WO1993021911A1

WO1993021911A1 - Method and means for preventing constriction of the pupil in the eye

Info

Publication number: WO1993021911A1
Application number: PCT/SE1993/000391
Authority: WO
Inventors: Birgitta ALMEGÅRD; Anders Bill; Johan Stjernschantz
Original assignee: Kabi Pharmacia Ab
Priority date: 1992-05-06
Filing date: 1993-05-05
Publication date: 1993-11-11
Also published as: AU4031193A; SE9201425D0

Abstract

The use of antagonists to fragments of calcitonin gene-related peptide and derivatives of these for preventing miosis in the eye, for example during surgery and in cases of uveitis. The invention also comprises ophthalmological compositions containing an active amount of antagonists to fragments of CGRP as well as their use for preparing such a composition for preventing miosis in the eye.

Description

METHOD. AND MEANS FOR PREVENTING CONSTRICTION OF THE PUPIL IN

THE EYE

* The invention relates to the use of antagonists to calcitonin gene related peptide (CGRP) , and fragments thereof, for preventing miosis in the eye, for example during surgery and in cases of uveitis.

The size of the pupil in the eye is governed by two muscles in the iris, opposite in character in respect of their mode of action. One of these muscles when undergoing contraction will produce a dilatation of the pupil (dilatator muscle) ; it is controlled by nerve fibres from the sympathetic nervous system. The other muscle (sphincter muscle situated near the iris edge region, i.e. near the pupil) will cause a diminution of the pupil. This muscle is governed by parasympathetic nerves which utilize acetylcholine as transmitter.

It has been known for a long time that constriction of. the pupil, i.e. miosis, can be caused by mechanisms other than acetylcholine release. In particular this occurs as part of the eye's response to various kinds of irritation. Such miosis cannot be prevented by antagonists of acetylcholine such as e.g. atropine. Clinically miosis of the type produced by irritation often implies substantial complications, e.g. in intraocular surgery. In cases of uveites (inflammation of the uvea), especially iritis, this type of miosis will sometimes give rise to very undesirable synechiae between the. iris and the lens. Miosis caused by irritation has been studied thoroughly in experimental animals and it has been found for instance that in rabbits - such irritation is caused by a peptide very similar to or identical with substance P released in the eye (Bill et al., 1979) . When synthetically produced substance P was injected into the anterior chamber this produced a substantial contractive-response of the sphincter muscle of the iris, thus suggesting that this miosis is brought about by the said peptide or a closely related substance.

It should be noted, however, that there are considerable variations from species to species as regards to pupillary responses to substance P — despite the fact that this neuropeptide has been detected immunohistochemically in the eyes of many species, including primates. It has been found inter alia that this response is totally absent in primates (Mandahl et al., 1980) and that therefore in primates irritation-caused miosis must be due to some other substance. Thus, the effect of certain neuropeptides is dependent on the presence or absence of receptors in the tissue innervated by sensory nerves, and such presence or absence of receptors di fers very much between animal species. For this reason it is entirely impossible to predict whether conditions valid in e.g. rabbit iris will be valid to the iris of monkeys or humans, and vice versa, even though the same peptide may have been identified in sensory nerves of the iris in different species.

In the rabbit, irritation will cause substance P to be released from the sensory nerve fibres in the iris. The iris sensory nerves contain a number of other potential trans¬ mitters, one of these being cholecystokinin (CCK) or a substance related to CCK; see Kuwayama et al., 1987. Another substance in this group is calcitonin gene-related peptide (CGRP) (ϋnger et al. , 1985).

While looking for the substance that is responsible in primates for the miosis response to local irritation we have unexpectedly found that certain active fragments of CGRP, which has the amino acid sequence CGRP

, s S j

Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-

Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-

Thr-Asn-Val-Gly-Ser-Gly-Ala-Phe-H2N

have potent miotic effects in monkeys. Such active fragments are in particular fragments 32-37 and 30-37 with the amino acid sequences

CGRP 32-37

Val-Gly-Ser-Glu-Ala-Phe-H₂N, and

CGRP 30-37

Thr-Asn-Val-Gly-Ser-Glu-Ala-Phe-H2N

The mechanism of action of a CGRP fragment (e.g. 32-37) induced miosis could be that CGRP which is known to be released from sensory nerves in the eye (Wahlestedt et al. , 1986) is cleaved by proteases in the iris rendering fragments acting on specific receptors which mediate pupillary constriction in the eye.

A dose of about 37 pmoles or fragment 32-37 injected into the anterior chamber will give a half-maximal effect.

Experiments carried out moreover show that Loxiglumide, a cholecystokinin (CCK) antagonist, antagonizes CGRP fragment 32-37 in vivo with respect to the miosis response. Upon injection of 1.2 nanomoles of Loxiglumide into the anterior chamber the dose-response curve of CGRP 32-37 was shifted to the right by several orders of magnitude- (Figure 1) . It now seems possible that the miosis seen in human beings in irritative conditions ,such as iritis, uveitis, and in cases of surgery in the anterior chamber, is due to the release of one or more fragments of CGRP. Consequently it ought to be possible to prevent this miosis by means of antagonists to such CGRP fragments. Antagonists to CGRP receptors in general, such as CGRP 8-37, may be beneficial also for blocking the effect of shorter CGRP fragments.

Unexpectedly, we have found that at least certain antagonist to cholecystokinin such as Loxiglumide are also active against the pupillary contraction induced by fragments of CGRP, such as CGRP 32-37.

While CGRP fragment antagonists so far are poorly known, several antagonists to CCK-receptors have been developed and at least some of these might also, as indicated above, exert an antagonistic effect on fragments of CGRP, which cause miosis. The use of antagonists to CCK for preventing pupil constriction in the eye is one aspect of EP422181 and the use of substances disclosed in that patent application is therefore excluded from the present application.

The possibility of preventing undesired noncholinergic miosis by means of antagonists to fragments of CGRP, particularly fragment 32-37 is now presenting itself as a highly interesting, novel concept for potential therapies of practical importance, in the first place in such contexts where the lens is being extracted and this is then followed by insertion of an artificial lens, but also in contexts of other intraocular surgeries, for instance surgery in the posterior segment. Blockage here can be effected by way of local and/or optionally general administration of the antagonist. The present invention thus relates to antagonists of CGRP and active fragments thereof, particularly fragments 32-37 and 30-37 and derivatives and analogues of such fragments, for preventing miosis as is liable to occur as a consequence of intraocular surgery, trauma, or uveitis and iritis. Only pharmaceutically active and physiologically acceptable antagonists to above mentioned peptide fragments are of course intended to be used according to this invention.

The invention accordingly relates to compositions which contain an effective amount of antagonists to CGRP and/or active fragments thereof, particularly fragments 30-37 and 32-37, or derivatives or analogues of these fragments in an ophthalmologically compatible vehicle for preventing miosis during intraocular surgery or as a consequence to trauma and uveitis or iritis. The term "surgery" includes also treatment of the eye with laser beams of various kinds. The term "effective amount" here means that the composition contains from 10 ng to 10 mg of one or more antagonists, depending on whether it is introduced directly into the anterior chamber of the eye, for instance after an operation, or whether it is applied topically on the cornea or is applied subconjunctivally. If the antagonist is administered systemically the dose range is preferentially about 0.01-50 mg/kg body weight.

The ophthalmologically compatible vehicle that may be employed for preparing compositions according to this invention consists of aqueous solutions such as for example physiological salines for compositions to be inserted into the eye, e.g. into the anterior chamber or subconjunctivally.

The ophthalmologically compatible vehicle that may be employed for preparing compositions for topical use consists of aqueous solutions such as for instance physiological salines, oil solutions or ointments. Furthermore the vehicle may contain - especially in cases where the composition is intended for topical use - ophthalmologically compatible preservatives such as e.g. benzalkonium chloride, sur¬ factants, liposomes or polymers, e.g. methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, hyaluronic acid, which may be employed for the purpose of increasing viscosity. Also soluble and insoluble drug inserts may be used to administer the antagonists to CGRP fragments. The invention is in a further aspect related to the use of antagonists to CGRP and/or active fragments thereof, as defined above, for preparing a composition for preventing miosis in the eye.

The invention moreover also relates to a method of preventing miosis induced by surgery (including also laser treatment) , trauma, uveitis or iritis. The method comprises administration of a therapeutically active amount of a composition containing at least one of the substances defined above. In a preferred embodiment a composition as described above is contacted with the eye so as to prevent miosis. The composition contains antagonists to CGRP related substances, including active fragments thereof, inducing miosis. The examples of such CGRP related substances include, besides fragments described above, also derivatives or analogues of CGRP fragments.

The invention is illustrated by a series of non-1imitating examples; results are set forth in Figures l and 2 as follows:

Figure 1:

Four different C-terminal fragments of CGRP studied for contractile effects on the cynomolgus monkey iris in vivo. EC-50 of fragment 32-37 was 37xl0-12 moles. Figure 2:

Antagonistic effect of Loxiglumide on the miotic effect of CGRP fragment 32-37.

Experiments

Example 1; Monkeys employed in these experiments (Macaca fascicularis) were anaesthetized with pentobarbital. The anterior chambers of the eyes were cannulated with 2 special needles each. Substance could be injected through one of the needles which was connected via a polyethylene tube to a syringe for small volumes (lOOμl) . A corresponding volume of aqueous humour could be drawn off via the other needle to thus avoid eye pressure alterations due to the intracameral injection of the test substances. The test substances, various fragments of CGRP, were dissolved in isotonic saline.

The animals were first treated with atropine to induce maximal dilatation of the pupil. The dose-response relation¬ ship was then determined for one eye. Pupil size was recorded as obtained by measuring the horizontal diameter of the pupil by means of a graduated ruler, these measurements being made at regular intervals and under standard light conditions. Typical examples of these experiments are shown in Figure 1.

Example 2: Dose-response determination of CGRP fragment 32- 37 was carried out on. one eye of atropine treated, pentobarbital anaesthetized monkeys in accordance with the description in Example 1. Thereafter 1.2 nanomoles of Loxiglumide dissolved in isotonic saline was injected into the anterior chamber of the other eye. The CGRP 32-37 dose- response relationship was then determined in the same way as in- the case of the first eye. Loxiglumide produced a shift of the dose-response curve by several orders of magnitude (Figure 2) .

Our experiments thus show that fragments of CGRP, particularly fragments 32-37 and 30-37-, induce miosis in primates and therefore in all likelihood also in humans, by stimulating specific receptors in the iris. Blockade of said receptors, using any of a variety of antagonists, will counteract this miosis. Consequently it seems possible that the mechanism responsible for irritation miosis in primates resides in a release of CGRP or a closely related substance which subsequently is activated by enzymatic cleavage to shorter fragments which produces miosis by way of stimulating specific receptors on smooth muscle cells in the iris sphincter muscle. This miosis is independent of acetylcholine.

REFERENCES

Bill, A. Stjernschantz, J. , Mandahl, A., Brodin, E. & Nilsson, G. 1979. Substance P: Release on trigeminal nerve stimulation, effects in the eye. Acta Physiol Scand 106:371- 373.

Kuwayama, Y., Terenghi, G . , Polak, J.M., Trojanowski, J.Q. & Stone, R.A. 1987. A quantitative correlation of substance P-, calcitonin gene-related peptide- and cholecystokinin- like immunoreactivity with retrogradely labelled trigeminal ganglion cells innervating the eye. Brain Res. 405:220-226.

Mandahl, A., Brodin, E. , Nilsson, G. & Bill, A. 1980. Substance P, release and effects in the eye. Acta Physiol Scand. 108:18A.

Unger, W.G., Terenghi, G. , Ghatei, M.A. , Ennis, K.W. , Butler, J.M., Zhang, Q.S., Too, H.P., Polak, J.M. and Bloom, S.R. (1985) . Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response. J. Ocular Pharmacol 1, 189-199.

Wahlestedt, C. , Beding, B. , Ekman, R. , Oksala, 0., Stjernschantz, J. and Hakansson, R. (1986). Calcitonin gene- related peptide in the eye: Release by sensory stimulation and effects associated with neurogenic inflammation. Regulatory peptides. 16: 107-115.

Claims

1. Use of an antagonist to calcitonin gene-related peptide (CGRP) or active fragments thereof, with the exclusion of cholecystokinin antagonist, for the manufacture of a composition for preventing pupil constriction in the eye.

2. Use according to claim 1, wherein the antagonist is an antagonist to CGRP fragment 30-37 or 32-37

3. Ophthalmological composition for the prevention of pupil constriction, characterized by containing a therapeutically active amount of a pharmaceutically active and physiologically acceptable antagonist to CGRP or an active fragment thereof, with the exclusion of cholecystokinin antagonists, in an ophthalmologically compatible vehicle.

4. Ophthalmological composition according to claim 3, characterized by containing an antagonist to CGRP fragment 30-37 or 32-37.

5. Ophthalmological composition according to claims 3 or 4, characterized in that the ophthalmologically compatible vehicle is a physiological salt solution, an oil solution or ointment, and optionally contains also ophthalmologically compatible preservatives, surfactants, liposomes or polymers.

6. A method for preventing pupil constriction in the eye which comprises administration of a pharmaceutically active amount of an antagonist to CGRP or an active fragment thereof, with the exclusion of cholecystokinin antagonists, to the eye.

7. A method according to claim 6, characterized by using an antagonist to CGRP fragment 30-37 or 32-37.