JP2003525206A - Method of inducing rescue of photoreceptor cells without retinal dysplasia by low dose IL-1β - Google Patents

Abstract

  (57) [Summary] The present invention discloses that low doses of IL-1β of <50 μg / ml, preferably <10 μg / ml, allow photoreception while minimizing the destructive sequelae of IL-1β such as retinal dysplasia and intraocular inflammation. It relates to the discovery that rescue of neurons, such as cells, can be triggered.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from US Provisional Application 60/132855 filed May 6,1999. This is described here for reference.

[0002]     (Statement regarding federal research or development)

FIELD OF THE INVENTION The present invention relates to methods of rescue photoreceptor cells and other neuronal cells susceptible to disease or injury.

BACKGROUND OF THE INVENTION Researchers have long focused on studying the causes of many neurological disorders that develop due to neuronal cell death due to injury or pathological processes. One of the fields of study was retinal photoreceptor cells (PRC). Retinal photoreceptor cells are a very specific cell type and are susceptible to damage in a wide range of microenvironments and various specific genetic abnormalities (J. Stone et al.). Mutations associated with photoreceptor cell degeneration in both rodents and humans include genes specific for the PRC external segment (Allikmets et al., Bird, Kohl et al.) Or adjacent retinal pigment epithelium. Genes important for the supporting role of cells (PRE) and Bruch's membrane (Mull
en, Chaitin et al., Weber et al., SM Stone et al., Cayouette et al., etc.) are often involved. The molecular events that trigger PRC apoptosis (cell death) by specific mutations are not yet fully understood (Adler, Travis).
, Cideci et al., Jomary et al., J. Stone et al.).

Although there are many causes of PRC degeneration, few models have been developed in which the apoptotic process is effectively ameliorated or avoided. Of note among these models are the transgenic mouse strains in which gene therapy was effective at the germline level (Chen et al., Hafezi et al., Tsang et al.). However, even if effective, the transformation method is not a means to prevent existing diseases.

Another approach has been to consider the use of exogenous trophic factors. Rat experiments have demonstrated that various recombinant gene products including representatives of numerous families (growth factors, cytokines and neurotropins) can increase PRC survival (Faktorovich et al., 1990 and 1992; LaVail). Etc.). The most effective of these were acidic fibroblast growth factor (αFGF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor ( CNTF) and interleukin-1 beta (IL-1β).
To date, bFGF and P by both extrinsic and endogenous induction by local injury
Special attention has been paid to the association of RC relief (Faktorovich et al., 1
990 and 1992; LaVail et al; Rakoczy et al, Hackett et al, Wen et al, Fontaine et al, Ak.
imoto et al.). However, the usefulness of trophic factors is currently limited due to destructive side effects.

[0007] For example, another damage-related mediator, interleukin-1 beta (IL-1β), has been shown to be involved in the rescue of photodamaged photoreceptor cells. However, administration of high doses of IL-1β resulted in extensive destruction of retinal cell architecture, which may be associated with the potent pro-inflammatory effects of this cytokine (LaVail et al., IL-1β 0.5 μg / μl. 1.0 μl intravitreal injection). Intravitreous and intraocular administration of IL-1β reveals retinal inflammatory response with disruption of vascular blood-retinal barrier (BRB) through increase of macrophage mononuclear / polymorphonuclear leukocytes. became. Therefore, IL-1β is considered to be a factor primarily in the pathogenic development of human retinitis (Bamforth et al., 1997.
, And 1997a), used to induce experimental retinitis. Considering that the therapeutic effect is clearly limited, it was abandoned to examine its role in the phenomenon of neuronal cell rescue from the findings regarding the destructive sequelae by IL-1β administration obtained in these conventional techniques. .

Surprisingly, the first evidence that IL-1β, when used at sufficiently low doses, outweighs its pro-inflammatory effects, is presented here.

Brief Summary of the Invention The present invention shows that when IL-1β is used at sufficiently low doses, rescue of photoreceptor cells is induced and the destruction previously observed at 50-1000-fold higher doses. Related to the discovery that common aftereffects are minimized. In the present invention, an IL designed to reduce or prevent its destructive sequelae, which shows a sufficient cell rescue activity at a low dose
-1 is used. In particular, the present invention provides a sufficiently low dose of interleukin-1 beta (IL-) that is effective to rescue neuronal cells without causing substantial dysplasia of neural tissue at dystrophic sites. The present invention relates to a method of locally administering a composition comprising 1β) to at least one dystrophic nerve cell site to rescue neuron cells at the dystrophic nerve cell site.

DETAILED DESCRIPTION OF THE INVENTION The present invention rescues neuronal cells at dystrophic neural tissue sites, while at the same time, is effective in preventing or minimizing the deleterious side effects of high doses of IL-1β. A method of locally administering a dose of interleukin-1 beta (IL-1β) to a dystrophic nerve tissue site. In particular, low doses of IL-1 effective to rescue neuronal cells without causing substantial dysplasia or damage of neural tissue at the dystrophic site.
It relates to a method of locally administering a composition comprising β. Dystrophic nerve tissue sites include dystrophic retinas, spinal cord defects or injuries, brain lesions due to stroke or lesions or injuries within the CNS. The term "dystrophic" encompasses not only damaged tissue, but genetically or pathogenic lesioned tissue.

In the treatment of dystrophic retina, low doses (<about 50 μg / ml) of IL-1β cause dysplasia such as retinal rosettes and retinal folds and other dysplasias in the outer granular layer (ONL). Retinal photoreceptor cells that are otherwise destined to undergo apoptosis can be rescued substantially without causing them. By "rescue" is meant the protection of the remaining functional neuronal cells, ie the prevention or delay of programmed cell death (apoptosis) in these cells.

The dose of IL-1β is preferably <about 50 μg / ml, more preferably <about 20.
μg / ml, conveniently <10 μg / ml. The concentration of low dose IL-1β is 0.01
It may be in the range of 10 μg / ml. These doses correspond to human recombinant IL-1β
(R & D Systems, USA). Special IL-1 to administer
It is within the skill of the artisan to adjust the concentration-specific effects of β.

Intravitreal injection may be used for topical administration of low dose IL-1β to the eye. As an example, a large dose of about 0.1-10 μl, preferably 0.5-2 μl is administered. Intrathecal injection may be used for spinal cord treatment and intracranial injection for brain lesion treatment. The low dose IL-1β composition may also be administered locally to the dystrophic nerve site by direct injection or application. Alternatively, a targeted drug delivery vehicle, such as an oral or intravenous formulation, designed to release ≦ 50 μg / ml IL-1β only upon reaching the nerve tissue site or its surrounding areas. It can also be used. Alternatively, topical administration of the composition can be carried out using a sustained release drug delivery vehicle that locally releases <50 μg / ml IL-1β over time at the dystrophic nerve tissue site.

IL-1β is an isolated and purified product or a recombinant product. Although not always necessary, the IL-1β used is preferably derived from the same animal species as the subject to be treated. However, any mammalian IL-1β may be used when treating a mammal in accordance with the present invention.

The use of low dose IL-1β as a general neuroprotective agent is associated with neuronal loss within the central nervous system, whether it is genetic, degenerative, post-traumatic, ischemic or virulent. Can be applied to the symptoms. Specific diseases that may benefit from treatment by 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 injury. Low-dose IL-1β is also a common adjuvant therapy for surgery, especially C
As an adjunctive therapy for NS tumor treatment, it promotes neuroprotection of normal neuronal cells and exerts a superior effect over the use of invasive measures or cytocidal methods.

Examples of retinal dystrophy susceptible to treatment with low dose IL-1β are based on all types of retinitis pigmentosa, all pyramidal dystrophy, all known or unknown RPEs. Other retinal degenerations such as dystrophy and macula are included. Loss of retinal ganglion cells also develop in glaucoma, retinal detachment and other optic neuropathy. These diseases also
It may be improved by local administration of low dose IL-1β. In the case of retinal detachment, administration of IL-1β is considered to be an adjunct therapy for protecting the retinal PRC before or simultaneously with retinal detachment.

Local administration of low-dose IL-1β is also a) an adjunctive therapeutic method for reducing destructive inflammatory sequelae (eg, a method of selectively inhibiting cytotoxic T lymphocyte or NK cell activity). Or b) an adjunctive therapy that enhances neuroprotective action (expression of transcription factor NFkB, function of NFkB gene product or selective “downstream” neuroprotective gene manipulation, etc.) ) Is also useful in combination.

Further, the present invention comprises a packaging material and a composition comprising IL-1β having an effect of rescuing neuronal cells in a dystrophic nerve tissue site which is a local administration site contained in the packaging material. It also includes industrial products. This composition is <about 50 μg /
It is preferably composed of low dose IL-1β in ml. The composition of the packaging material rescues the neuronal cells remaining in the dystrophic nerve tissue site, which is the local administration site, and minimizes the dysplasia of nerve tissue in the dystrophic nerve tissue site caused by administration. It is composed of a label indicating that it can be used to suppress For the purpose of treating retinal dystrophy, the packaging material states that the composition is used to rescue photoreceptor cells without causing substantial retinal dystrophy. Or you may notify.

The present invention is described in detail using the following non-limiting examples. The present invention is not limited to the disclosed embodiments but covers all variants which come within the spirit and scope of the invention as claimed.

Example I Method Material

The Royal College of Surgeon (RCS) rat: 3 to 4-week-old dystrophic colored rats (rdy ^- p ^+), dystrophic pink eye rat (rdy ^- p ^-) and congenic rat (rdy ⁺ p ^- ). In accordance with NIH and ARVO laboratory animal guidelines,
The animals were acclimatized. The RCS rat is a model of congenital retinal dystrophy due to apoptosis and photoreceptor cell degeneration (Tso et al.).

Experimental Procedure Intravitreal injection was performed using a flat glass micropipette with a fine gradient coated with Sigma Coat®. The pipette was connected to a 10 μl Hamilton syringe through a polyethylene tube, and the instrument was filled with PBS. Prior to injection, the pipette was fitted with an airlock to prevent concentration dilution.

In Experimental Example 1, three types of RCS rats were used, namely, dystrophic colored rats, dystrophic albino rats or non-dystrophic congenic rats. All rats
He was 3 weeks old.

For each of 3 animals of each animal species, one dose of any one of 3 doses (0.5, 2 and 5 μg / ml) was injected into the vitreous of the left eye in a large amount of 1 μl. A total of 36 animals were dosed. IL-1β was derived from mouse (R & D Sy
stems, USA). Four weeks after injection, the animals were euthanized with an overdose (2 g / kg body weight) of sodium pentobarbital and the eyes were enucleated. The extracted eye was fixed by immersion in 2% glutaraldehyde / 1% paraformaldehyde overnight at 4 ° C. Next, the anterior chamber portion (cornea, iris, lens, etc.) was peeled off and discarded. Transfer the eyecups to a 30% sucrose / PBS solution for cryoprotection and let stand overnight to allow OCT (Tiss
ue-Tek®) and frozen for cryosectioning. A 6 μm-thick section was cut out from the central part of the eye where the optic nerve was present, and stained with hematoxylin and eosin (H & E). The slides were then observed and the number of photoreceptor cells counted.

In Experimental Example 2, 4-week-old dystrophic RCS rats were treated with IL-1β (5 μm).
Intravitreal injection (0.5-5.0 μl, preferably 1.0-2.0 μl in large volume) of either g / ml; n = 5) or bFGF (1000 μg / ml; n = 5) .
IL-1β and bFGF were human recombinants (R & D Systems, U respectively).
SA and Promega). Four weeks after injection, the animals were euthanized with an overdose of sodium pentobarbital and the eyes were enucleated. Extracted eye 4% overnight at 4 ℃
Immersion fixation was carried out in paraformaldehyde. The eye cup was peeled off, transferred to a 30% sucrose / PBS solution for cryoprotection, left overnight, embedded in OCT (Tissue-Tek (registered trademark)), and frozen for low temperature cutout. . Thickness 6 μm from the center of the eye
The section was cut out, stained with H & E, and the number of photoreceptor cells was counted. The sections obtained in Experimental Example 2 were immunostained for CD45, which is a marker for all hematopoietic cells except red blood cells (1:50; Pharmingen, USA). CD45 staining was visualized using Cy3 immunofluorescence (1: 150; Jackson, USA).

In order to observe the signs of inflammatory reaction after IL-1β injection, IL-3β (5 μg / ml) was injected into the vitreous of the right eye of three 3-week-old dystrophic RCS rats. did. The left eye served as a time-matched control. Injection 24, 48 and 72
Animals were euthanized at time, eyes were enucleated and immersion fixed in 4% paraformaldehyde overnight at 4 ° C. The isolated eye is semi-hemisectioned and half of it
a Instruments GmbH, Germany), the other half is a CD for low temperature cutting
45 immunohistochemistry. Furthermore, for another animal, IL-1β (
5 μg / ml) or bFGF (1000 μg / ml) was injected, and after 4 weeks, they were euthanized, and the eyes were treated by the method described above.

Counting Protocol FIG. 1 is a three-dimensional view of the sampling process used to count the number of photoreceptor cells in each retina. The number of photoreceptor cell profiles is three 50 for each of the six retinal regions (upper periphery, upper, upper center, lower center, lower and lower periphery).
Counted using μm bins. Three sections of analysis were performed for each eye. A total of 9 samples for each area and a total of 54 samples for each eye were analyzed. The first sample was taken from the upper area of the nasal section and the last sample from the lower area of the temporal section. All collected sections contained the optic nerve and were relatively centrally located.

Analysis of the effects of all IL-1β concentrations and sham injection on the number of photoreceptor cells by ANOV.
A was used for comparison. This statistical study was also used to compare the effects of IL-1β on bFGF.

Results IL-1β Dose-Dependent Rescue of Photoreceptor Cells FIGS. 2A-B show the average number of photoreceptor cells in dystrophic colored rats dosed with various agents. FIG. 2A shows the average number of photoreceptor cells per 50 μm in the 5 treatment groups consisting of dystrophic colored rats. The number of photoreceptor cells in the IL-1β 5 μg / ml administration group was significantly higher than that in all other administration groups (* = p <0.0
5; Student-Newman-Keuls post-hoc test). In the IL-1β 2 μg / ml administration group,
The number of photoreceptor cells was significantly higher than that in the non-administration group (+ = p <0.05; Student
-Newman-Keuls post-hoc test). The number of photoreceptor cells in the IL-1β 0.5 μg / ml-administered group was significantly higher than that in the vehicle-administered group or the non-administered group (‡ = p <0.0
5; Student-Newman-Keuls post-hoc test).

FIG. 2B shows each region of the retina (upper region to lower peripheral region) in all administration groups.
3 shows the average number of photoreceptor cells per 50 μm contained in the sample. ANO for each area
When the VA test was performed, the peripheral region of the upper retina (* = p = 0.0009; df4, 11;
F = 10.748) and the lower peripheral region (+ = p = 0.0029: df4, 11;
F = 7.962), a significant increase in the number of photoreceptor cells due to IL-1β was observed. Regarding the upper peripheral region, the number of photoreceptor cells was significantly higher in the 5 μg / ml administration group than in all other administration groups (* = p <0.05; Student-Newman-Keuls pos).
t-hoc test). Regarding the lower peripheral region, the number of photoreceptor cells was significantly higher in the 5 μg / ml administration group than in the non-administration group, the vehicle administration group and the IL-1β 0.5 μg / ml administration group. In the ml-administered group, the number of photoreceptor cells was significantly higher than that in the non-treated group (* = p <0.05; Student-Newman-Keuls post-hoc test).

In summary, observation of the eyes of colored RCS rats at 4 weeks post-injection revealed that IL
A statistically significant difference was observed in the number of surviving photoreceptor cells in the -1β-injected eye (p <0.
0001; F = 9.488; df = 4, 91; see FIG. 2A). The number of photoreceptor cells in the IL-1β high-dose group (5 μg / ml) was significantly higher than that in all other administration groups (Student-Newman-Keuls post-hoc test at a significance level of 5%). In the IL-1β medium-dose group (2 μg / ml) and the low-dose group (0.5 μg / ml), more photoreceptor cells were rescued than in the non-administered group (Student-Newman-Keuls post-hoc test). ). Also, I
An increase in the number of photoreceptor cells was also observed in the L-1β low dose group as compared with the vehicle administration group. No significant effect was observed in the vehicle-administered group (Student-Newman-Keuls pos.
t-hoc test).

These effects were still significant to some extent when the numbers of cells in each region of the retina were compared (FIG. 2B). In the high dose IL-1β group (5 μg / ml), the number of photoreceptor cells was significantly higher in both the upper and lower peripheral regions of the retina. Two I
The L-1β low-dose group (0.5 or 2 μg / ml) also showed an increase in photoreceptor cells, and the degree in each case had a gradual relationship with that observed in the high-dose group. Admitted. However, no significant effect in a specific area was observed in any of these administration groups.

Since no effect was observed when intravitreally injecting only the buffer solution (although it was not significant), the PRC number of the medium-injected eyes was determined for the purpose of comparison with pink eye rats. Used as a baseline value (see Figures 3A-B for results). Using this approach, the number of PRCs in the eyes of dystrophic pink-eyed RCS rats was still significant (p <0.0006; F = 6.507; df =
3, 68; Figure 3A). FIG. 3A shows the average number of photoreceptor cells per 50 μm in the four treatment groups consisting of dystrophic albino rats. In the IL-1β 5 μg / ml administration group, the number of photoreceptor cells was significantly higher than that in the vehicle administration group or the IL-1β 0.5 μg / ml administration group (* = p <0.05; Student-Newman- Keuls post-hoc test). The number of photoreceptor cells in the IL-1β 2 μg / ml-administered group was significantly higher than that in the vehicle-administered group (+ = p <0.05; Student-Newman-Keuls post-hoc test).

The two IL-1β high-dose groups had significantly higher numbers of photoreceptor cells in both groups than vehicle-administered groups; the IL-1β highest-dose group rescued compared to the lowest-dose group. The number of photoreceptor cells was significantly higher (Student-Newman-Keuls post-hoc test). These effects were also reflected in the differences in each region of the retina (Fig. 3B). FIG. 3B shows the average number of photoreceptor cells per 50 μm contained in each region of the retina (upper region to lower peripheral region) in all administration groups. AN for each area
When the OVA test was performed, the lower retina area (+ = p = 0.0278; df = 3, 8
F = 5.196) and the area around the lower retina (* = p = 0.0425; df = 3, 8
F = 4.363), a significant increase in the number of photoreceptor cells due to IL-1β was observed. In both the lower retina and the lower peripheral region, in the 5 μg / ml administration group,
The number of photoreceptor cells was significantly higher than that in the vehicle administration group (* and + = p <0.05)
; Student-Newman-Keuls post-hoc test).

However, also in this case, although a tendency was recognized between the retinal regions, a significant effect was recognized only in the peripheral region in the analysis of each region. Specifically, in the lower region (p = 0.0278; F = 5.196; df = 3, 8) and the lower peripheral region (p = 0.0425; F = 4.363; df = 3, 8), IL The effect of -1β administration was observed, and the number of photoreceptor cells was significantly higher in the highest dose group than in the vehicle administration group (Student-Newman-Keuls post-hoc test).

IL-1β and bFGF Induced Rescue of Photoreceptor Cells Comparison As shown in FIGS. 4A-C (H & E stained historesin-embedded dystrophic colored RCS rat retina sections), IL-1β and bFGF Were both associated with significant PRC salvage. FIG. 4A is an example of a medium-injected eye 4 weeks after medium injection. Substantial disappearance of photoreceptor cells is observed, and the outer granular layer (ONL) shows a decrease of 2-3 cells in thickness. FIG. 4B shows the highest dose of IL-1 (5 μg / ml).
This is an example of the 4th week after β injection, and a good storage state of ONL is observed. Figure 4C shows
This is an example of a bFGF-injected eye 4 weeks after the bFGF injection, and a rescue level equivalent to that of the IL-1β-injected eye is observed.

Thus, in the IL-1β highest dose group (5 μg / ml), a significant increase in the number of photoreceptor cells was observed as compared with the buffer injection control (ANOVA; p <0.0001;
F = 21.646; df = 2, 72). Furthermore, the number of photoreceptor cells was high in all 6 retinal areas. Two low dose groups (2 μg / ml and 0.5 μg / ml)
But PRC rescue was triggered.

In terms of maximal effect, IL-1β has a clear advantage (p
<0.0001; F = 20.675; df = 2, 87; data not shown), but the results were comparable for these two molecules. It is important to note here that heparin was not added to bFGF. Therefore, the bFGF data obtained this time do not match the previous report of LaVail et al.

In addition, this study also examined the dose-response relationship of low dose IL-1β. As shown in FIGS. 2 and 3, the PRC rescue level obtained in this study showed a positive correlation with the intravitreal dose. It should be noted that low dose IL-1β was associated with significant PRC in spite of the 100-1000 fold lower dose used by LaVail et al.
It was related to the relief level. More importantly, the low dose of IL-1β used in this study was associated with retinal dysplasia (rosette, retina), a complication observed after LaVail et al. Used high doses of IL-1β or bFGF. Folds and other localized destruction of the extraretinal granular layer).

Transientity of Inflammatory Response by Intravitreal Administration of IL-1β In the present study, the maximum dose (5 μg / ml) of IL-1β was administered in order to investigate the extent and duration of the inflammatory response when administered intravitreally. Animals were observed early in the latter period.

Twenty-four hours after the administration, significant leukocyte infiltration was observed in the vitreous in the retina and around the injection site (upper peripheral region). This infiltrate was composed of numerous small CD45-positive (CD45 +) cells (FIGS. 5A-B). In contrast, at this time point, the retina remote from the injection site contained relatively few CD45 + cells (FIGS. 5C-D). (FIG. 5: CD45-stained (A and C) and H & E-stained (B and D) dystrophic RC at 24 hours after IL-1β injection.
3 shows a retina cryosection of an S rat. Leukocyte infiltration is very prominent in the infused and upper peripheral regions (A and B), but in the central retinal region (C and D).
Is not so noticeable)

By 48 hours post-injection, leukocyte infiltration was significantly increased in the retina (FIGS. 6A-D).
(FIG. 6 shows retinal cryosections of CD45-stained (A and C) and H & E-stained (B and D) RCS rats 48 hours after IL-1β injection. Leukocyte infiltration had high levels throughout the retina. Retina central region (C and D)
, The maximum change from the 24th hour was observed, and a significant increase in white blood cell count was observed.)

72 hours after the injection, the number of CD45 + cells began to settle down. This was also observed in the central region, but it was particularly remarkable in the peripheral region of the retina (Figs. 7A-D).
. (FIG. 7 shows VD45 staining (A and C) 72 hours after IL-1β injection.
And H & E stained (B and D) retinal cryosections of RCS rats.
White blood cell counts began to decrease in both the periretinal area (A and B) and the central retinal area (C and D). However, still quite a lot of CD45 +
Cells are visible (A & C)).

At 4 weeks post-infusion, CD45 remaining in the vitreous or retina of IL-1β-injected eyes
No + cells were observed (Fig. 8B). At the time of this measurement, no CD45 + cells were observed in the bFGF-injected eyes (Fig. 8C). (FIGS. 8A-C show retinal cryosections of CD45-stained dystrophic RCS rats. FIG. 8A shows vehicle-injected eyes 4 weeks after injection, FIG. 8B shows IL-1β injection 4 weeks after injection. Eyes, Figure 8C, show bFGF-injected eyes at 4 weeks post-injection, with low levels of CD45 in all cases, indicating very few infiltrating leukocytes).

The results presented here show that both exogenous IL-1β and bFGF induce substantial photoreceptor cell rescue in the rat. However,
These results are the first to show that low dose IL-1β rescues PRC without causing the concomitant retinal dysplasia seen in the prior art at high dose infusion. "Low dose" IL-1β is <about 50 μg / ml, preferably <2
Refers to a concentration upon topical administration of 0 μg / ml, conveniently <10 μg / ml. These concentrations are based on the specific action of human recombinant IL-1β (R & D Systems, USA).

Administration of the low dose IL-1β described herein is 100-1 less than administration of bFGF.
It is a potent inducer that rescues photoreceptor cells at concentrations as low as 000 times (Faktorovic
h et al., 1990).

The role of bFGF as a neurotrophic factor (Echenstein, et al.), As well as the role of IL-1β in the inflammatory cascade (Dimarello), is well established.
The mechanism by which pro-inflammatory signals trigger the expression of new cytokines and trophic factors important for wound repair is just beginning to be elucidated. In this stream, IL-1β is a potent inducer of bFGF in various settings including the CNS (Aravjo et al., Rivera et al.). The presence of IL-1β promotes bFGF production by glial cells. This results in I, as observed here.
There is one possible explanation for L-1β being neuroprotective even at low doses.

Furthermore, intravitreal injection of IL-1β or bFGF has been observed to increase the expression rate of mononuclear cells in the retina (Faktorovish, 1990; LaVail et al.). Mononuclear cells, especially activated macrophages, secrete both IL-β and bFGF (Avron et al., Baird et al.). There is also evidence that these factors, once present, are capable of inducing their own expression (Mauviel et al., Flott-Rahme et al.). Thus, (a) topical administration of either factor (IL-1β or bFGF) to the periretinal region induces an increase in the concentration of both factors, and (b) in vivo.
Therefore, the action of one factor is considered to reflect the existence of the other factor to some extent.

Interestingly, although IL-1β and bFGF are both present in a variety of animal species, injury-related PRC rescue is associated with relatively mild microenvironmental injury, and It is particularly prominent in the rat, an animal in which significant expression of bFGF occurs (Wen et al., Cao et al.). The cases for the role of bFGF are widespread, and due to the ubiquity, potency, and ability to upregulate trophic factors (including bFGF) of IL-1β, IL-1β was able to reduce PRC even at low doses.
It is suggested that it will significantly contribute to salvation.

Effect of IL-1β on cells The IL-1 family of cytokines shares considerable sequence homology with the FGF family, and both families are proposed to result from replication of a common ancestral gene. (Zhang et al.). IL-1β is a secreted IL-1 family member that plays a major role in the early stage of the inflammatory cascade (Dinarello
). Under typical circumstances, although hyperproliferative inflammation is itself responsible for tissue damage (Geiger et al., Jeohn et al., Andersson et al., Theofilopoulos et al.), The inflammatory response is an early process of tissue regeneration and repair. It can also be considered. In this flow, it is not necessary to consider that the neurotoxic effect and the neuroprotective effect of IL-1β are contradictory.

To date, IL-1β has been implicated in neuropathological processes in a series of studies (Jeohn et al., Rothwell et al., Toulmond et al., Downen et al., Downen et al., Pearson et al.).
IL-1β potentiates the destructive effects of various other drugs. In particular, when IL-1β and nitric oxide (NO) are used together, cytotoxicity to cultured neurons occurs (Ch
ao et al., 1996; Hu et al.). Several researchers have studied in vitro (Gahring et al.) And in vi
Some neuroprotective effects of this cytokine in vo (LaVail: Klusman et al .; Wang et al.) have been reported but have been shown to be associated with the destructive effects of IL-1β. In contrast, the results obtained here demonstrate that low doses of IL-1β exert a major neuroprotective effect without causing substantial dysplasia of the neural tissue at the site of administration. This is the first result.

The mechanism by which IL-1β exerts a neuroprotective action is the IL-1RI receptor (Ohtsuk
i)), the transcription factor NFkB (Yu et al.), and possibly the expression of the "downstream" gene, albeit conservatively. Interestingly, NFkB was also involved in the expression of numerous pro-inflammatory cytokines by activated macrophages (Kelly et al.), Some of which were also involved in neuroprotection (Carlson et al., Klusman). Etc.).

Thus, one of the many functions that IL-1β has is the early warning or response of cells that play a role in promoting cell survival in the face of various noxious stimuli, including the inflammatory response itself. It is thought to be a function of stress signals (Neta et al.,
Lee et al., Galcheva-Gargova et al., Han et al.). It is likely that cells that did not enter this protective mode, probably due to malfunction, were at increased risk of cell death with an increased inflammatory response. ⁷⁸ Central nervous system (CNS) neurons (exemplified by PRC cells) are less susceptible to replacement and thus respond to low doses of IL-1β and show marked anti-apoptotic responses. For other effects of this cytokine,
It is likely that multiple new genes are triggered as part of the amplification cascade, resulting in a protective response.

Photoreceptor Cell Rescue Promoting PRC survival by low-dose IL-1β is of particular interest for retinal dysplasia, which, due to genetic error, causes extensive photoreceptor cell apoptosis and severe visual impairment (Bird; Stone J et al.).

Although not critical to the efficacy of the low dose IL-1β administration taught herein, it is believed that the observed PRC rescue effect is indicative of prolonged survival of pre-existing photoreceptor cells. References

[Brief description of drawings]

FIG. 1 is a three-dimensional view of a sampling process used for counting the number of photoreceptor cells in each retina.

FIG. 2A-B shows the mean number of photoreceptor cells in dystrophic colored rats treated with various agents.

FIGS. 3A-B show mean PRC numbers in dystrophic albino rats dosed with various agents.

Figures 4A-D show retinal sections of H & E stained historesin-embedded dystrophic RCS rats.

FIG. 5: CD45 staining (A and C at 24 hours after IL-1β injection.
) And H & E stained (B and D) retinal cryosections of dystrophic RCS rats.

6A-D show retinal cryosections of CD45-stained (A and C) and H & E-stained (B and D) RCS rats 48 hours after IL-1β injection.

7A-D show retinal cryosections of CD45-stained (A and C) and H & E-stained (B and D) RCS rats 72 hours after IL-1β injection.

8A-C show retinal cryosections of CD45 stained dystrophic colored RCS rats after vehicle (A), IL-1β (B) or bFGF injection.

─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor White Ray, Simon, Jay.             United States 02174 Massachusetts             Arlington Teal Strey             To 48 Apartment 1 (72) Inventor Young, Michael, Jay.             United States 01930 Massachusetts             Gloucester The Heights 1002 F-term (reference) 4C084 AA02 BA44 DA13 MA58 NA14                       ZA012 ZA332                 4H045 AA30 BA10 CA40 DA03 EA20

Claims (19)

[Claims] 1. Without causing substantial dysplasia of dystrophic nerve tissue,
Low dose of interleukin 1-β (IL-1 showing effects in neuronal cell rescue
A method of rescue of neuronal cells in a dystrophic nerve tissue site, which comprises locally administering a composition comprising β) to at least one dystrophic nerve tissue site. 2. The method of claim 1, wherein the at least one dystrophic neural tissue site is the dystrophic retina. 3. The method of claim 1 or claim 2, wherein said composition comprises IL-1β at a concentration ≦ about 50 μg / ml. 4. The method of claim 1 or claim 2, wherein said composition comprises IL-1β at a concentration ≦ about 20 μg / ml. 5. The method of claim 1 or claim 2, wherein the composition comprises IL-1β at a concentration ≦ about 10 μg / ml. 6. The method of claim 1 or 2, wherein the composition comprises IL-1β at a concentration of about 0.1-10 μg / ml. 7. The method of claim 2, wherein the composition comprises an IL-1β solution at a concentration ≦ about 50 μg / ml and the composition is administered intravitreally in a volume of about 0.1-10 μl. 8. The method of claim 2, wherein the composition comprises an IL-1β solution at a concentration ≦ about 20 μg / ml and the composition is administered intravitreally in a volume of about 0.1-10 μl. 9. The method of claim 2, wherein the composition comprises an IL-1β solution at a concentration ≦ about 10 μg / ml and the composition is administered intravitreally in a volume of about 0.1-10 μl. 10. Retinal dystrophy is pigmented retinitis, pyramidal dystrophy, dystrophy based on retinal pigment epithelium (RPE), retinal degeneration, macular degeneration, retinal ganglion cell loss, glaucoma, optic nerve. 3. The method of claim 2, wherein the method is the result of a member selected from the group consisting of disorders and retinal detachment. 11. Use of a targeted drug delivery vehicle for local administration of the composition, which locally releases ≦ 50 μg / ml of IL-1β only upon reaching the nerve tissue site or surrounding area. A method according to claim 1 or claim 2. 12. The topical administration of the composition uses a sustained release drug delivery vehicle that locally releases ≦ 50 μg / ml IL-1β at a dystrophic nerve tissue site for an extended period of time. The method according to claim 1 or claim 2. 13. A packaging material and a composition contained in the packaging material,
The composition is effective in salvaging neuronal cells at its local site, dystrophic nerve tissue site, wherein the composition has a low dose of IL-1β in the range of ≦ about 50 μg / ml.
Wherein the packaging material rescues neuronal cells remaining at the dystrophic nerve tissue site, which is the site of local administration of the composition, and minimizes dysplasia of the nerve tissue at the site caused by the administration. Consisting of a label that can be used for
Industrial products. 14. The industrial product according to claim 12, wherein the dystrophic nerve tissue site is a dystrophic retina. 15. A packaging material and a composition contained in the packaging material,
Low dose of IL-1β, wherein the composition is effective in rescue photoreceptor cells without causing substantial dysplasia of at least one retina of its topical administration site
Wherein the low dose IL-1β is ≦ about 50 μg / ml, and the packaging material is
A method of rescuing at least one photoreceptor cell in a dystrophic retina while minimizing the substantial retinal dysplasia caused by IL-1β administration to the retina, to a person with a dystrophic retina An industrial product, which comprises the instructions to administer the composition. 16. The dystrophic retina is pigmented retinitis, cone dystrophy, dystrophy based on retinal pigment epithelium (RPE), retinal degeneration, macular degeneration, retinal ganglion cell loss, glaucoma, 16. An industrial product according to claim 14 or claim 15 which is the result of a member selected from the group consisting of optic neuropathy and retinal detachment. 17. The method of claim 1, wherein the dystrophic nerve site is the diseased or damaged spinal cord. 18. The method of claim 1, wherein the dystrophic nerve site is a CNS site having a disease or injury. 19. The method according to claim 1, wherein the dystrophic nerve site is a brain lesion.