Petitioner: Haag-Streit AG
`Petitioner: Haag-Streit AG
`
`Ex. 100(cid:28)
`
`EX. 1009
`
`

`

`.
`Unlted States Patent
`
`Peyman et al.
`
`[19]
`
`.
`
`lllllllllllllllllllllllllllllllllllll|||||Illllllllllllllllllllllllllllllll
`USOOS346689A
`[11] Patent Number:
`
`5,346,689
`
`[45] Date of Patent:
`
`Sep. 13, 1994
`
`[54] METHOD OF PERFORMING
`ANGIOGRAPHY
`
`. ” Ophthalmic Surgery, vol.
`.
`Dyes for Angiography .
`21(4), Apr. 1990, pp. 250-257.
`
`[76]
`
`Inventors: Gholam A- Peyman;'Bahram
`Khoobehi, both of 2020 Gravier St.,
`Ste. B: New Orleans, La. 70112'2234
`[21] Appl. No.: 111,294
`
`Primary Examiner—Gary E. Hollinden
`Assistant Examiner-John D. pak
`Attorney, Agent, or Firm—Haverstock, Garrett &
`Roberts
`
`[22] Filed:
`
`Aug. 24, 1993
`
`[57]
`
`ABSTRACT
`
`[60]
`
`Related U-S- Application Data
`Division of Ser. No. 880,301, May 9, 1992, Pat. No.
`5,266,302, which is a continuation of Ser. No. 592,190,
`OCt- 37 1990’ abandoned.
`
`Int. 01.5 ................................... A61K 49/00
`[51]
`[52] us. c1. ......................................... 424/7.1; 424/4;
`424/9; 128/654; 514/453; 514/411
`....... 424/9, 7.1, 4; 514/453,
`514/411; 128/654
`
`[58] Field of Search
`
`[56]
`
`References Clted
`PUBLICATIONS
`
`A method of performing angiography of the occular
`fundus of an eye of a patient includes the steps of inject-
`ing intravenously calcein into the patient in an effective
`amount immediately prior to angiography and perform—
`ing angiography on the patient. The invention further
`discloses a method of performing photoooagulation
`therapy and/or photodynamic therapy and angiogra-
`phy of the ocular fundus of an eye of a patient which
`includes the steps of performing photocoagulation ther-
`apy, injecting intravenously calcein into the patient in
`an effective amount immediately prior to angiography,
`and performing angiography on the patient.
`
`Fang et a1. “Comparative Study of Three Fluorescent
`
`7 Claims, No Drawings
`
`

`

`1
`
`5,346,689
`
`2
`
`METHOD OF PERFORMING ANGIOGRAPHY
`
`SUMMARY OF THE INVENTION
`
`In order to increase the circulation decay time and to
`permit simultaneous angiography and photocoagulation
`therapy a method of performing angiography is pro-
`vided which includes injecting intravenously calcein
`into the patient and performing angiography on the
`patient. The method of the present invention also allows
`laser photocoagulation therapy to be performed prior to
`angiography when calcein is used as the fluorescent
`dye.
`The flourescent indicator substance to be used in the
`method of performing angiography of the present in—
`vention is calcein. Calcein is a water-soluble, self-
`quenching compound that is inexpensive, stable, and
`highly fluorescent. Calcein is prepared by the interac-
`tion of fluorescein, formaldehyde and iminodiacetic
`acid. The resulting compound has the structure:
`
`R
`
`OH
`
`R
`
`HO
`
`0
`
`O
`
`C\
`/
`C
`\O
`
`\ \
`
`Wherein R is: —CH2—N
`
`CHz—COZH
`
`CHz—COZH
`
`Calcein has a maximum abxorption occurring at 495
`nm and maximum emission at 515 nm. Calcein has a
`molecular weight of 622 and a lipid solubility that is less
`than that of sodiuum fluorescein. Calcein is a highly
`negatively charged molecule in neutral pH and does not
`easily leave the wall of the blood vessel. A detailed
`description of the properties of calcein can be found in
`Diehl, Calcein, Calmagite and 0-0’-dihydroxyazoben-
`zene: Titrz'metric, Colormetric and Fluorometric Reagents
`for Calcium and Magnesium, G. Frederick Smith Chem-
`ical Company, Columbus, Ohio, 1964.
`Accordingly, it is an object of the invention to pro-
`vide a method which permits simultaneous photocoagu-
`lation therapy and angiography to be performed.
`It is a further object of the present invention to use
`calcein in a method of performing angiography of the
`ocular fundus of the eye.
`These and other objects and advantages of the pres-
`ent invention will become apparent to those skilled in
`the art after considering the following detailed specifi-
`cation.
`
`DETAILED DESCRIPTION OF PREFERRED
`EMBODIMENTS
`
`The following sets forth summaries of a number of
`laboratory and animal studies which demonstrate the
`principles of the present invention, namely, that use of
`calcein as the fluorescent dye in angiography provides
`unexpected results because calcein has a longer decay
`time than other dyes used for this purpose and the use of
`calcein as the fluorescent dye in angiography after pho-
`tocoagulation therapy provides unexpected results be-
`cause calcein has less leakage or staining than other
`
`“This is a divisional of copending application Ser.
`No. 07/880,301 filed on May 9, 1992, which was a con-
`tinuation of application Ser. No. (17/592,190 filed on
`Oct. 3, 1990, now abandoned.”
`
`BACKGROUND OF THE INVENTION
`
`The present invention relates to a method of perform-
`ing angiography by using calcein as the fluorescent
`indicator substance and also to a method of performing
`photocoagulation therapy and angiography.
`Photography of the circulatory system of the eye and
`angiography of the ocular fundus require the absorption
`of a fluorescent indicator substance or dye into the
`blood as it flows through the vasculature of the eye.
`Investigators have reported the use of soduim fluores-
`cein (Naf), carboxyfluorescein (CF), indocyanine green,
`lissamine green, patent blue, Evans blue, and acridine
`orange as dyes suitable for angiography. Fluorescein
`angiography is one of the most improtant tools in diag-
`nosing retinal-chloroidal diseases. Sodium fluorescein
`and indocyanine green are the two agents currently
`used as indicators for angiography of the retinal and
`choroidal vasculature. Sodium fluorescein is the only
`fluorescent dye currently in clinical use.
`Sodium fluorescein has its light absorption peak near
`490 nm and it fluoresces maximally at 514 nm to 520 nm.
`The molecular weight of sodium fluorescein is 376 and
`it has a relatively high lipid solubility. Sodium fluores-
`cein is readily metabolized to fluorescein glucuronide,
`which is weakly fluorescent and easily crosses the
`blood—ocular barrier. Given these factors, angiograms
`taken with sodium fluorescein have a relatively short
`decay time in the retinal vasculature. Also, with sodium
`fluorescein, fluorescein leakage into the vitreous tends
`to obscure retinal and choroidal structures in later pha-
`ses which hinders or prohibits photocoagulation ther-
`apy prior to or after angiography.
`Carboxyfluroescein is another fluorescent dye which
`has been used for fundus angiography. Carboxyfluore-
`scein is a hydrophilic derivative of fluorescein. The
`light absorption peak of carboxyfluorescein is 490 nm
`and it fluoresces maximally at 520 nm. Carboxyfluore-
`scein has a molecular weight of 373. The main distinc-
`tion between carboxyfluorescein and sodium fluores-
`cein is that carboxyfluorescein has 1/1000 the lipid
`solubility of sodium fluorescein and thus is less likely to
`penetrate cell membranes. Studies of carboxyfluroes—
`cein used in fluorophotometry to investigate blood-ocu-
`lar barriers indicate that it may delineate certain abnor-
`malities of these barriers better than sodium fluorescein.
`
`Because carboxyfluorescein is not as readily glucuro-
`nated as sodium fluorescein, carboxyfluorescein also
`has been used as a tracer in quantitative physiological
`studies of the anterior chamber of human eyes.
`Although sodium fluorescein and carboxyfluorescein
`are suitable for angiography there is a need for a fluores-
`cent dye which has a longer circulation decay time than
`that of these and any other known fluorescent indicator.
`Additionally, there is a need for a fluorescent dye which
`has limited leakage to permit simultaneous angiography
`and photocoagulation therapy without obscuring the
`fundus view with leaking dye from the photocoagulated
`structure.
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`3
`dyes used for this purpose. The laboratory and animal
`studies discussed herein have been described in articles
`
`by the inventors herein which have appeared in Oph—
`thalmic Surgery, April 1990, Vol. 21, No. 4, pages
`250—257 and International Ophthalmology 142 245~250,
`1990, with both articles being incorporated herein by
`this reference.
`
`Calcein to be used as fluorescent dye for fundus angi-
`ography was obtained from Sigma Chemical Company,
`St. Louis, M0. The calcein is repurified by column
`chromatography. The method for repurifying calcein
`comprises the following steps. A 10 m1 of 100 mg/ml
`solution of calcein, having a pH of between 7.4 and 7.6,
`is applied to a 2.5 X40 cm column packed with Sepha—
`dex LH—20 to remove any hydrophobic impurities. Dis-
`tilled, deionized water is used to elute the dye at a flow
`rate of 1.0—1.2 ml/min. After elution, fractions of ap—
`proximately 2.0 ml are collected and the clear deep red
`fractions are combined. The fractions that preceded and
`followed the deep red fractions contained brown and
`green contaminants are discarded. After the deep red
`fractions are combined, the concentration of the com-
`bined fractions is determined by measurement of the
`absorption at 495 nm of aliquots of the dye diluted in
`methanol. The concentration of the dye is then adjusted
`to 50 mg/ml.
`Three experiments were performed using monkeys
`and rabbits. The first experiment consisted of perform-
`ing normal angiography in monkeys. The second exper-
`iment consisted of performing normal angiography in
`rabbits. The third experiment consisted of performing
`angiography in rabbits after laser photocoagulation of
`the eyes of the rabbits. Although laser photocoagulation
`therapy is described, it is also possible to perform photo-
`dynamic therapy. Photodynamic therapy includes in-
`jecting a dye into the eye and using a laser, such as a red
`or infrared laser, directed at the eye for the dye inside
`the eye to absorb the laser energy. It is to be understood
`that laser photocoagulation therapy includes photody—
`namic therapy. Two squirrel monkeys weighing 595 g
`and 620 g,
`respectively and six pigmented rabbits
`weighing from 1,265 g to 2,050 g were used in the ex-
`periments. A mixture of ketamine hydrochloride (30
`mg/kg body weight) and xylazine (3 mg/kg body
`weight) was used for intramuscular anesthesia. Propara-
`caine hydrochloride 0.5% was instilled into the eye
`prior to laser treatment. The pupils were dilated using
`phenylephrine hydrochloride 10%, tropicamide 1.0%,
`and cyclopentolate hydrochloride 0.5%. Sodium fluo-
`rescein was prepared from Fluorescite obtained from
`Alcon Laboratories of Fort Worth, Tex. Carboxy—
`fluorescein was obtained from Molecular Probes of
`Eugene, Oreg. Calcein was obtained from Sigma Chem-
`ical Company of St. Louis, M0. The sodium fluorescein
`and carboxyfluorescein were purified by column chro-
`matography according to a method reported by Ralston
`et al. in Biochim biophys Acta. 1981;649:133—137. The
`concentrations used in the experiments were sodium
`fluorescein 7 mg/ml, carboxyfluorescein 7 mg/ml, and
`calcein 11.3 mg/ml. All of the dyes had a molarity of 18
`mM. One ml/kg of each dye was injected into the ear
`vein in the rabbits and into the leg vein in the monkeys.
`The following equipment was utilized for the experi—
`ments. Fundus angiograms were taken using a Topcon
`fundus camera, Model TRC—WT. Although a fundus
`camera was used, a video camera may also be used.
`Filter devices were used for routine fluorescein angiog-
`raphy. Transmittance of these filters was measured
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`5,346,689
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`4
`using a spectrophotometer, such as Model DU40 manu-
`factured by Beckman of Fullerton, Calif. A flash inten-
`sity of 25 was used for the rabbits and a flash intensity of
`300 was used for the monkeys. Kodak TRI—X film
`(ASA 400) was used. All films were developed with
`identical processes. Fundus photocoagulation was per-
`formed using a Coherent Model920 laser which is an
`argon-blue green laser. Other lasers having other fre—
`quencies, such as red or infrared, may also be used. In
`all experiments at least 72 hours elapsed before angiog-
`raphy was repeated in any one animal.
`The first experiment, as previously discussed, con-
`sisted of performing normal angiography of monkeys.
`Angiography was performed following the administra-
`tion of an intravenous injection of one of the three dyes,
`sodium fluorescein, carboxyfluorescein, and calcein.
`After injection sequential photographs of the fundus
`were taken every few seconds during the first minute,
`the 2, 3, 5, 10, 15, 20, 30, 45, 60, 75, and 90 minutes.
`The second experiment, as discussed above, consisted
`of performing normal angiography of rabbits. The pro-
`cedure was similar to that of the first experiment. Angi—
`ograms were taken at the same intervals up to and in-
`cluding the one done 60 minutes after injection. Then
`photographs were taken up to 4 hours after injection.
`The third experiment consisted of performing angiog-
`raphy of rabbits after laser photocoagulation of the eyes
`of the rabbits. Three rabbit eyes were used for this
`experiment. Each eye was photocoagulated in two dif-
`ferent areas. The first area had multiple laser lesions
`created close to the optic disc in an attempt to occlude
`the major retinal vessels. The laser spot size was 200
`micrometers, the power was 500 to 600 mW, and the
`exposure time was 200 ms. The second area consisted of
`a group of 16 to 20 lesions of 200 micrometers apart.
`The power ranged from 120 to 200 mW and the expo-
`sure duration was 200 ms. This resulted in a homoge—
`nous whitish-yellow lesion. Thirty minutes after laser
`photocoagulation angiography was performed as in the
`second experiment.
`The results of the three experiments are as follows.
`Anglograms in the first experiment (normal angiogra-
`phy in monkeys) taken 17 seconds after the injection of
`sodium fluorescein showed a choroidal flush that be-
`came very bright and somewhat diminished visualiza-
`tion of the overlying retinal vessels. The choroidal flush
`was less intense with carboxyfluorescein and calcein
`which provided better contrast for visualization of the
`retinal vascular tree. Three minutes after injection the
`intensity of the choroidal flush was reduced in the eyes
`injected with sodium fluorescein and carboxyfluore—
`scein. The choroidal flush in the eyes injected with
`calcein was practically invisible at three minutes after
`injection. Retinal circulation was barely visible in any
`of the angiograms taken after 10 minutes, regardless of
`the dye used. Sixty minutes after injection the angio-
`grams taken with sodium fluorescein were completely
`dark. However, some fluorescence still could be seen in
`the retina vessels in the angiograms taken with carboxy-
`fluorescein and calcein.
`In the second experiment (normal angiography in
`rabbits) the choroidal flush in the early phase of angiog—
`raphy (O to 25 seconds) was most intense in soduim
`fluorescein injected eyes,
`less intense with carboxy-
`fluorescein injected eyes, and least intense with calcein
`injected eyes. One minute after injection all dyes pro-
`vided good visualization of the retinal and choroidal
`vasculature. The intensity of choroidal fluorescence in
`
`

`

`6
`to effectively extend the time for circulation decay to
`render the dye effective and too much calcein may have
`harmful side effects. The amount of calcein used in this
`
`invention will depend on the body weight of the mam-
`mal on which angiography is being performed. The
`calcein will be injected in a water solution which may
`or may not include other dyes. An effective amount of
`calcein will generally be at least 5 mg of calcein per kg
`of mammal body weight. Preferably, about 7 mg of
`calcein per kg of mammal body weight will be opti-
`mum.
`There has thus been shown and described a novel
`
`method of performing angiography which fulfills all of
`the objects and advantages sought therefor. It will be
`apparent to those skilled in the art, however, that many
`changes, modifications, variations, and other uses and
`applications of the subject method of performing angi-
`ography are possible and contemplated. All such
`changes, modifications, variations, and other uses and
`applications which do not depart from the spirit and
`scope of this invention are deemed to be covered by the
`invention, which is limited only by the claims which
`follow.
`What is claimed is:
`
`1. A method of performing photocoagulation therapy
`and angiography of the ocular fundus of an eye of a
`patient comprising the steps of:
`performing photocoagulation therapy on the eye of
`the patient;
`injecting intravenously a solution of purified calcein
`into the patient in an effective amount immediately
`prior to angiography; and
`performing angiography on the patient.
`2. The method of claim 1 wherein the step of per-
`forming angiography comprises the step of using a fun-
`dus or video camera.
`3. The method of claim 1 wherein the calcein is puri—
`fied by performing column chromatography.
`4. The method of claim 1 wherein the performing
`photocoagulation therapy step comprises using a laser.
`5. The method of claim 4 wherein the laser is an
`argon-blue green laser.
`6. The method of claim 1 wherein said effective
`amount is about 7 mg per kilogram of mammal body
`weight.
`7. The method of claim 1 wherein said calcein is in a
`water solution.
`
`*
`
`*
`
`*
`
`*
`
`*
`
`5,346,689
`
`5
`the sodium fluorescein injected eyes diminished only
`after five minutes, whereas it diminished after 16 sec-
`onds with carboxyfluorescein, and after 43 seconds with
`calcein. In the rabbits there was no difference in the
`circulation decay times of the three dyes.
`In the third experiment (angiography after laser pho-
`tocoagulation) anglograms taken from 22 to 33 seconds
`after dye injection showed more intense leakage around
`the occluded vessels (the first area) in the sodium fluo-
`rescein and the carboxyfluorescein injected eyes than in
`those injected with calcein. In the calcein injected eyes
`it was possible to detect the occluded vessel. Ten min-
`utes after injection a significant amount of dye leakage
`occurred in the first area of the sodium fluorescein
`injected eyes. In the carboxyfluorescein and calcein
`angiograms leakage was present but was less intense
`than it was in the sodium fluorescein angiograms. Angi-
`ograms of fresh lesions of the periphery of the retina
`(the second area) taken at 30 minutes after injection
`showed that soduim fluorescein had leaked into the
`tissues and it was difficult to visualize individual laser
`lesions. Similar changes occurred with carboxyfluore-
`scein but to a lesser degree. With calcein, the dye stain-
`ing in the photocoagulated area was minimal and indi-
`vidual lesions could be visualized distinctly.
`The experiments also showed that when calcein was
`injected into the anesthetized monkeys there was no
`injection reaction nor were acute toxic effects observed
`in the monkeys after repeated injections. Other experi-
`ments have been performed on rats in which a dosage at
`or below 170 mg/kg of calcein was found to be non-
`toxic.
`The above—described studies and experiments have
`shown that the method of performing angiography on
`the eye by injecting intravenously calcein into the pa-
`tient overcomes the disadvantages of the more com—
`monly used fluorescent indicator dyes. These experi-
`ments demonstrate the distinct characteristics found in
`anglograms which use calcein as the fluorescent indica-
`tor substance. Calcein has been shown to have a longer
`circulation decay time which permits detailed visualiza-
`tion of the retinal vasculature in the early phase of angi-
`ography. Additionally, calcein was shown to reduce
`leakage and staining of tissues immediately after photo-
`coagulation therapy.
`Calcein preferably may be used alone or may be used
`in combination with other dyes in combination with
`performing angiography. Enough calcein must be used
`
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`

`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`CERTIFICATE OF CORRECTION
`
`PATENTNO.
`
`: 5,316,689
`
`DATED
`
`: September 13, 1994
`
`|NVENTOR(S)
`
`: Peyman et a1
`
`It is certified that error appears in the above-indentified patent and that said Letters Patent is hereby
`corrected as shown below:
`
`
`
`Col. 4, line 42 "Anglograms" should be --Angiograms--.
`
`Col. 5, line 7 "anglograms" should be «angiograms».
`
`Col. 5, line 39 "anglograms" should be --angiograms--.
`
`Signed and Sealed this
`
`Fifieenth Day of November, 1994
`
`[Law
`
`BRUCE LEEMAN
`
`Commissioner of Parent: and Trademark:
`
`