic neurons of
the cornea.8 After epinephrine treatment,
most of the radioactive material which re-
mains in the cornea for the 3 hr tested is
unmetabolized, suggesting that it is pro-
tected from metabolism. The smaller percent
of radioactive material appearing in the cor-
nea as unmetabolized epinephrine 3 hr after
dipivefrin treatment may reflect saturation of
uptake due to the greater amount of drug
present after dipivefrin treatment.
Previous experiments3 have shown that 10
times as much radioactive drug is absorbed
into ocular tissues after dipivefrin treatment
as after epinephrine treatment. In the exper-
iments reported here, we have shown that
the amount of epinephrine present in ocular
tissues is also greater after dipivefrin treat-
ment, about nine times more at 30 min after
treatment. The results support the conten-
tion that the greater efficacy of dipivefrin in
reducing intraocular pressure compared to
epinephrine is due to the increased amount
of active drug present in ocular tissues after
dipivefrin treatment.
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