Petitioner: Haag-Streit AG
`Petitioner: Haag-Streit AG
`
`Ex. 100(cid:24)
`
`EX. 1005
`
`

`

`United States Patent
`Laties et a1.
`
`[19]
`
`[54]
`
`OPHTHALMIC USE OF
`CARBOXYFLUORESCEIN
`
`[76]
`
`Inventors: Alan M. Laties, 2403 Spruce St.,
`Philadelphia, Pa. 19103; Richard A.
`Stone, 1720 Sue Ellen Dr.,
`Havertown, Pa. 19083
`
`[21]
`
`Appl. No.: 265,934
`
`[11]
`
`[45]
`
`4,350,676
`
`Sep. 21, 1982
`
`.
`
`[56]
`
`‘
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,306,820 2/1967 Krezanoski ............................. 424/7
`
`OTHER PUBLICATIONS
`
`Grimes et al., Biol. Abs., vol. 70, Nov. 15, 1980, Ab. No.
`68201.
`
`Pitet et al., Chem. Abs., vol. 78, 1973, Ab. No. 70919r.
`
`Primary Examiner—Anna P. Fagelson
`Attorney, Agent, or Firm—Caesar, Rivise, Bernstein &
`Cohen, Ltd.
`
`[22]
`
`Filed:
`
`May 21, 1981
`
`[57]
`
`ABSTRACI
`
`[51]
`
`[52]
`[58]
`
`Int. c1.3 ..................... A61K 49/00; GOIN 21/25;
`. GOIN 21/64
`............................................ 424/7; 424/9
`as. C].
`Field of Search ........................................ 424/7, 9
`
`A method of using carboxyfluorescein in ophthalmic
`studies comprising applying of the eye an effective
`amount of at least one active isomer of carboxyfluore-
`g scein.
`
`3 Claims, No Drawings
`
`

`

`1
`
`4,350,676
`
`OPHTHALMIC USE OF CARBOXYFLUORESCEIN
`
`This invention relates to the ophthalmic use of car-
`boxyfluorescein. A principal aspect of the present in-
`vention involves the use of carboxyfluorescein as an
`angiographic .dye to study the eye in health and disease.
`However,
`the present
`invention contemplates other
`ophthalmic uses of carboxyfluorescein, such as in topi-
`cal applications to the eye. The present invention fur-
`ther contemplates the use of carboxyfluoresceinin renal
`physiology andin liver function studies.
`The internal structures of the eye are transparent,
`allowing light to pass unimpeded from the cornea in the
`front to the light sensitive cells at the back, the outer
`part, of the retina. In order for light to go through to the
`light sensitive cells, the retina, for practical purposes, is
`transparent. The retinal blood vessels are both on the
`surface and within the retina; they are visible through
`the front of the eye when properly illuminated. They
`appear red1n color because of their content of blood.
`For the retina to obtain a proper supply of nutrients,
`such nutrients must either pass through the walls of the
`retinal blood vessels or through or between the walls of
`cells just outside the retina, called the pigment epithe-
`lium. In each case, different substances do or do not pass
`thrbugh. The fact that many substances are held back
`from passage is denoted by the term “blood-retinal
`barrier”. Thus, it might be said that the structures in-
`volved1n the blood-retina barrier are the lining cells of
`blood vessels and a continuous sheet of epithelial cells at
`the outer surface of the retina. The more general term
`“blood-ocular barrier” includes the blood-retinal bar-
`rier, but also includes blood vessels and epithelia in
`other parts of the eye that separate the intraocular con-
`tents from the blood, such as theiris blood vessels or
`ciliary body epithelium.
`The main clinical tool currently available for study-
`ing the functional integrity of the blood ocular barriers
`is the dye fluorescein. Fluorescein, has the chemical
`property of glowing bright green when illuminated
`with a blue light. Therefore, fluorescein can be injected
`into a vein in the arm, and as the fluorescein circulates
`it can be seen to flow through the blbod vessels of the
`eye. Using appropriate blue illumination, the blood
`vessels of the eye can be visualized with high contrast as
`the fluorescein flows through them This clinical test is
`called fluorescein angiography. This technique has been
`used to study retinal diseases and has recently been
`appliedin an attempt to study diseases of the front part
`of the eye as well.
`.
`It1s an essential property of the normal retinal blood
`vessels and the epithelium underneath the retina that
`they impede the diffusion of fluorescein into the eye. In
`short, fluorescein illustrates some properties of the
`blood-retinal barrier. In certain disease states, however,
`permeability may be altered; fluorescein leaks from the
`blood vessels of the retina or through the epithelium of
`the retina. The existence of such leakage is important
`diagnostically and therapeutically.
`Fluorescein has a long history of use in medicine but
`the prime reasons it has been applied to ophthalmology
`are that it is, (a) safe and (b) easily visualized. During
`the past twenty years fluorescein angiography has de-
`veloped into a powerful tool to study the blood-ocular
`barriers.
`.
`The question, of fluorescein permeability, however, is
`a complex one.
`
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`2
`Anatomical disruption of these epithelial and vascu-
`lar structures leads to abnormal fluorescein leakage. For
`instance, recently, with the application of vitreous
`fluorophotometry,
`it has been noted that fluorescein
`accumulation within the eye may occur in the absence
`of a specific anatomical defect.
`Instead, fluorescein
`penetration can result from functional alterations ofthe
`barrier cells.
`There are four main characteristics of a chemical that
`determine whether it will pass across the blood-ocular
`barriers. First, molecular size is important; smaller mol-
`ecules cross more readily Second, the tendency of a
`chemical to bind to plasma proteins affects passage.
`Third, substances with high lipid solubility pass more
`easily than those with a low lipid solubility Fourth, ,
`there are transport mechanisms at the cellular level for
`moving metabolites or nutrients one way or the other
`across these barriers; a chemical. structurally related to
`a naturally occurring chemical may move by such a
`transport mechanism.
`The physical properties of the fluorescein molecule
`and the nature of its interactions with the barrier cells
`complicate the interpretation of dye leakage. By virtue
`of size, that portion of intravascular fluorescein bound
`to plasma proteins or absorbed to red blood cells does
`not cross the blood-ocular barriers. In contrast unbound
`dye can diffuse to some extent through the plasma mem-
`branes of barrier cells. The tendency of fluorescein or
`any organic electrolyte to penetrate cell membranes1s
`related to its partition coefficient which reflects lipid
`solubility, to its dissociation coefficient (Kg); to its mo-
`lecular weight, and to pH (3——.5) Fluorescein, equili-
`brated between octanol and artificial sea water, has a
`partition coefficient of 0.6 indicating considerable dye
`solubility in the non-polar phase, and by inference con-
`siderable permeability in cell membranes. This rela-
`tively high partition coefficient is consistent with the
`observation that the half-time of fluorescein leakage
`from liposomes is only 5 minutes. Further, the pKa of
`the equilibrium between the divalent and monovalent
`forms of the fluorescein anion is 6.7, a value not far
`removed from physiological pH. Although most of the
`dye is in the divalent form at this pH, a significant
`amount of the less polar monoanion is also present.
`Small pH changesin the physiological range, by alter-
`ing the relative concentrations of the two ionic species,
`can be expected to change the membrane penetration.
`. Passive diffusion is not the only factor affecting trans-
`fer of unbound fluorescein across the blood-ocular bar-
`riers. The dye also serves as a substrate for an active
`transport system in the anterior uvea and probably in
`the posterior segment as well. In both regions the direc-
`tion of transport appears to be outward, that is, from
`eye to blood. This active process presumably opposes
`inward diffusion of fluorescein through barrier cell
`membranes, minimizing net transfer of dye from blood
`to intraocular structures in normal eyes.
`Fluorescein is handled by these mechanisms. There-
`fore, from the foregoing, it can be seen that the question
`of fluorescein permeability at the blood-ocular barriers
`and the true meaning of fluorescein “leakage” is indeed
`complex. As an indicator of barrier function fluorescein
`has a major drawback. It has a high lipid solubility at
`blood pH.
`Carboxyfluorescein has now been determined to
`overcome this shortcoming of fluorescein. Carboxy-
`fluorescein differs from fluorescein by the addition of a
`carboxyl group to the basic fluorescein molecule. As a
`
`

`

`3
`result, carboxyfluorescein is much more water soluble
`and much less lipid soluble than fluorescein. The factor
`here is approximately 1,000. This makes carboxyfluore-
`scein considerably less likely to cross the blood retinal
`barrier by solubility. The ability of carboxyfluorescein
`to pass the blood ocular barriers by specific cellular
`transport mechanisms seems to parallel fluorescein. The
`molecular size of fluorescein and carboxyfluorescein is
`about the same. Finally, the fluorescent properties of
`fluorescein, both wavelength and quantum efficiency
`are also present to an equal degree in carboxyfluore-
`scein, so that both dyes can be visualized as easily in the
`eye.
`
`Thus, carboxyfluorescein is a dye with better solubil-
`ity properties than fluorescein in the definition of bar-
`rier integrity while being similar both in visibility and
`transport quality. Since lipid solubility is the biggest
`drawback to fluorescein, carboxyfluorescein has now
`been determined to be a worthwhile replacement in
`ophthalmic diagnosis.
`Carboxyfluorescein has the following structural for-
`mula:
`
`HO
`
`¢o
`
`COOH
`
`COOH
`
`Carboxyfluorescein as sold commercially (Kodak) is
`a mixture of 4 carboxyfluorescein and 5 carboxyfluore-
`scein isomers. Either or both of these are active (active
`isomer) and it is conceivable that other isomers are
`active. Some commercial literature apparently incor-
`rectly names the compound as 5 (6) carboxyfluorescein
`or 6-carboxyfluorescein, but in any event the present
`invention involves only active isomers of carboxy-
`fluorescein.
`Prior uses for carboxyfluorescein include a technique
`wherein carboxyfluorescein is incorporated into small
`lipid vesicles (lipsomes) and the interaction of these
`lipsomes with cells in culture is studied. Another prior
`use involves performing microinjections of carboxy-
`fluorescein into individual cells in culture and studying
`the movement of this dye across open cell junctions.
`Both of these applications involve the use of cells in
`tissue culture and do not contemplate the potential ap-
`plication and development of. carboxyfluorescein as a
`dye for clinical diagnosis
`To determine whether a less membrane permeable
`dye than fluorescein has value as a probe of the struc-
`tural and functional integrity of the blood-ocular barri-
`ers we have undertaken studies of carboxyfluorescein, a
`compound with the same fluorescence characteristics as
`fluorescein, but with lower lipid solubility at physiolog-
`ical pH (7). In particular, the lipid solubility of carboxy-
`fluorescein was compared with fluorescein by measur-
`ing dye partitioning between octanol and aqueous
`buffer at different pH to determine solubility behavior
`relevant to the permeability of cell membranes in vivo.
`Studies were then made of the ocular distribution car-
`boxyfluorescein in rats after intravenous injection using
`quantitative fluorescence microscopy; and compared to
`the results for fluorescein. The plasma binding of car-
`
`4,350,676
`
`4
`boxyfluorescein was also studied and compared to fluo-
`rescein.
`
`Octanol/buffer partition ratios: The ratio of concen-
`trations of a given solute in equilibrium distribution
`between two_immiscible solvents is termed the partition
`coefficient. This expression properly refers only to the
`distribution of a single molecular species between the
`two phases. Both solutes studied exist as a mixture of
`ionized forms within the pH range tested. Total dye
`concentration in the two solvent phases was measured
`without correction for ionization or self-association and
`the recommended term, “partition ratio”,
`is used to
`refer to these uncorrected distributions.
`
`Carboxyfluorescein (Eastman Kodak Co., Rochester)
`or sodium fluorescein (Sigma Chemical Co., St. Louis)
`was dissolved at concentrations of 10—3 M or 10-5 M in
`octanol-saturated 0.1 M phosphate buffer at different
`pH values between 6.40 and 8.03. The aqueous dye
`solutions were equilibrated with equal volumes of buff-
`er-saturated octanol by 100 inversions during 5 minutes,
`and the phases were separated by centrifugation. Dye
`concentrations were measured spectrofluorophotomet-
`rically at excitation and emmision wavelengths of 487
`and 512 nm, respectively, with an Aminco-Bowman
`Ratio Spectrofluorophotometer.
`Two methods were used to calculate partition ratios.
`In the first, the dye concentration in the aqueous phase
`was measured before (Cb) and after (Ca) partitioning,
`and the ratio was obtained from the equation:
`
`RR. = Coot/Caq = (Cb~ Ca)/Ca,
`
`where Coct and Caq are the dye concentration in the
`octanol and aqueous phases respectively after equilibra-
`tion. In the second method, concentration in the octanol
`phase was determined by extracting the dye from octa-
`nol with an equal volume 0.1 N NaOH. Concentration
`in the octanol phase could not be measured directly
`because of the marked loss of fluorescence of both dyes
`in this solvent. A single base extraction of the octanol
`recovered 99% of the dissolved fluorescein and 99.9%
`of the dissolved carboxyfluorescein. Therefore, dye
`concentration in the first base extract was accepted as
`the concentration in the octanol phase and calculated
`the partition ratio from the equation:
`
`P.R.=Coct/(Cb—Coct)
`
`Both methods for determining the partition ratio gave
`similar results when more than 10% of the initial dye
`concentration was removed from the aqueous phase by
`partitioning. When very small quantities of dye were
`removed, measurement of concentration differences in
`the aqueous phase before and after partitioning became
`unreliable, and we used only the second method to
`calculate the ratio.
`Intra-ocular dye distribution: Young male Wistar rats
`(180—250 g) under pentobarital anesthesia (50 mg/kg
`i.p.) were injected intravenously with carboxyfluore-
`scein or fluorescein solutions (pH 7.4) at doses of 12.5,
`62.5 and 125 mg/kg. One minute after dye injection, the
`eyes were removed and immediately frozen in isopen-
`tane cooled to —— 110° C. by a liquid nitrogen bath. Be-
`tween 2 and 4 minutes after injection a blood sample
`from the abdominal aorta was drawn into a heparinized
`syringe for determination of the concentration of un-
`bound dye in plasma. The frozen eyes were dried under
`vacuum at —35° C. and embedded in paraffin. After
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`

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`4,350,676”
`
`6 .
`fluorescein and sodium fluoreScein solutions to plasma
`separated'from heparinized human blood so that the
`final dye concentration in the plasma was between
`10—3 M and 10‘6 M. The plasma/dye mixture was
`incubated for 2 hours at 37° C. Then using the dialysis
`and measuring procedures described above, the ratio of
`unbound dye to total dye in the plasma mixture was
`calculated.
`
`RESULTS
`
`Partition ratios: the measured octanol/buffer parti-
`tion ratios (Table 1) demonstrate that, over the pH
`range tested, carboxyfluorescein is approximately 1000
`times less soluble than fluorescein in the octanol phase.
`A hundred-fold change in dye concentration has no ,
`effect on the equilibrium distribution.
`TABLE I
`—-—-————___._—_____
`PARTITION RATIOS 0F CARBOXYFLUORESCEIN AND
`FLUORESCEIN AT DIFFERENT pH
`pH—-——————__.____________
`
`DYE
`CONCENTRATION
`CARBOXY
`,
`FLUORESCEIN
`
`7.38
`
`8.03
`
`6.42
`
`6.87
`
`10—3M
`10-5M
`FLUORESCEIN
`
`.033
`.037
`
`.0028
`.0030
`
`.0008
`.0007
`
`.00006
`*
`
`.038
`.51
`5.1
`35
`10-3M
`
`10-5M .030 33 4.9 .64
`
`
`
`
`Variation of pH, however, profoundly laters the parti-
`tion ratios; as pH falls, octanol solubility of both dyes
`increases. When the logarithm of the partition ratio is
`plotted against pH, linear relationships of similar slope
`are demonstrated for both carboxyfluorescein and fluo-
`rescein. Despite the exponential increase of partition
`ratios with decreasing pH, the ratios for carboxyfluoree
`scein remain well below 0.1 even at pH 6.4, the lowest
`value tested. Fluorescein, on the other hand, reaches a
`partition ratio of 1.0 at pH 7.25, not far removed from
`normal plasma pH.
`
`INTRAVENOUS USE OF CARBOXY
`FLUORESCEIN
`
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`embedding the eyes were stored under vacuum at —20° '
`C. to minimize the development of autofluorescence. '
`Additional eyes from normal rats not injected with dye
`were processed and stored identically for measurement
`of tissue autofluorescence. Immediately prior to exami-
`nation with the fluorescence microscope, sections were
`cut at a thickness of 12 microns, placed on glass slides,
`and mounted with xylene.
`,
`Fluorescence intensity in small areas of the tissue
`sections was measured with a 450 u fiber optic probe
`placed in the object plane of an ocular and connected to
`a photomultiplier tube. An amplified signal from the
`photomultiplier was recorded with a digital voltmeter.
`The illumination system consisted of a 50 W high pres-
`sure mercury light source and an epifluorescence con-
`densor equipped with a 455—490 nm exciter filter, a 510
`nm dichroic beam splitter, and a 520 nm barrier filter.
`All measurements were performed using a 63X oil ob-
`jective (N.A. 1.4). With this magnification, an aperture
`in the object plane restricted the area of illumination to
`acircle, 24 microns in diameter. The fiber optic probe
`covered a specimen field, 6 microns in diameter, cen-
`tered in the larger illuminated field.
`,
`Fluorescence intensity of four tissue layers was re-
`corded in the posterior segment: the inner plexiform,
`outer nuclear, and photoreceptor outer segment layers
`of the retina, and the choriocapillaris. Each of these
`layers is of sufficient width so that the illuminated area
`encompassed a homogeneous field. Foci of intense fluo-
`rescence, such as dye filled retinal capillaries, within the
`illumination field but outside the detection area of the
`fiber optic probe, can increase the recorded signal by
`light scatter into the detection area. Fields containing
`such bright foci were avoided. Examination of the sec-
`tion, location of the area for measurement, and focusing
`of the image Was accomplished under dim green illumi-
`nation from a tungsten lamp. The illumination was then
`switched to the fluorescence system, and the maximum
`value of fluorescence intensity was recorded as voltage.
`Measurements were made at 12 sites in each layer to
`obtain the average fluorescence intensities for an indi-
`vidual eye. These values were then corrected‘for tissue
`autofluorescence by subtracting the average fluores-
`cence intensity of each layer in eyes from animals not
`injected with dye.
`The response characteristics of the recording system
`are such that in the time required to‘reach a peak read-
`ing, significant fading occurs; the recorded value, there-
`fore, does not represent the maximum fluorescence
`intensity of the tissue. It was established, however, that
`the peak reading is linearly proportional to dye concen-
`tration in solution, and we assume that it is similarly
`proportional to dye concentration in tissue sections.
`The measurements obtained represent relative fluores-
`cence intensity only, and not absolute dye concentra-
`tion in tissue.
`Measurement of unbound dye in plasma: Plasma was
`separated by centrifugation from the heparinized blood
`obtained from rats injected with sodium fluorescein or
`carboxyfluorescein in the intraocular dye distribution
`study. The plasma was dialyzed for 24 hours at 37° C.
`against 0.1 M phosphate buffer at pH 7.4 using cellulose
`dialysis tubing with a 3500 molecular weight cut-off.
`Dye concentration was measured in the dialysate spec-
`trofluorophotometrically and from this value calculated
`the unbound plasma dye concentration.
`To study the fraction of unbound dye in human
`plasma,
`there was added small aliquots of carboxy-
`
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`
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`
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`
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`
`Intraocular dye distribution: the histological localiza-
`tion of fluorescence in sections from eyes of rats in-
`jected with carboxyfluorescein differs markedly from
`that found with fluorescein. At one minute after injec-
`, tion of either dye, brilliant fluorescence is seen within
`all blood vessels and throughout the stroma of the cho-
`roid and ciliary processes. In the anterior segment,
`however, cells of the ciliary epithelium which are
`brightly stained by fluorescein, show no detectable
`' fluorescence with carboxyfluorescein though the adja-
`cent intercellular spaces open to the stroma are well-
`filled by the dye. The iris stroma contains some carbox-
`yfluorescein, but the intensity of fluorescence is less
`than that seen with fluorescein; the iris epithelium, like
`the ciliary epithelium remains dark in these short-term
`experiments. In the posterior segment, no fluorescence
`is seen in retinal tissue following carboxyfluorescein
`injection, whereas, in comparison, a diffuse dim fluores-
`cence is seen in all layers of the retina in animals given
`fluorescein.
`
`Quantitative measurements of fluorescence intensity
`in the retina extend the impressions gained from visual
`inspection (Table II) herein below. Animals injected
`with carboxyfluorescein have concentrations of un-
`
`'
`
`’
`
`

`

`4,350,676
`
`.55 i- .03
`.29 i .01
`
`8
`7
`than for fluorescein, and ratios for both dyes are very
`bound dye in plasma which are significantly higher than
`sensitive to pH changes. Precise description of the parti—
`in animals injected with fluorescein. However, despite
`tioning behavior of such charged molecules is compli-
`high fluorescence intensity levels measured in the cho-
`cated not only by pH effects, but also by possible solute
`roid, there is virtually no detectable fluorescence in
`retinal tissue. The negligible values that are recorded in 5 association and variable hydration in different phases.
`the retinal layers could indicate a very low tissue con-
`The partition ratio does not fully characterize the distri-
`centration of carboxyfluorescein, more likely, they are
`bution of these two dyes, but the magnitude of the dif-
`an artifact caused by light scattered from flecks of dye
`ference between the partition ratios for carboxyfluore—
`displaced along the surface of the tissue section from cut
`scein and fluorescein is not only a valid reflection of the
`blood vessels.
`10 relative lipid solubilities, it also presages the compartive
`TABLE II
`FLUORESCENCE INTENSITY IN CHOROID AND RETINA AFTER INJECTION OF
`CARBOXYFLUORESCEIN AND FLUORESCEIN
`PLASMA UNBOUND DYE
`TISSUE RELATIVE FLUORESCENCE INTENSITY
`DOSE
`CONCENTRATION
`[MEAN i S.E.M.!‘I
`
`(MG/KG)
`(MG/ML)
`CHOROID
`OUTER SEGMENTS
`ONL
`IPL
`n
`CARBOXYFLUORESCEIN
`.006 i .003
`7.8 i .8
`125
`.008 i .001
`8.0 i .4
`62.5
`‘
`_
`FLUORESCEIN
`6
`.150 i .034
`.196 i .011
`.452 i .021
`5.7 i .5
`.29 i .01
`125
`6
`.076 i .004 .086 i .008
`.153 i .008
`2.7 i .2
`.18 i .01
`62.5
`
`
`
`
`
`12.5 6 .04 i .03 .57 i- .03 .032 i- .003 .011 i .001 .021 i .002
`
`°Corrected for tissue autofluorescence as described in the text.
`
`.003 i .001
`.002 i .001
`
`.002 i .001
`.001 i- .001
`
`4
`5
`
`In contrast to carboxyfluorescein, significant fluores- 25
`.
`cence intensity is measured in the retinas of all animals
`tissue distribution of the two dyes.
`injected with fluorescein. Importantly, the fluorescein
`The partition ratios for fluorescein are of particular
`levels of choriocapillaris and the retinal layers are pro-
`interest because of their high values in the physiological
`portional to the administered dose. Values recorded in
`the outer segment layer are 6-8% of those measured in 30 pH range. In studying the partitioning of fluorescein
`the choroid, and are, in turn, twice as high as those of
`between octanol and artificial sea water, a distribution
`the deeper retinal layers. Although fluorescence inten-
`ratio of 0.6, was found very close to what we measured
`sity to dye concentration cannot be precisely related,
`at pH 7.38. In aqueous solution, fluorescein can exist in
`the high levels recorded at the photoreceptor outer
`four forms; the dianion, monoanion, neutral molecule,
`segment. layer indicate a concentration gradient across 35 and the cation, with respective pKa values of 6.7, 4.4,
`the retina, and suggest that much of the dye enters the
`and 2.2. The dianion and monoanion forms predominate
`retina by Way of the pigment epithelium.
`within the pH range of 6.4 to 8.0, and the marked in-
`Though the measurements of plasma unbound dye
`crease of partition ratio with decreasing pH in this inter-
`concentration in rats (Table II) precludes extensive
`val indicates that the monoanion is very much more
`plasma binding as the cause of the very low ocular 40 lipid soluble than the dianion. However, the rate of
`penetration of carboxyfluorescein, the binding of the
`increase of the partition ratio is greater than would be
`two dyes in plasma obtained from human blood (Table
`expected simply from an increase in concentration of
`III) hereinbelow was further related. Large amounts of
`the monoanion as estimated from pKa and pH. This
`free carboxyfluorescein and free fluorescein are present
`discrepancy suggests that other undetermined factors
`over the entire dye concentration range tested, and 45 increase dye transfer to the non-polar phase. Ionization
`carboxyfluorescein is consistently less bound to human
`constants for carboxyfluorescein are not available to
`plasma proteins than fluorescein.
`our knowledge, but, on the basis of partition ratios, the
`
`TABLE III
`additional carboxyl substituent probably has-a pKa low
`enough to provide
`increased molecular polarity
`throughout the pH range studied. The presence of an-
`other ionized carboxyl group also may explain the re-
`duced binding of carboxyfluorescein to plasma protein
`in view of the hydrophobic nature of the fluorescein
`binding site on albumin.
`Intraocular dye distribution at one minute after intra-
`venous dye injection reflects well the difference in lipid
`solubility of carboxyfluorescein and fluorescein. Very
`little carboxyfluorescein penetrates the barrier layers of
`the anterior and posterior segments. Thus, it cannot be
`The blood-ocular barriers are markedly less permea— 60 seen within the cells of the ciliary epithelium and ir is
`ble to intravascular carboxyfluorescein than to fluores-
`epithelium, nor in retinal tissue. In contrast, fluorescein
`cein. The relative inability of carboxyfluorescein to
`brightly stains the cells of the ciliary and iris epithelium,
`cross these barriers is not caused by more extensive
`and can be seen to a lesser extent in all retinal layers.
`binding to plasma proteins; rather, the lower lipid solu-
`Quantitative measurements of fluorescence intensity
`bility of carboxyfluorescein significantly reduces pene- 65 in the choroid and retina confirmed that, in comparison
`tration through barrier cell membranes.
`to fluorescein, (even when very high doses of dye are
`Octanol/buffer partition ratios, used to estimate lipid
`given) carboxyfluorescein enters retinal
`tissue, or in
`solubility are 1000 times lower for carboxyfluorescein
`negligible amounts. Much greater amounts of fluores-
`
`DIALYZABLE FRACTION OF CARBOXYFLUORESCEIN
`AND FLUORESCEIN IN HUMAN PLASMA
`DIALYZABLE FRACTION
`(lg) MEAN .._. S.E.M.
`pLASMA
`CARBOXY-
`.
`
`CONCENTRATION
`FLUORESCEIN
`FLUORESCEIN
`10-3M
`(4) .61 i .02
`(4) '30 i. .02
`10-4M
`(4) .50 i .01
`(2) .33 -+_- .03
`10‘5M
`(6) ~51 1' ~03
`(4) ~33 i ‘01
`
`10—5M
`(2) .46 i .01
`(2) .32 i .02
`
`50
`
`55
`
`

`

`cein, on the other hand, were detected throughout the
`retina with fluorescence intensity levels proportional to
`the dose administered. The highest fluorescein readings
`in the retina were recorded in the outer segment layer
`suggesting significant penetration across the pigment
`epithelium. Some diffusion of 'fluorescein should also
`occur through the walls of retinal vessels.
`It is believed that carboxyfluorescein has potential for
`experimental and clinical use as a probe of the blood-
`ocular barrier. It is similar to fluorescein in molecular
`weight, in spectral properties, including quantum effi-
`ciency, and, like fluorescein, a significant proportion of
`carboxyfluoreseein is not bound to plasma proteins. The
`lower lipid solubility of carboxyfluorescein, however,
`significantly reduces diffusion across barrier mem-
`branes. This property may yield better definition of the
`nature of barrier abnormalities than is now possible with
`fluorescein.
`
`TOPICAL USE OF CARBOXYFLUORESCEIN
`
`Carboxyfluorescein was prepared as a 1% solution in
`water, buffered to pH 7.4. Carboxyfluorescein was
`made into strip form, using strips of #1 filter paper,
`impregnated with carboxyfluorescein and allowed to
`dry. The carboxyfluorescein was applied to rabbit eyes
`both with the paper impregnated strip and also as a‘l%
`topical drop. The topical application of carboxyfluore-
`scein caused no apparent hyperemia or corneal changes
`within 5 to 10 minutes of application. In addition, when
`applied to an awake rabbit, the rabbit did not react in a
`negative fashion, thus suggesting no subjective discom-
`fort associated with the application of carboxyfluore-
`scein.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`45
`
`35
`When applied topically to rabbit eyes, carboxyfluore- ,
`scein is seen in the tear film and has its characteristic
`green fluorescence when illuminated by a blue light. An
`experimentally produced corneal abrasion, was made
`on the eye of a rabbit under general anaesthesia, and
`carboxyfluorescein densely stained the corneal epithe-
`lial defect. The stain persisted but lessened in intensity
`during an observation period of 30 to 45 minutes. When
`the aqueous humor from the anterior chamber of the
`eye is removed approximately 45 minutes after the cor-
`neal abrasion and the application of carboxyfluoresCein,
`with the rabbit under general anesthesia, the aqueous
`humor is seen to contain the dim green fluorescence of
`carboxyfluorescein when examined under a blue light.
`In the control eye where the corneal epithelium was not
`removed experimentally, markedly less carboxyfluore—
`scein was seen in the aqueous humor of the eye.
`‘
`
`9
`
`4,350,676
`
`10
`These observations with carboxyfluorescein are en-
`tirely similar to observations made clinically in humans
`with fluorescein, the dye currently used topically to
`visualize the tear film and to outline defects in the cor-
`neal epithelium. It is believed that penetration of the
`cornea by carboxyfluorescein would be less than the
`penetration of the cornea by fluorescein. One of the
`major disadvantages of topical fluorescein is that its
`ready penetration through the cornea into the aqueous
`humor creates an artificial aqueous flare, commonly
`called “fluorescein flare.” This flare can be confused
`with a condition seen in ocular inflammation and on
`occasion can lead to some difficulties in diagnosis. The
`lower lipid solubility, and presumably lower penetra-
`tion of carboxyfluorescein through the cornea, would _
`reduce this problem. For this reason, it is believed that
`carboxyfluorescein is not only an adequate substitute
`for fluorescein in topical use, but may indeed prove to
`have a substantial advantage over fluorescein.
`The current topical uses of fluorescein include; (1)
`visualization of the tear film (in diagnosing dry eyes,
`fitting contact lenses, studying lacrimal duct function,
`recording intraocular pressure in humans by the tech—
`nique of applanatiOn tonometry, etc.); (2) staining of
`abnormalities of the outer layer of the cornea called the
`corneal epithelium, (as in diagnosing corneal abrasions,
`assessing various infections or other disorders of the
`superficial cornea, etc.); (3) assessing ocular integrity
`after surgery or penetrating injuries ,(Seidel test); (4) use
`as a market for aqueous humor dynamics (a research
`application in which fluorescein is applied to the cornea
`and aided in its pentration into the aqueous humor by
`the application of a very weak electrical current after
`which the disappearance of fluorescein from the eye is
`used to study the flow rates of aqueous humor); and (5)
`as a method of studying corneal permeability. The in-
`vention contemplates the use of carboxyfluorescein for
`all these purposes.
`Without further elaboration the foregoing will so
`fully illustrate our invention that others may, by apply—
`ing current or future knowledge, readily adapt the same
`for use under various conditions of service.
`What is claimed as the invention is:
`1. The method of performing ophthalmic studies
`comprising applying to the eye an effective amount
`therefdr of at least one active isomer of carboxyfluore-
`seem.
`
`50
`
`2. The method of claim 1 wherein said carboxyfluore-
`, scein is administered intraveneously.
`3. The method of claim 1 wherein said carboxyfluore-
`scein is administered topically.
`Ill
`*
`i
`*
`*
`
`55
`
`65
`
`