Prostaglandin F2a-l-Isopropylester Lowers Intraocular
`Pressure Without Decreasing Aqueous Humor Flow
`
`J. R. Kerstetter, M.S., R. F. Brubaker, M . D, S. E. Wilson, M . D .,
`and L. J. Kullerstrand, B.S.
`
`fluorophotometry, we performed a
`Using
`randomized, dose-response study of the ef­
`fects of a prostaglandin derivative on aqueous
`humor
`flow. Prostaglandin F 2(I-l-isopropyl-
`ester, 0.224 p,g, 0.448 p.g, and 1.120 p.g, in
`saline with polysorbate 80 was instilled into
`one eye of 20 subjects in three separate dose
`studies. Polysorbate 80 in saline was instilled
`in the fellow eye as a control. The drug had no
`measurable effect on aqueous humor flow or
`corneal endothelial permeability. Intraocular
`pressure measured eight hours after adminis­
`tration of the highest dose, 1.120 fig, was 20%
`lower in the treated eye as compared to the
`fellow eye (P < .001).
`
`PROSTAGLANDINS administered topically re­
`duce intraocular pressure in several species. 1
`For example, topical administration of prosta­
`glandin F2a (tromethamine salt) reduces intra­
`ocular pressure in rabbits.2 Lee, Podos, and
`Severin3 noted reductions in intraocular pres­
`sure in rabbits, cats, and monkeys after topical­
`ly administered prostaglandin F2„ (trometha­
`mine salt) in doses ranging from 250 to 1,000
`|xg. They observed no changes in aqueous
`humor flow as measured by fluorophotometry,
`but did observe significant increases in outflow
`facility as measured by tonography. Additional
`studies
`in cats and rhesus monkeys have
`shown that topically applied prostaglandin F2„
`(tromethamine salt) produces a transient (15- to
`
`Accepted for publication Sept. 15, 1987.
`From the Mayo Medical School (Mr. Kerstetter) and
`the Department of Ophthalmology (Drs. Brubaker and
`Wilson and Ms. Kullerstrand), Mayo Clinic and Founda­
`tion, Rochester, Minnesota. This study was supported
`by National Institutes of Health Grant EY 00634, Re­
`search to Prevent Blindness, Inc., and the Mayo Founda­
`tion.
`Reprint requests to Richard F. Brubaker, M.D., De­
`partment of Ophthalmology, Mayo Clinic, Rochester,
`MN 55905.
`
`30-minute) increase in intraocular pressure, 4
`followed by a persistent decrease in intraocular
`pressure without development of tachyphy-
`laxis when twice-daily doses are given.5
`A study of owl monkey eyes showed that
`1,000-u.g doses of prostaglandin F2o (trometha­
`mine salt) can reduce intraocular pressure by
`4.7 mm Hg in normal eyes and 25 mm Hg in
`glaucomatous eyes. 6 Crawford, Gabelt, and
`Kaufman7 showed that 100-u.g doses of topical­
`ly administered prostaglandin F2a (form not
`specified) produced an 8-mm Hg reduction in
`intraocular pressure and a reduction in aque­
`ous humor flow in some but not all of their
`experiments using cynomolgus monkeys. 7
`Camras and associates8,9 noted a 10-mm Hg
`reduction of intraocular pressure in the same
`species without an accompanying reduction of
`aqueous humor flow after a 250-u.g topical dose
`of prostaglandin F2a (tromethamine salt). These
`investigators attributed the effect to changes of
`outflow facility.
`In a study of 18 nonglaucomatous subjects,
`Giuffre10 administered a 200-u.g topical dose of
`salt) and
`prostaglandin F2a (tromethamine
`noted
`significant
`reductions of
`intraocular
`pressure in treated eyes as compared to control
`eyes. The studies cited here have shown consis­
`tent reductions of intraocular pressure in sever­
`al species with high doses of prostaglandin F2a.
`Bito and Barody11 have shown that the corne­
`al epithelium acts as a barrier to prostaglandin
`F2a. Bito12 has subsequently studied several es­
`ters of prostaglandin F2a and found that methyl,
`ethyl, and isopropyl esters reduce intraocular
`pressure in cats at much lower doses
`than
`prostaglandin F2(1. He also observed that the
`isopropyl ester of prostaglandin F2a provided
`longer-term effects than the methyl or ethyl
`esters.
`In a study of 0.1-, 0.5-, and 10-(xg doses of
`prostaglandin F 2t<-l-isopropylester in six nor­
`mal male volunteers, a dose-related reduction
`of intraocular pressure with a maximum reduc-
`
`30
`
`©AMERICAN JOURNAL OF OPHTHALMOLOGY 105:30-34, JANUARY, 1988
`
`Micro Labs Exhibit 1033
`Micro Labs v. Santen Pharm. and Asahi Glass
`IPR2017-01434
`
`

`

`Vol. 105, No. 1
`
`Lowered Intraocular Pressure With Prostaglandins
`
`31
`
`tion eight hours after instillation was noted (A.
`Aim, M.D., and J. Villumsen, M.D., personal
`communication, 1985). The mean reduction of
`intraocular pressure eight hours after instilla­
`tion was 5.7 mm Hg (P < .02). Villumsen and
`Aim13 also studied 0.5-u.g doses of prostaglan-
`din F 2a-l-isopropylester administered at 8 A.M.
`and 8 P.M. for one day to 12 glaucomatous
`patients. They observed intraocular pressure at
`eight, 12, and 24 hours after the first dose and
`noted mean reductions in pressure of 5.7 mm
`Hg (P < .001), 7.9 mm Hg (P < .001), and 7.3
`mm Hg (P < .005), respectively.
`The purpose of this study was to measure the
`rate of flow of aqueous humor in the human eye
`after the topical administration of a range of
`doses of prostaglandin F 2o-l-isopropylester
`known to lower intraocular pressure.
`
`Subjects and Methods
`
`We selected 20 volunteers (11 men and nine
`women), ranging in age from 21 to 60 years,
`with no history of eye disease or significant
`systemic disease for the study. Subjects were
`excluded from the study for any of the follow­
`ing: (1) active ocular disease, (2) anatomic dif­
`ferences in the size and color of their eyes, (3)
`systemic drug use, (4) inability to undergo
`satisfactorily tonometry or fluorophotometry,
`(5) suspected hypersensitivity to drugs, or (6)
`participation in a trial of any other investiga-
`tional drug within two weeks. The screening
`examination included a medical and ophthal­
`mic history, visual acuity testing, slit-lamp ex­
`amination, Goldmann applanation tonometry,
`and direct ophthalmoscopy. Written, informed
`consent according to federal guidelines was
`obtained from all subjects. Preliminary
`tests
`included photogrammetry to measure the vol­
`ume of the anterior chamber. 14 Autofluores-
`cence of each cornea was also measured. 10 Re­
`sults of blood tests for pregnancy were negative
`two to three days before beginning the study in
`all female subjects.
`Prostaglandin F 2o-l-isopropylester was used
`as an ophthalmic solution at a concentration of
`22.4 u.g/100 ml. The drug was instilled by mi-
`cropipette in 1-, 2-, and 5-JJLI doses, which
`contained 0.224, 0.448, and 1.12
`|xg of
`the
`investigational drug, respectively. The study
`was conducted under investigational new drug
`exemption No. 28,391 of the Food and Drug
`Administration.
`
`Ten left and ten right eyes were randomly
`assigned to the study group. Each subject re­
`ceived a single 1-, 2-, and 5-|xl dose of the test
`drug in the selected eye and a placebo (polysor-
`bate 80 in saline) in the fellow eye in three
`separate studies spaced by a minimum of two
`weeks.
`At 2 A.M. on the day of the study each subject
`instilled 2% fluorescein
`into each eye every
`three minutes for a total of five to ten instilla­
`tions per eye. The number of instillations was
`individualized for each subject to produce the
`optimal concentration of fluorescein
`in
`the
`stroma.
`Subjects reported to the testing area at 8 A.M.
`on the day of the study. Intensity and polariza­
`tion of fluorescein were measured in both cor­
`neas and anterior chambers using the two-
`dimensional
`scanning
`fluorophotometer of
`McLaren and Brubaker. 16 After these measure­
`ments, the drug and placebo were instilled into
`the lower cul-de-sac of the subjects by micropi-
`pette; a total volume of 1, 2, or 5 u.1 was
`delivered containing 0.224 u.g, 0.448 (jig, and
`1.120 (jig of prostaglandin F2„-l-isopropylester,
`respectively. The subjects were instructed to
`keep their eyelids closed for four minutes after
`instillation to retard washout of the drug by
`tears. Hourly measurements of fluorescein in
`the cornea and anterior chamber were
`then
`made on all subjects.
`Polarization of fluorescence was measured in
`each eye four to eight hours after instillation of
`the drug. At 4 P.M. on the day of the study
`(eight hours after drug instillation), intraocu­
`lar pressure was measured
`in each eye by
`Goldmann applanation tonometry by one of
`us (S.E.W.) who did not know which eye had
`been treated. The measurements were taken
`three times, beginning with the right eye and
`alternating between eyes, and then averaged.
`Pretreatment intraocular pressures were meas­
`ured in the same manner during the screening
`examination. Pretreatment
`intraocular pres­
`sures were not measured on the study day be­
`cause of the confounding effect that fluores-
`cein-containing topical anesthetics would have
`had on the measurement of aqueous humor
`flow by fluorophotometry. At the conclusion
`of the study, an ophthalmic examination was
`again performed. Each subject was queried
`about symptoms, and any symptoms reported
`were recorded.
`Aqueous humor flow was calculated by a
`modification17 of method 2 of Jones and Mau­
`rice.18 The rate of clearance of fluorescein was
`
`Micro Labs Exhibit 1033-2
`
`

`

`32
`
`AMERICAN JOURNAL OF OPHTHALMOLOGY
`
`January, 1988
`
`calculated as the rate of disappearance of fluo-
`rescein from the combined cornea and anterior
`chamber divided by the average fluorescent
`intensity in the anterior chamber during that
`interval. The rate of diffusional clearance of
`fluorescein was assumed to be 0.25 ui/minute. 19
`
`Results
`
`All 20 subjects completed the study of the 1-
`and 2-ui doses. However, two subjects com­
`plained of pain and photophobia and were
`noted to have marked conjunctival hyperemia
`for several hours after instillation of the 2-|xl
`dose. They were excluded from tests of the 5-uJ
`dose.
`Neither of the two smaller doses caused a
`significant reduction of intraocular pressure
`eight hours after
`instillation (Table 1). The
`highest dose produced an ocular hypotensive
`effect
`that was significant eight hours after
`instillation. Intraocular pressure was 11.8 ± 2.8
`mm Hg in the treated eye and 14.4 ± 3.0 mm Hg
`in the untreated eye, a difference of approxi­
`mately 20% (P < .001).
`We were unable to demonstrate any effect of
`the drug on the rate of aqueous humor flow.
`The flow was in the range of 2.5 to 2.9 |xl/
`
`TABLE 1
`EFFECTS OF PROSTAGLANDIN F2o-1-ISOPROPYLESTER
`ON INTRAOCULAR PRESSURE
`
`TABLE 2
`EFFECTS OF PROSTAGLANDIN F2(,-1-ISOPROPYLESTER
`ON AQUEOUS HUMOR FLOW
`(jLlL/MIN)*
`
`MORNING
`(8 A.M.
`-NOON)
`
`AFTERNOON
`(NOON-8 P.M.)
`
`AVERAGE
`(8 A.M
`-8 P.M.)
`
`DOSE
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`1 ix\ (N = 20)
`Drug
`Placebo
`2 id (N = 20)
`Drug
`Placebo
`5 M I (N = 18)
`Drug
`Placebo
`
`2.72
`2.86
`
`2.88
`2.78
`
`2.67
`2.52
`
`0.72
`0.77
`
`0.45
`0.62
`
`0.48
`0.47
`
`2.65
`2.66
`
`2.67
`2.65
`
`2.68
`2.50
`
`0.47
`0.50
`
`0.49
`0.47
`
`0.53
`0.40
`
`2.69
`2.76
`
`2.78
`2.72
`
`2.68
`2.51
`
`0.60
`0.64
`
`0.47
`0.55
`
`0.51
`0.44
`
`'Differences between drug-treated and placebo-treated eyes
`were not significant.
`
`minute under all conditions of the experiment.
`The rates of flow were generally higher in the
`morning than the afternoon in both the treated
`eye and the fellow eye (Table 2), a difference
`that can be attributed to the diurnal variation of
`aqueous humor flow in humans.
`The permeability of the corneal endothelium
`to fluorescein was not significantly altered by
`(Table 3),
`prostaglandin F 2a-l-isopropylester
`nor was there any evidence from polarization
`of fluorescence in the cornea or anterior cham-
`
`INTRAOCULAR PRESSURE (MM
`
`HG)
`
`PRETREATMENT
`
`8 HOURS
`POSTTREATMENT
`
`TABLE 3
`EFFECTS OF PROSTAGLANDIN F 2„-1-ISOPROPYL!
`ESTER
`ON ENDOTHELIAL PERMEABILITY
`x 10"4)
`(CM/MIN
`
`14.7
`14.1
`= 19
`N =
`
`2.6
`2.3
`
`14.4
`14.4
`= 19
`N =
`
`2.4
`2.5
`
`14.4
`14.6
`= 17
`N =
`
`2.3
`2.7
`
`12.9
`14.0
`
`2.6
`2.9
`■■ 20
`N =
`
`13.6
`13.4
`
`3.6
`2.7
`
`= 20
`N =
`
`2.8
`11.8*
`3.0
`14.4*
`■■ 18
`N =
`
`MORNING
`(8 A.M.-NOON)
`
`AFTERNOON
`(NOON-8 P.M.)
`
`AVE
`RAGE
`(8 A.M.
`-8 P.M.)
`
`DOSE
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`1 ill (N = 20)
`Drug
`Placebo
`2 ill (N = 20)
`Drug
`Placebo
`5 / il (N = 18)
`Drug
`Placebo
`
`3.56
`3.69
`
`3.79
`3.70
`
`3.57
`3.52
`
`0.96
`0.68
`
`0.63
`0.70
`
`0.58
`0.62
`
`4.00
`3.94
`
`3.66
`3.65
`
`3.99
`3.91
`
`0.72
`0.63
`
`0.58
`0.46
`
`0.48
`0.66
`
`3.78
`3.82
`
`3.73
`3.68
`
`3.78
`3.72
`
`0.84
`0.66
`
`0.61
`0.58
`
`0.53
`0.64
`
`DOSE
`
`1 ,x\ (0.224 /xg)
`Drug
`Placebo
`
`2 /nl (0.448 /xg)
`Drug
`Placebo
`
`5/il(1.12/ig)
`Drug
`Placebo
`
`'Difference between drug-treated and placebo-treated eyes
`was significant (P < .001) using two-tailed f-test.
`
`'Differences between drug-tested and placebo-treated eyes
`were not significant.
`
`Micro Labs Exhibit 1033-3
`
`

`

`Vol. 105, No. 1
`
`Lowered Intraocular Pressure With Prostaglandins
`
`33
`
`TABLE 4
`EFFECTS OF PROSTAGLANDIN F2„-1-ISOPROPYLESTER ON POLARIZATION OF FLUORESCENCE*
`
`PRETREATMENT
`
`POSTTREATMENT
`
`CORNEA
`
`ANTERIOR
`CHAMBER
`
`CORNEA
`
`ANTERIOR
`CHAMBER
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`MEAN
`
`S.D.
`
`0.167
`0.165
`
`0.154
`0.157
`
`0.171
`0.172
`
`0.036
`0.030
`
`0.022
`0.023
`
`0.023
`0.026
`
`0.031
`0.025
`
`0.023
`0.027
`
`0.026
`0.028
`
`0.023
`0.016
`
`0.016
`0.020
`
`0.014
`0.017
`
`0.176
`0.174
`
`0.175
`0.184
`
`0.188
`0.189
`
`0.043
`0.025
`
`0.023
`0.038
`
`0.022
`0.021
`
`0.033
`0.027
`
`0.019
`0.023
`
`0.026
`0.026
`
`0.018
`0.012
`
`0.011
`0.013
`
`0.015
`0.012
`
`DOSE
`1 Ail (N = 20)
`Drug
`Placebo
`2 id (N = 20)
`Drug
`Placebo
`5 MI (N = 18)
`Drug
`Placebo
`
`•Differences between drug-treated and placebo-treated eyes were not significant.
`
`ber that the drug had caused a breakdown of
`the blood-aqueous barrier (Table 4). In
`this
`experiment, polarization of
`fluorescence
`is
`used to measure any changes in the polariza­
`tion characteristics of the cornea or anterior
`chamber that would occur in response to the
`presence of additional proteins from break­
`down in the blood-aqueous barrier.
`Two subjects had moderate redness for sever­
`al hours and ocular discomfort after the 2-|i.l
`dose. Otherwise, the drug was well tolerated.
`All subjects had conjunctival hyperemia that
`lasted one to several hours after instillation.
`
`Discussion
`
`This study confirms the observations of oth­
`ers11 that low doses of topically applied prosta-
`glandin esters can reduce intraocular pressure.
`This pressure lowering effect must be the result
`of effects on outflow since we did not observe
`any decrease in the rate of clearance of fluores-
`cein. Since no significant changes in the rate of
`aqueous humor flow were noted, we calculated
`the statistical power of the experiment based on
`a previous study of 71 normal, untreated sub­
`jects, in whom the variance of our measure­
`ment technique was determined. In the present
`experiment where n = 20, the probability of
`detecting an 18% change in flow is 95%. There
`is a 50% chance that we would have detected
`
`changes in flow of approximately 10%. We be­
`lieve, as did Camras and associates 89 and Lee,
`Podos, and Severin, 3 that prostaglandin F 2a has
`no clinically significant effect on aqueous
`humor flow and that its ocular pressure lower­
`ing effect must be mediated via the outflow
`system.
`Compared to the four major classes of thera­
`peutic ocular hypotensive agents currently
`used
`for
`the
`treatment
`of
`glaucoma—
`cholinergics, adrenergic agonists, adrenergic
`blockers, and carbonic anhydrase inhibitors—
`prostaglandin F2a appears to have effects that
`are most similar to adrenergic agonists, such as
`epinephrine. First, epinephrine's effect may be
`the result of endogenous prostaglandin re­
`lease.2021 Second, both epinephrine 22 and pros­
`7 are thought to enhance uveo-
`taglandin F2a
`scleral outflow. However, ocular hypotensive
`response to prostaglandins may also be the
`result of unique mechanisms not shared by any
`currently used therapeutic agents.
`
`ACKNOWLEDGMENT
`Pharmacia AB, Uppsala, Sweden, supplied
`the prostaglandin F 2tt-l-isopropylester.
`
`References
`
`1. Bito, L. Z.: Prostaglandins, other eicosanoids,
`and
`their derivatives as potential antiglaucoma
`
`Micro Labs Exhibit 1033-4
`
`

`

`34
`
`AMERICAN JOURNAL OF OPHTHALMOLOGY
`
`January, 1988
`
`agents. In Drance, S. M. (ed.): Glaucoma. Applied
`Pharmacology in Medical Treatment. Orlando, Fla.,
`Grune and Stratton, Inc., 1984, chap. 20, pp. 477-
`505.
`2. Camras, D. B., Bito, L. Z., and Eakins, K. E.:
`Reduction of intraocular pressure by prostaglandins
`applied topically to the eyes of conscious rabbits.
`Invest. Ophthalmol. Vis. Sci. 16:1125, 1977.
`3. Lee, P. Y., Podos, S. M., and Severin, C: Effect
`of prostaglandin F2 alpha on aqueous humor dynam­
`ics of rabbit, cat, and monkey. Invest. Ophthalmol.
`Vis. Sci. 25:1087, 1984.
`4. Stern, F. A., and Bito, L. Z.: Comparison of the
`hypotensive and other ocular effects of prostagland­
`ins E2 and F2 alpha on cat and rhesus monkey eyes.
`Invest. Ophthalmol. Vis. Sci. 22:588, 1982.
`5. Bito, L. Z., Draga, A., Blanco, J., and Camras,
`C. B.: Long-term maintenance of reduced intraocular
`pressure by daily or twice daily topical application of
`prostaglandins to cat or rhesus monkey eyes. Invest.
`Ophthalmol. Vis. Sci. 24:312, 1983.
`6. Camras, C. B., and Bito, L. Z.: Reduction of
`intraocular pressure in normal and glaucomatous
`primate (Aotus trivirgatus) eyes by topically applied
`prostaglandin F2 alpha. Curr. Eye Res. 1:205, 1981.
`7. Crawford, K., Kaufman, P. L., and Gabelt,
`B. T.: Effects of topical PGF2(, on aqueous humor
`dynamics in cynomolgus monkeys. Curr. Eye Res.
`6:1035, 1987.
`8. Camras, C. B., Bhuyan, K. C, Rosenthal, J. S.,
`Podos, S. M., and Bhuyan, D. K.: Pathophysiolog-
`ical effects of prostaglandin (PG) F2o or epinephrine
`applied topically in monkeys. ARVO Abstracts. Sup­
`plement to Invest. Ophthalmol. Vis. Sci. Philadel­
`phia, J. B. Lippincott, 1985, p. 226.
`9. Camras, C. B., Podos, S. M, Rosenthal, J. S.,
`Lee, P. Y., and Severin, C. H.: Multiple dosing of
`prostaglandin F2o or epinephrine on cynomolgus
`monkey eyes. Invest. Ophthalmol. Vis. Sci. 28:463,
`1987.
`10. Giuffre, G.: The effects of prostaglandin F2
`alpha in the human eye. Graefes Arch. Clin. Exp.
`Ophthalmol. 222:139, 1985.
`
`11. Bito, L. Z., and Barody, R. A.: The penetration
`of exogenous prostaglandin and arachidonic acid
`into, and their distribution within, the mammalian
`eye. Curr. Eye Res. 1:659, 1982.
`12. Bito, L. Z.: Comparison of the ocular hypoten­
`sive efficacy of eicosanoids and related compounds.
`Exp. Eye Res. 38:181, 1984.
`13. Villumsen, J., and Aim, A.: The effect of pros­
`taglandin F2„ eye drops in open angle glaucoma.
`ARVO Abstracts. Supplement to Invest. Ophthal­
`mol. Vis. Sci. Philadelphia, J. B. Lippincott, 1987, p.
`378.
`14. Johnson, S., Passmore, J. A., and Brubaker,
`R. F.: The fluorescein distribution volume of the
`anterior chamber. Invest. Ophthalmol. 16:633, 1977.
`15. Brubaker, R. F.: The flow of aqueous humor in
`the human eye. Trans. Am. Ophthalmol. Soc. 80:391,
`1982.
`16. McLaren, J. W., and Brubaker, R. F.: A two-
`dimensional scanning ocular
`fluorophotometer.
`In­
`vest. Ophthalmol. Vis. Sci. 26:144, 1985.
`17. Brubaker, R. F., and McLaren, J. W.: Uses of
`fluorophotometry
`in glaucoma research. Ophthal­
`mology 92:884, 1985.
`18. Jones, R. F., and Maurice, D. M.: New meth­
`ods of measuring the rate of aqueous flow in man
`with fluorescein. Exp. Eye Res. 5:208, 1966.
`19. Brubaker, R. F.: Clinical evaluation of the cir­
`culation of aqueous humor. In Duane, T. D. (ed.):
`Clinical Ophthalmology. Philadelphia, Harper and
`Row Publishers, 1986, chap. 46.
`20. Camras, D. B., Feldman, S. G., Podos, S. M.,
`Christensen, R. E., Gardner, S. K., and Fazio, D. T.:
`Inhibition of the epinephrine-induced reduction of
`intraocular pressure by systemic indomethacin in
`humans. Am. J. Ophthalmol. 100:169, 1977.
`21. Bhattacherjee, P., and Hammond, B. R.: Effect
`of indomethacin on the ocular hypotensive action of
`adrenalin in the rabbit. Exp. Eye Res. 24:307, 1977.
`22. Townsend, D. J., and Brubaker, R. F.: Immedi­
`ate effect of epinephrine on aqueous formation in the
`normal human eye as measured by fluorophotome­
`try. Invest. Ophthalmol. Vis. Sci. 19:256, 1980.
`
`OPHTHALMIC MINIATURE
`One case of acute and two cases of chronic glaucoma were treated by
`excisions of a portion of the iris in each eye. The acute case completely
`recovered with capillary hemorrhage; the two chronic cases are improving.
`In cases in which the operation has been successfully performed,
`the
`aqueous humour is not accumulated in the anterior chamber, but appears
`to escape through the wound as it is secreted. No active
`inflammatory
`symptoms have occurred after treatment as one would expect after so
`severe an operation.
`
`British Medical Journal, March 6, 1858, p. 181
`
`Micro Labs Exhibit 1033-5
`
`