WO2000068267A1 - LOW-DOSE IL-1β-INDUCED PHOTORECEPTOR CELL RESCUE WITHOUT RETINAL DYSPLASIA - Google Patents

Abstract

The present invention relates to the discovery that low levels of IL-1β, administered at 50 νg/ml or less, preferably 10 g$(m)g/ml or less, can still induce rescue of neuronal cells such as photoreceptor cells, while minimizing IL-1β's destructive sequelae, such as retinal dysplasia and intraocular inflammation.

Description

TITLE OF THE INVENTION LOW-DOSE IL-lβ-INDUCED PHOTORECEPTOR CELL RESCUE WITHOUT

RETINAL DYSPLASIA

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 60/132,855, filed on May 6, 1999, which is fully incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

BACKGROUND OF THE INVENTION The invention relates to the rescue of photoreceptor cells and other neuronal cells prone to disease or injury.

Researchers have long focused on studying the causes of many neurological dysfunctions, which occur due to injury to or pathological processes resulting in neuronal cell death. One area of study has been retinal photoreceptor cells (PRCs). Retinal photoreceptors are a highly specialized cell type, vulnerable to a wide range of microenvironmental insults and numerous specific gene defects (e.g., J. Stone et al . ) . In both rodents and humans, mutations associated with photoreceptor cell degeneration frequently involve genes that are specific to the PRC outer segment (e.g., Allikmets et al . ; Bird; Kohl et al.) or are important to the supportive role of the adjacent retinal pigment epithelium (RPE) and Bruch' s membrane (e.g., Mullen; Chaitin et al . ; Weber et al . ; Weber et al; E.M. Stone et al.; Cayouette et al.) However, the molecular events by which specific mutations lead to PRC appoptosis (i.e., cell death), have yet to be fully elucidated (Adler; Travis; Cideci et al.; Jomary et al., J. Stone et al.).

At the same time, although the causes of PRC degeneration are many, researchers have developed few models in which the apoptotic process has been effectively ameliorated or circumvented. Notable among these are transgenic murine strains in which gene therapy has been effected at the germline level (Chen et al . ; Hafezi et al.; Tsang et al.) Even when efficacious, however, a transgenic strategy does not represent a means of arresting pre-existing disease. Another strategy has been to investigate the use of exogenous trophic factors. Work in the rat has demonstrated that PRC survival can be significantly enhanced by a variety of recombinant gene products, including representatives from a number of families: i.e., growth factors, cytokines and neurotrophins (Faktorovich et al. 1990 and 1992; LaVail et al.) Among the most efficacious of these are acidic fibroblast growth factor (αFGF) , basic fibroblast growth factor (bFGF) , brain-derived neurotrophic factor (BDNF) , ciliary neurotrophic factor (CNTF) and interleukin-1 beta (IL-lβ) . Attention has, until now, focused particularly on bFGF' s association with PRC rescue, both by exogenous introduction and by endogenous induction by local injury (e.g., Faktorovich et al . , 1990 and 1992; LaVail et al.; Rakoczy et al., Hackett et al . , Wen et al.; Fontaine et al.,; Akimoto et al.). However, to date, the usefulness -3-

of some trophic factors has been limited by destructive side effects.

For instance, interleukin 1 beta (IL-lβ), another injury-related mediator, has been found associated with rescue of light-damaged photoreceptor cells. However, the amount of IL-lβ administered also produced extensive disruption of the retinal cytoarchitecture, likely related to the potent pro-inflammatory actions of this cytokine (LaVail et al., injecting a 1.0-μl bolus of 0.5 μg/μl IL-lβ intravitreally) . The prior art has found that intravitreal and intraocular administration of ILlβ causes retinal inflammatory response accompanied by breakdown of the vascular blood-retinal barrier (BRB) , via recruitment of macrophages mononuclear and polymorphonuclear leukocytes. Thus, IL-lβ has been considered primarily as a factor in the pathogenesis of human retinal inflammation (Bamforth et al. 1997 and 1997a) , and has been used to induce experimental retinal inflammation. These prior art findings of the destructive sequelae of IL-lβ administration have discouraged consideration of its role in the phenomenon of rescuing neuronal cells, given its apparent limited therapeutic potential.

Reported here is the first evidence that, surprisingly, the neuroprotective role of IL-lβ predominates over its inflammatory effects when used at sufficiently low doses. BRIEF SUMMARY OF THE INVENTION The present invention relates to the discovery that significantly lower levels of IL-lβ can still induce photoreceptor cell rescue while minimizing the destructive sequelae previously observed at 50-1000 fold higher doses. The invention uses lower but rescue- effective levels of 11-1 designed to reduce or eliminate its destructive sequelae. Specifically, the invention provides a method of rescuing neuronal cells in a dystrophic neural tissue site, by locally administering to at least one dystrophic neural tissue site in an individual, a composition comprising a low-dose amount of interleukin 1-beta (IL-lβ) that is effective to rescue neuronal cells without causing substantial dysplasia of the neural tissue at the dystrophic site.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

Figure 1 is a topographical representation of the sampling process used in counting photoreceptor number for each retina;

Figures 2A-B show the mean photoreceptor cell counts in pigmented dystrophic rats after different treatments; Figures 3A-B show the mean PRC counts in albino dystrophic rats after different treatments;

Figures 4A-D shows historesin-embedded sections of dystrophic pigmented RCS rat retinas, stained with H&E;

Figure 5 shows cryostat sections of retina from dystrophic pigmented RCS rat, 24 hours after injection with IL-lβ, stained for CD45 (A and C) and H&E (B and D) ; Figures 6A-D show cryostat sections of retina from IL-lβ-injected RCS rat, 48 hours after injection, stained for CD45 (A and C) and H&E (B and D) ;

Figures 7A-D show cryostat sections of retina from IL-lβ-injected RCS rat, 72 hours after injection, stained for CD45 (A and C) and H&E (B and D) ; and

Figures 8A-C show cryostat sections of dystrophic pigmented RCS rat retinas, stained for CD45, after injection with media (A), IL-lβ (B) , or bFGF (C) .

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the local administration of low-dose administration of interleukin 1-beta (IL-lβ) to a dystrophic neural tissue site in an individual, in an amount effective to rescue neuronal cells at the site while avoiding or minimizing the damaging side-effects of higher doses of IL-lβ. Specifically, one locally administers a composition comprising a low-dose amount IL-lβ of that is effective to rescue neuronal cells without causing substantial dysplasia or disfiguration of the neural tissue at the dystrophic site. The dystrophic neural tissue site may be a dystrophic retina, a spinal cord defect or injury, a brain lesion from a stroke, or other diseased or injured site within the CNS . The term "dystrophic" encompass injured as well as genetically or pathogenically diseased tissue.

In the case of treating a dystrophic retina, administration of low-dose IL-lβ (about 50 μg/ml or less) "rescues" retinal photoreceptor cells that are otherwise destined for apoptosis, without substantially triggering dysplasias such as rosettes, retinal folds and other malformations in the outer nuclear layer (ONL) . By "rescue" is meant the preservation of remaining, functional neuronal cells, i.e., the prevention or delay of programmed cell death (apoptosis) in these cells.

The amount of IL-lβ administered is preferably about 50 μg/ml or less, more preferably about 20 μg/ml or less, advantageously lOμg/ml or less. The low-dose ILlβ concentration may be within a range of 0.01-10 μg/ml. These dosages are made with reference to the specific activity of human recombinant IL-lβ (R&D Systems, USA) . It is within the purview of one skilled in the art to adjust the concentrations specific activity of the particular IL-1B administered.

Local administration of low-dose IL-lβ to the eye may be by intravitreal injection, e.g., in a bolus volume of about 0.1-10 μl, preferably 0.5-2 μl . Intrathecal administration may be used when treating a spinal cord, and intracranial delivery for treating brain lesions. The low-dose IL-lβ-comprising composition may also be locally administered to a dystrophic neural site by means of injection or application of the composition directly to the neural site. Alternatively, a targeted drug- delivery vehicle such as an oral or intravenous formulation could be used that is designed to release <50μg/ml of IL-lβ only upon reaching the neural tissue site or a vicinity thereof. Alternatively, local administration of the composition can be accomplished by a time-release drug-delivery vehicle that locally releases <50μg/ml of IL-lβ in the dystrophic neural tissue site over an extended period of time.

The IL-lβ may be an isolated and purified product or a recombinant product. Preferably, but not necessarily, the IL-lβ used is from the same species as the treated individual. However, any mammalian IL-lβ may be used when treating a mammalian according to the invention.

The use of low-dose IL-lβ as a general neuroprotective agent can be applied to conditions involving neuronal loss within the central nervous system, be they genetic in origin, degenerative, post- traumatic, ischemic, or toxic. Specific diseases that may benefit from treatment in accordance with the method of the invention include stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) , brain trauma, cerebral palsy, various cerebellar ataxias, and spinal cord trauma. Low-dose Il-lβ might also serve as an adjunct to surgery in general, and to the treatment of CNS tumors in particular, as a means of promoting neuroprotection of healthy neuronal cells, prior to an invasive procedure or the use of cytodestructive modalities .

Examples of retinal dystrophies amenable to treatment by low-dose IL-lβ include but are not limited to all forms of retinitis pigmentosa, all cone dystrophies, all known or unknown RPE-based dystrophies, other retinal degenerations including macular degeneration. Retinal ganglion cell loss also occurs in glaucoma, retinal detachment, and other optic neuropathies. These conditions might also be ameliorated by local administration of low-dose IL-lβ. In the case of retinal detachment, the IL-lβ administration would be an adjunct treatment, for preserving PRCs in the retina prior to or in conjunction with reattaching the retina. Local administration of low-dose IL-lβ would also be useful in combination with adjunct therapeutic modalities that either: a) decrease destructive inflammatory sequelae, e.g., selectively inhibit cytotoxic T- lymphocyte or NK cell activity; or b) increase neuroprotective efficacy, e.g., by manipulating gene expression of the transcription factor NFkB, NFkB gene product function, or selected "down-stream" neuroprotective genes, either directly or indirectly.

The invention also encompasses an article of manufacture, comprising packaging material and an IL-lβ- comprising composition contained within said packaging material, wherein the composition is effective for rescuing neuronal cells in a dystrophic neural tissue site to which the composition is locally administered. The composition preferably comprises a low-dose amount of IL-lβ in a range of about 50 μg/ml or less. The packaging material comprises a label that indicates that the composition can be used for rescuing neuronal cells remaining in a dystrophic neural tissue site to which the composition is locally administered, while minimizing dysplasia of neural tissue at said site resulting from said administration. When directed toward treatment of a dystrophic retina, the packaging material may indicate or instruct that the composition is for use in rescuing photoreceptor cells without causing substantial retinal dysplasias .

The invention is further described by way of the following, non-limiting example. The present invention is not limited to the particular embodiments disclosed, but encompasses all variations that are within the spirit and scope of the invention as defined by the claims.

Example I

METHODS Subjects

Royal College of Surgeon (RCS) rats: 3 and 4 week- old pigmented dystrophic (rdy^~p⁺) , pink-eyed dystrophic (rdy^~p^~) and congenic (rdy⁺p^~) . Animals were maintained according to NIH and ARVO animal guidelines. RCS rats are a model of inherited retinal dystrophy due to apoptosis and photoreceptor degeneration. (Tso et al.)

Experimental Procedure

Intravitreal injections were performed using fine, beveled, pulled glass micropipettes, coated with Sigmacote®. The pipette was connected to a lOμl Hamilton syringe via poly-ethylene tubing and the apparatus was filled with PBS. An air lock, introduced into the pipette before the volume to be injected, prevented dilution of the concentration.

In the first experiment, 3 strains of RCS rat were used; pigmented dystrophic, albino dystrophic, or congenic non-dystrophic animals, all 3 weeks of age. Three animals from each strain received 1 of 3 different dosages of IL-lβ (0.5μg/ml, 2μg/ml, and 5μg/ml) , or PBS, as a lμl bolus into the vitreous of the left eye, for a total of 36 animals. IL-lβ was mouse-derived (R&D Systems, USA) . Four weeks after injection, animals were sacrificed by sodium pentobarbitone overdose (2g/kg body weight) and their eyes removed. The eyes were immersion fixed in 2% gluteraldehyde / 1% paraformaldehyde at 4°C overnight and then the anterior part of the eye (cornea, iris, lens etc.) dissected away and discarded. The eyecups were transferred to 30% sucrose in PBS overnight for cryoprotection, embedded in OCT (Tissue-Tek®) and frozen for cryostat sectioning. Sections were cut at 6μm thickness from the middle of the eye, where the optic nerve was present, and stained with Haemotoxylin and Eosin (H&E) . Slides were then examined for photoreceptor number.

In the second experiment, ten 4-week-old pigmented dystrophic RCS rats received intravitreal injections (of a bolus of 0.5-5.0 μl, preferably 1.0-2.0 μl) of either

IL-lβ (5μg/ml; n=5) , or bFGF (lOOOμg/ml; n=5) . IL-lβ and bFGF were human recombinant (R&D Systems, USA and Promega, respectively) . Four weeks after injection, animals were sacrificed by sodium pentobarbitone overdose and their eyes removed and immersion fixed overnight in 4% paraformaldehyde at 4°C. The eyecups were dissected away, transferred to 30% sucrose in PBS overnight for cryoprotection, embedded in OCT and frozen for cryostat sectioning. Sections were cut at 6μm thickness from the middle of the eye, stained with H&E and photoreceptor cell profiles counted. A second series of sections were taken to immunostain for CD45 (1:50; Pharmingen, USA), a marker of all cells of hematopoetic lineage except erythrocytes . CD45 staining was visualized using Cy3 immunofluorescence (1:150; Jackson, USA).

In order to examine signs of inflammatory response following injection of IL-lβ, three additional 3-week-old pigmented dystrophic RCS rats received intravitreal injections of IL-lβ (5μg/ml) to the right eye, with the left eye serving as a time-matched control. Animals was sacrificed at 24, 48, and 72 hours post-injection respectively, their eyes removed and immersion fixed overnight in 4% paraformaldehyde at 4°C. Eyes were hemi- sected, half the eye processed for Historesin (Leica Instruments GmbH, Germany) with H&E staining, the other half processed for cryostat with CD45 immunohisto- chemistry. Additional animals were injected with IL-lβ (5μg/ml) or bFGF (lOOOμg/ml) and sacrificed 4 weeks later, their eyes processed as described above.

Counting protocol

Figure 1 is a topographical representation of the sampling process used in counting photoreceptor number for each retina. The number of photoreceptor cell profiles were counted in three 50 μm bins for each of 6 retinal regions: Superior Peripheral, Superior, Superior Central, Inferior Central, Inferior and Inferior Peripheral. For each eye, 3 sections were analyzed, providing a total of 9 samples per region and 54 samples per eye. The first sample came from the superior region of the nasal section, and the last sample was from the inferior region of the temporal section. All sections sampled contained the optic nerve, thus ensuring their relatively central location.

Analysis

The effect of all IL-lβ concentrations and sham injections on photoreceptor number was compared by ANOVA. This statistical test was also used to compare the effects of IL-lβ against bFGF.

RESULTS

IL-lβ elicits photoreceptor dose-dependent rescue Figures 2A-B show the mean photoreceptor cell counts in pigmented dystrophic rats after different treatments. Fig. 2A shows the mean number of photoreceptors per 50μm for the 5 treatment groups of pigmented dystrophic rats. The group that received injections of 5μg/ml of IL-lβ had significantly more photoreceptors than all other the groups (* = p<0.05; Student-Newman-Keuls post-hoc analysis). The 2μg/ml IL-lβ group had significantly more photoreceptors than the group with untreated eyes (+ = p<0.05; Student-Newman-Keuls post-hoc analysis). The 0.5μg/ml lβ group had significantly more photoreceptors than the media-injected or uninjected eyes (Φ = p<0.05; Student-Newman-Keuls post-hoc analysis) .

Fig. 2B shows the mean number of photoreceptors per 50μm for all treatment groups, shown for each region of the retina from superior to inferior peripheries. ANOVA tests for each individual region revealed that IL-lβ significantly increased the number of photoreceptors in the superior periphery (* = p=0.0009; df4,ll; F=10.748) and the inferior periphery of the retina (+ = p=0.0029; df 4,11; F=7.962). In the superior periphery, the 5μg/ml group had significantly more photoreceptors than all other groups (* = p<0.05; Student-Newman-Keuls post-hoc analysis) . In the inferior periphery, the 5μg/ml group had significantly more photoreceptors than the untreated, media- and 0.5μg/ml IL-lβ-injected groups, while the 2μg/ml group had significantly more photoreceptors than the untreated group (+ = p<0.05; Student-Newman-Keuls post-hoc analysis) .

In summary, when the eyes of pigmented RCS rats were examined at 4 weeks post-injection, there was a significant difference in the number of photoreceptors surviving in IL-lβ-injected eyes (p<0.0001; F=9.488; df= 4,91; see Figure 2A) . The highest dose of IL-lβ (5μl/ml) was associated with significantly more photoreceptors than all other treatment groups (Student Newman-Keuls post-hoc analysis at 5% significance level) . The intermediate and low doses of IL-lβ (2μg/ml, 0.5μg/ml) both showed photoreceptor rescue relative to uninjected eyes (Student Newman-Keuls post-hoc analysis) and the low dose of IL-lβ also showed an increased number of photoreceptors compared to the media injected eyes. No significant effect was found for the media-injected eyes (Student Newman-Keuls post-hoc analysis) .

These effects were still apparent to some extent when cell number was compared for individual regions of the retina (Figure 2B) . The highest dose of IL-lβ (5μl/ml) was associated with significantly more photoreceptors in both superior and inferior peripheral regions of the retina. The two lower doses of IL-lβ (0.5μg/ml, 2μg/ml) showed a trend towards increased photoreceptors as well, the magnitude in each case bearing an incremental relationship to that seen with the highest dose. Neither of these groups showed significant effects at specific locations however. Because intravitreal injection of buffer alone was not entirely without effect (even though not significant) , PRC numbers from media-injected eyes were used as baseline values for purposes of comparison for the pink-eyed rats (results shown in Figures 3A-B) . Using this approach, the effects of intravitreal IL-lβ on PRC number in the eyes of pink-eyed dystrophic RCS rats were still significant (p<0.0006; F=6.507; df=3,68; Figure 3A) . Specifically, Figure 3A shows the mean number of photoreceptors per 50μm for the 4 treatment groups of albino dystrophic rats. The group that received injections of 5μg/ml of IL-lβ had significantly more photoreceptors than the groups which received injections of media or 0.5μg/ml of IL-lβ (* = p<0.05; Student- Newman-Keuls post-hoc analysis) . The 2μg/ml IL-lβ group had significantly more photoreceptors than the group with media-injected eyes (+ = p<0.05; Student-Newman-Keuls post-hoc analysis).

Both of the higher dose IL-lβ groups had significantly more photoreceptors than the media injected group; the highest IL-lβ dose produced significantly more rescue than the lowest dose group as well (Student Newman-Keuls post-hoc) . These effects were also reflected in the regional differences across the retina (Figure 3B) . Specifically, Figure 3B shows the mean number of photoreceptors per 50μm for all treatment groups, shown for each region of the retina from superior to inferior peripheries. ANOVA tests for each individual region revealed that IL-lβ significantly increased the number of photoreceptors in the inferior (+ = p=0.0278; df=3,8; F=5.196) and the inferior periphery of the retina (* = p=0.0425; df=3,8; F=4.363). In both the inferior and inferior periphery of the retina, the 5μg/ml group had significantly more photoreceptors than the media injected group (* and + = p<0.05; Student-Newman-Keuls post-hoc analysis) .

However, it was again the case that while trends were seen across the retina, it was only in the peripheral regions that any significant effects were found by individual regional analyses. Specifically, the inferior (p=0.0278; F=5.196; df=3,8) and inferior peripheral (p=0.0425; F=4.363; df=3,8) regions of the retina showed effects from IL-lβ treatment, with the highest dose having significantly more photoreceptors than the media-injected group well (Student Newman-Keuls post-hoc) .

Comparison of IL-lβ and bFGF-induced photoreceptor rescue

Both IL-lβ and bFGF were associated with prominent PRC rescue, as seen from Figures 4A-C: historesin- embedded sections of dystrophic pigmented RCS rat retinas, stained with H&E. Figure 4A is an example of a media-injected eye, 4 weeks post-injection. There is substantial photoreceptor loss, with the outer nuclear layer (ONL) reduced to 2 to 3 cells in thickness. Figure 4B is an example of the highest low-dose injection of Il-lβ used (5μg/ml), 4 weeks after injection, which gives good preservation of the ONL. Figure 4C is an example of bFGF-injected eye, 4 weeks post-injection, which gives comparable levels of rescue to IL-lβ-injected eyes.

Thus, at the highest dose of low-dose IL-lβ used here (5μg/ml), photoreceptor number was significantly elevated compared to buffer-injected controls (ANOVA; p<0.0001; F=21.646; df=2,72). Furthermore, higher numbers of photoreceptors were present in all 6 retinal regions. IL-lβ also induced PRC rescue at the two lower dosages used here (2μg/ml, 0.5μg/ml).

Compared to bFGF in terms of maximal efficacy, results were similar for these 2 molecules, although IL- lβ showed an apparent advantage (p<0.0001; F=20.675; df=2,87; data not presented). It is important to note that bFGF was used here without the addition of heparin. Our bFGF data are therefore not inconsistent with the previous report by LaVail et al . In addition, this study also examined the relationship between dose and response for low-dose IL- lβ. As seen in Figures 2 and 3, the level of PRC rescue obtained showed a positive correlation with the intravitreal dosage administered. Of note, low-dose IL- lβ was associated with significant levels of PRC rescue, despite being used at dosages 100-1,000 fold lower than in LaVail et al . More significantly, the low dosages of IL-lβ used in this experiment did not result in retinal dysplasia (i.e., rosettes, retinal folds, and other focal disruptions of the retina's outer nuclear layer), a complication seen after LaVail et al.'s treatment with IL-lβ or bFGF at higher dosages.

Transience of inflammation response to intravitreal IL-lβ To investigate the magnitude and duration of the inflammatory response evoked by the maximum dosage level (5μg/ml) of intravitreal IL-lβ used in this study, a subset of animals were examined early in the post- injection period. At 24 hours, a substantial leukocytic infiltrate was present in the retina and overlying vitreous in the vicinity of the site of injection (superior periphery) . This infiltrate consisted of numerous small, CD45 positive (CD45+) cells (Figures 5A-B) . In contrast, retina distant to the injection site contained relatively few CD45+ cells at this time point (Figures 5C-D) . (Figure 5 shows cryostat sections of retina from dystrophic pigmented RCS rat, 24 hours after injection with IL-lβ, stained for CD45 (A and C) and H&E (B and D) . Leukocyte infiltrate is very evident in the region of injection, the superior periphery (A and B) , but much less apparent in the central retina (C and D) . )

By 48 hours after injection, leukocytic infiltration of the retina had increased noticeably (Figures 6A-D) . (Figure 6 shows cryostat sections of retina from IL-lβ- injected RCS rat, 48 hours after injection, stained for CD45 (A and C) and H&E (B and D) . Leukocyte infiltrate has reached high levels throughout the retina, with the greatest change from 24 hours occuring in the central retina (C and D) , where a large increase in leukocyte number is apparent.)

At 72 hours post-injection, the number of CD45+ cells had begun to subside; this was particularly apparent in the peripheral retina, although also the case in the central region (Figures 7A-D) . (Figure 7 shows cryostat sections of retina from IL-lβ-injected RCS rat, 72 hours after injection, stained for CD45 (A and C) and H&E (B and D) . Leukocyte numbers have begun to decrease in both peripheral (A and B) and central (C and D) retina. However, substantial numbers of CD45 positive cells are still visible (A and C) . )

At 4 weeks after injection, eyes that had received IL-lβ 4 exhibited no remaining CD45+ cells in the vitreous or retina (Figure 8B) . Neither were there any CD45+ cells in bFGF-injected eyes at this time point (Figure 8C) . (Figures 8A-C show cryostat sections of dystrophic pigmented RCS rat retinas, stained for CD45. Fig. 8A: Media-injected eye, 4 weeks post-injection; Fig. 8B: IL-lβ-injected eye, 4 weeks after injection; and Fig. 8C: bFGF-injected eye, 4 weeks post-injection. In all cases, CD45 is at low levels, implying few infiltrating leukocytes. )

The results presented here show that exogenous IL-lβ and bFGF both induce substantial photoreceptor rescue in the rat. However, these results are the first showing that low-dose IL-lβ rescues PRCs without the concomitant retinal dysplasia seen at the higher doses used in the prior art. "Low-dose" IL-lβ refers to concentrations of about 50 μg/ml or less, preferably 20 μg/ml or less, advantageously 10 μg/ml or less, as low as 0.01 μg/ml, administered locally. These concentrations are with reference to the specific activity of human recombinant IL-lβ ) R&D Systems, USA) .

Administration of low-dose IL-lβ as described here is a potent inducer of rescue at dosages 100-1,000 fold lower than the levels used for bFGF administration (Faktorovich et al., 1990).

The role of bFGF as a neurotrophic factor is now firmly established (e.g., Eckenstein) , as is the role of IL-lβ in the inflammatory cascade (see Dinarello) . The mechanisms by which pro-inflammatory signals induce the expression of additional cytokines and trophic factors important for wound repair has begun to be eludicated. In this context, IL-lβ is a potent inducer of bFGF in a variety of settings, including the CNS . (Aravjo et al.; Rivera et al . ) . The presence of IL-lβ markedly enhances the production of bFGF by glial cells, thereby providing one possible explanation for the neuroprotective effects of even low-dose IL-β, as observe here.

In addition, intravitreal injection of either IL-lβ or bFGF has been observed to result in an increased incidence of mononuclear cells in the retina (Faktorovish 1990; LaVail et al.) . Cells of the monocyte lineage, particularly activated macrophages, in turn secrete both IL-lβ and bFGF (Avron et al.l; Baird et al.) There is also evidence that both factors, once present, are capable of inducing their own expression (Mauviel et al., Flott-Rahmel et al.) It therefore seems likely that: (a) by locally administering either factor in the vicinity of the retina, one induces increased concentrations of both factors (IL-lβ and bFGF) ; and (b) in vivo, the actions of one factor reflect, to some extent, the presence of the other .

Interestingly, although both IL-lβ and bFGF are found in a wide variety of species, the phenomenon of injury-related PRC rescue is particularly conspicuous in the rat, an animal with robust intraretinal expression of bFGF following relatively mild microenvironmental insults

(Wen et al.; Cao et al.) The case for bFGF' s role is extensive, yet IL-lβ' s ubiquity, potency and ability to upregulate trophic factors, including bFGF, all suggest a significant contribution of IL-lβ to PRC rescue even at low doses .

Cellular effects of IL-lβ

The IL-1 family of cytokines shares considerable sequence homology with the FGF family and it has been proposed that both evolved from duplication of a common ancestral gene (Zhang et al.) IL-lβ is a secreted member of the IL-1 family that plays a major role early in the inflammatory cascade (Dinarello) Although exuberant inflammation is itself a cause of tissue injury (e.g., Geiger et al.; Jeohn et al . ; Andersson et al.; Theofilopoulos et al . ) under more typical circumstances, the inflammatory response may be better viewed as the initial step in tissue regeneration and repair. It is within this context that the neurotoxic and neuroprotective effects of IL-lβ need not appear contradictory.

Up to now, a large body of work has associated IL-lβ with neuropathological processes (Jeohn et al . ; Rothwell et al . ; Toulmond et al.; Downen et al . ; Downen et al.; Pearson et al.). IL-lβ potentiates the destructive effects of a variety of other agents. In particular, IL- lβ in combination with nitric oxide (NO) is cytotoxic to cultured neurons (Chao et al 1996; Hu et al.). Although a few have reported some neuroprotective effects of this cytokine, in vi tro (Gahring et al.) and in vivo (LaVail; Klusman et al.; Wang et al.), these have been found to be concomitant with IL-lβ' s destructive effects. In contrast, the present results are the first showing that low-dose administration of IL-lβ can confer a primarily neuroprotective effect without causing substantial dysplasia of the neural tissue to which the low-dose IL- lβ is administered.

The mechanism by which IL-lβ exerts a neuroprotective influence appears to involve the IL-1RI receptor (Ohtsuki et al.), the transcription factor NFkB (Yu et al.) and, by implication, the expression of "downstream" genes. Interestingly, NFkB has also been implicated in the expression of a number of pro- inflammatory cytokines by activated macrophages (Kelly et al.), some of which have also been implicated in neuroprotection (Carlson et al . , Klusman et al . ) .

One of the many functions of IL-lβ may therefore be that of an early warning or "stress" signal, the cellular response to which serves to enhance cell survival in the face of various deleterious stimuli, including the inflammatory response itself (Neta et al., Lee et al., Galcheva-Gargova et al.; Han et al . ) Cells that have not entered into this protective mode, presumably because they are functioning too poorly to do so, would be at increased risk for cell death as the inflammatory response intensifies.⁷⁸ Central nervous system (CNS) neurons (as exemplified by PRC cells) , being less amenable to replacement, apparently can react to low levels of IL-lβ with a robust anti-apoptotic response. As with other functions of this cytokine, the protective response likely results from the induction of multiple additional genes, perhaps as part of an amplifying cascade .

Photoreceptor Rescue

The promotion of PRC survival by low-dose ILl-β is of particular interest for retinal dystrophies, in which genetic errors can result in widespread photoreceptor apoptosis and severe visual disability (see Bird; Stone J et al . ) .

While not critical to the effectiveness of the low- dose IL-lβ administration taught herein, it is believed that the PRC rescue effects observed represent the prolonged survival of pre-existing photoreceptor cells.

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Claims

1. A method of rescuing neuronal cells in a dystrophic neural tissue site, comprising locally administering to at least one dystrophic neural tissue site in an individual, a composition comprising a low-dose amount of interleukin 1-beta (IL-lβ) that is effective to rescue neuronal cells without causing substantial dysplasia of thedystrophic neural tissue.

2. The method of claim 1, wherein the at least one dystrophic neural tissue site is a dystrophic retina.

3. The method of claim 1 or 2, wherein the composition comprises IL-lβ in a concentration of about <50 μg/ml.

4. The method of claim 1 or 2, wherein the composition comprises IL-lβ in a concentration of about <20 μg/ml.

5. The method of claim 1 or 2, wherein the composition comprises IL-lβ in a concentration of about <10 μg/ml.

6. The method of claim 1 or 2, wherein the composition comprises IL-lβ in a concentration of about 0.1 μg/ml - lOμg/ml .

7. The method of claim 2, wherein the composition comprises a solution of IL-lβ in a concentration of about <50 μg/ml and the composition is administered intravitreally in a volume in a range of about 0.1-10 μl .

8. The method of claim 2, wherein the composition comprises a solution of IL-lβ in a concentration of about

<20 μg/ml and the composition is administered intravitreally in a volume in a range of about 0.1-10 μl .

9. The method of claim 2, wherein the composition comprises a solution of IL-lβ in a concentration of about

<10 μg/ml and the composition is injected intravitreally in a volume in a range of about 0.1-10 μl .

10. The method of claim 2, wherein the retinal dystrophy is a result of a member selected from the group consisting of: a retinitis pigmentosa, a cone dystrophy, a retinal pigmented epithelium (RPE) -based dystrophy, a retinal degeneration, a macular degeneration, a retinal ganglion cell loss, a glaucoma, an optic neuropathy; and a retinal detachment .

11. The method of claim 1 or 2, wherein local administation of the composition is accomplished by a targeted drug-delivery vehicle that locally releases <50μg/ml of IL-lβ only upon reaching the neural tissue site or a vicinity thereof.

12. The method of claim 1 or 2, wherein local administration of the composition is accomplished by a time-release drug-delivery vehicle that locally releases <50μg/ml of IL-lβ in the dystrophic neural tissue site over an extended period of time.

13. An article of manufacture, comprising packaging material and a composition contained within said packaging material, wherein the composition is effective for rescuing neuronal cells in a dystrophic neural tissue site to which the composition is locally administered, wherein said composition comprises a low-dose amount of IL-lβ in a range of about 50 μg/ml or less, and wherein the packaging material comprises a label that indicates that the composition can be used for rescuing neuronal cells remaining in a dystrophic neural tissue site to which the composition is locally administered, while minimizing dysplasia of neural tissue at said site resulting from said administration.

14. The article of manufacture of claim 12, wherein the dystrophic neural tissue site is a dystrophic retina.

15. An article of manufacture, comprising packaging material and a composition contained within said packaging material,

wherein the composition comprises a low-dose amount of IL-lβ that is effective to rescue photoreceptor cells without causing substantial dysplasia of at least one retina to which the composition is locally administered, said low-dose amount of IL-lβ being about <50 μg/ml; and

wherein the packaging material comprises instructions for administering the composition to an individual having a dystrophic retina in a manner that rescues at least one photoreceptor cell within the dystrophic retina while minimizing any substantial retinal dysplasia caused by administering Il-lβ to the retina.

16. The article of manufacture of claim 14 or 15, wherein the dystrophic retina is a result of a member selected from the group consisting of: a retinitis pigmentosa, a cone dystrophy, a retinal pigmented epithelium (RPE) -based dystrophy, a retinal degeneration, a macular degeneration, a retinal ganglion cell loss, a glaucoma, an optic neuropathy; and a retinal detachment.

17. The method of claim 1, wherein the dystrophic neural site is a diseased or injured spinal cord.

18. The method of claim 1, wherein the dystrophic neural site is a diseased or injured CNS site.

19. The method of claim 1, wherein the dystrophic neural site is a brain lesion.