WO1992017175A1

WO1992017175A1 - Ophthalmic uses of sigma site binders

Info

Publication number: WO1992017175A1
Application number: PCT/US1992/001847
Authority: WO
Inventors: Ronald D. Schoenwald; Charles F. Barfknecht; Roger E. Newton
Original assignee: University Of Iowa Research Foundation
Priority date: 1991-03-29
Filing date: 1992-03-05
Publication date: 1992-10-15
Also published as: AU1747992A

Abstract

Tear stimulants which stimulate natural tear production by topically applying to the eye compounds wich are sigma site binders and therefore activate tear production.

Description

OPHTHALMIC USES OF SIGMA SITE

BINDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Serial No. 557,581, filed July 24, 1990, which itself is a continuation-in-part of Serial No. 257,826 filed October 14, 1988, which is a continuation of Serial No. 168,680 filed March 16, 1988, now U.S.

Patent 4,820,737 dated April 11, 1989, which itself is a continuation of Serial No. 015,117 filed

February 17, 1987, now abandoned.

BACKGROUND OF THE INVENTION

Tear film disturbances account for eye symptoms in millions of Americans. At the present time, treatment primarily consists of replacing a

defective tear film with artificial tear substitutes which are sold over-the-counter. The major

limitation of these products is their short retention time in the eye. Patients must apply drops as often as every hour to obtain comfort from these products. Recent accomplishments have focused on developing aqueous solutions containing components which will stabilize the tear film or replace specific

deficiencies.

A normal tear film is the product of: (1) aqueous secretion by the lacrimal gland and accessory lacrimal glands; (2) secretion of mucous primarily by the goblet cells of the conjunctiva; and, (3) lipids secreted by the meibomian gland and the glands of Zeis and Moll in the lids. Mucin, the innermost layer (0.035 μm), wets the lipophilic epithelial surface of the cornea with the middle aqueous layer. The aqueous layer (7 μm) contains dissolved proteins, carbohydrates, glycoproteins, oxygen, and inorganic salts. The outer lipid layer (0.1 μm) retards evaporation of the aqueous component.

Dry eye syndrome or keratoconjuctivitis sicca (KCS) can occur secondarily to many autoimmune diseases, and as a result of abnormalities in the precorneal tear film physiology. Besides an

awareness of a chronically irritable sore eye by the patient, clinicians can diagnose dry eye syndrome by various measurements. These include a tear breakup time of 10 seconds or less in the absence of

blinking, or a Schirmer test value of 5 mm or less. The latter involves leaving a standard strip of filter paper under the lower lid for 5 minutes.

Other measurements are also helpful and include an observation of a smaller than normal marginal tear strip upon slitlamp examination, and/or a positive rose bengal stain which detects the presence of precipitated mucin and devitalized cells. The stimulation of aqueous tears by a drug acting on the autonomic nervous system is an approach that had in the past limited success via a systemic route and little success via a topical route of administration. For example, some ophthalmologists have recommended oral ingestion of very dilute solutions of the cholinergic, pilocarpine, to

stimulate tear secretion. However, unpleasant side effects have discouraged widespread use of ingested pilocarpine.

The stimulation of aqueous tears by the systemic or oral route has the undesirable side effect of causing systemic drug reactions by materials such as pilocarpine and other cholinergics. Moreover, by the time the active drug transfers itself through the body to the eye, its effect is significantly diluted. To date, there is no known effective composition for topical route of administration to treat dry eye syndrome.

As earlier indicated, the treatment with tear replacement compositions is not totally satisfactory because of their short retention time in the eye.

Often the use by sufferers of dry eye syndrome of tear replacement products must continually apply drops even as often as every hour to obtain eye comfort. Moreover, especially for wearers of contact lenses, this problem of short time retention becomes quite real, rendering tear replacement unsatisfactory. In short, sufferers of dry eye syndrome are currently, for all practical purposes, excluded from the possibility of wearing contact lenses, since those lenses and their effective use, to say nothing of their comfortable use, necessarily depends upon adequate tear production.

U.S. Patent 4,820,737 issued April 11, 1989 to two of the present case inventors relates to certain low toxicity compounds as tear stimulants.

Certain compounds including those of low

toxicity of the '737 patent have been discovered to bind to specific molecular recognition sites (drug receptor or neurotransmitter receptor sites) within tissue. The primary area in which early work on receptor site discoveries have occurred has been the brain due to the high population and diversity of such sites within nervous tissue although such sites are present throughout the body. Awareness of

receptor sites in other parts of the body has emerged from pharmacological studies originally intended to discover the bases of action for drugs empirically found to be useful in the treatment of various

disorders. It is believed that many disorders, particularly those of the nervous system, are due to altered activity in various receptor populations. Through study of the regional distribution and activity of these varied receptor systems a picture of tissue function can be derived. The initial step in any such study involves the selection of compounds which will selectively interact with the sites of interest.

One neurotransmitter-receptor system of interest is the dopaminergic system. The relationship of the activity of this system to psychopathological

symptoms is not entirely clear, however, its

importance is inferred through the effectiveness of dopamine receptor antagonists in abating psychotic symptoms. A number of compounds of different

classes have this desired effect and would lend themselves to adaptation for study of their sites of activity. These would include drugs of the

butyrophenone class such as spiperone, haloperidol (Haldol) and droperidol; drugs of the phenothiazine class such as trifluoperazine and thiothixene; and drugs of the benzamide class such as sulpiride.

Alternatively, dopamine receptor agonists, such as bromocriptine, might serve as useful ligand

substrates for study of the dopaminergic system, particularly among individuals suffering from

parkinsonism. The term "ligand" is used herein to generally describe the tissue binding portion of the active molecules referred to above. It can be seen that for the most part the molecules are psychoactive drugs. However, from time to time the broader term "ligand" is used because many of the useful compounds may well be derived from psychoactive drugs, but they themselves may not be the active drug form.

Pharmacological, biochemical and behavioral characteri-zation of sigma binding sites is currently the focus of intense, widespread investigation.

While the precise nature of sigma binding sites in cells is not quite known, many studies have

suggested that it represents the site of action for a number of important drugs. For example, haloperidol, a butyrophenone antipsychotic, exhibits high

affinity for sigma binding sites and several

psychotominetics, including PCP and benzomorphantype compounds (pentazocine), also bind at this site.

Thus, strong binding sigma agents are indicative of usefulness in the treatment of schizophrenia.

In fact, several sigma compounds have been developed as antipsychotics. It is also believed that strong sigma site binding may also indicate therapeutic targets for epilepsy and brain ischemia. In sum, the discovery of sigma binding sites has prompted investigation into the functional role of the sites. While the functional role is not

precisely understood, it is nevertheless true that binding studies have revealed sigma sites which may exhibit a unique pharmacological profile, and have provided evidence favoring the existence of a

multiplicity of sigma binding sites in the central nervous system. There is therefore a continuing investigation and search for psychoactive compounds having a strong affinity for sigma binding sites. A prior application of the present applicants in Serial No. 557,581, filed July 24, 1990 relates to use of the tear stimulant compounds of U.S. Patent 4,820,737 anti-psychotics.

As earlier stated, the prior invention of joint applicants Schoenwald and Barfknecht in U.S. Patent 4,820,737 generally relate to use of certain

arylalkylamines as natural lacrimal secretion

stimulants. The prior application of joint inventors Schoenwald, Barfknecht and Newton relates to the discovery that those same lacrimal secretion

stimulants are psychoactive drugs binding as sigma site binders.

The present invention is predicated upon the discovery that sigma binding sites are present in lacrimocytes, and therefore as a general matter, sigma site binding activity means ophthalmic

activity, particularly as lacrimal secretion

stimulants.

The primary objective of the present invention is to provide as effective tear stimulant tear compositions, which can be administered as topical compositions which contain active sigma site binders.

Another objective is to provide a protocol for selecting ophthalmically active compounds, by

screening such compounds for sigma site binding activity.

Another objective of the present invention is to provide to the art the fundamental knowledge that sigma site binders are present in certain ophthalmic cells, and thus sigma site binding compounds are ophthalmically active as a general matter.

Other objectives of the present invention are wholly consistent with the objectives of the earlier U.S. Patent of joint inventors Schoenwald and

Barfknecht, namely U.S. Patent 4,820,737 and the earlier application of the three joint inventors for this application, namely Schoenwald, Barfknecht and Newton, identified as PSYCHOACTIVE DRUG AND

TREATMENT, Serial No. 557,581 filed July 24, 1990.

The entire disclosure of U.S. Patent 4,820,737 and of patent application Serial No. 557,581 are

incorporated herein by reference, since both

logically relate to significant parts of the present invention.

SUMMARY OF THE INVENTION

This invention relates to a method and

composition for stimulating lacrimal secretion. The method comprises topically applying to the eye an ophthalmically effective amount of a sigma site binder. Because the treatment involves stimulation of tear production by the eye itself, as opposed to tear replacement, there is not a continuing need for eye drop addition on a nearly hourly basis, as there is with current tear replacement compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a drawing showing treatment of the normal rabbit eye with some of the active compounds of the present invention and with a certain prior art compound bromhexine hydrochloride, which has been reported in published literature, especially German literature as having some effective use as a tear stimulant, see Prause, Acta Ophthalmologica 62,

(1984) 489-497, entitled "Lacrimal And Salivery

Secretion In Sjogrens Syndrome: The Effect of Systemic Treatment With Bromhexine" and U.S. Patent No. 4,436,091, issued March 20, 1984 to Gruber et al.

Figure 2 shows a graph illustrating test results for a known sigma site binding psychoactive drug when used in a topical installation of isotonic buffered drug in comparison with a non-treated eye, and demonstrates that psychoactive sigma binding

derivatives have ophthalmic activity as lacrimal stimulants.

DETAILED DESCRIPTION OF THE INVENTION

In its broadest sense, this invention provides a method of topically applying to the eye for treatment of dry eye syndrome and for tear stimulation of a small but ophthalmically effective amount of a sigma site binder. Sigma site binders which may be used are selected from the group consisting of natural and synthetic opioids, butyrophenones, arylamines, sigma site hallucinogens and sigma site binding anti-psychotics.

The arylalkylamine compounds useful for the present invention as sigma site binders, and

therefore natural tear stimulants are those disclosed in earlier U.S. Patent 4,820,737. In particular, they are compounds that have the general formula:

wherein Y is selected from the group consisting of hydrogen, hydroxy, amino, C₁ to C₅ alkyl, C₁ to C₅ hydrogen, hydroxy, amino, C₁ to C₅ alkyl, C₁ to C₅ hydroxyalkoxy and C₁ to C₅ alkoxy; Z is selected from the group consisting of C. to C₆ alkylene, C₁ to C₆ oxyalkylene, and C₁ to C₆ aminoalkylene; R is

selected from the group consisting of C₁ to C₆ alkyl and C₃ to C₇ cycloalkyl; R' is selected from the group consisting of hydrogen, C₁ to C₆ alkyl and C₃ to C₇ cycloalkyl; R" is selected from the group of hydrogen and C₁ to C₆ alkyl; and X is a

pharmaceutically acceptable counteranion.

Y can be in either an ortho-, meta- or paraposition and is preferably hydrogen. Z likewise represents a moiety which can be ortho-, meta- or para- with respect to the Y, but is preferably parapositioned with respect to Y, and is preferably a C₁ to C₆ alkylene, and most preferably C₂ to C₄ alkyl, R is preferably a C₁ to C₃ alkyl, and most preferably methyl. R' is preferably cycloalkyl, most preferably cyclohexyl. R" is preferably hydrogen or C₁ to C₃ alky. X, as earlier mentioned, represents any pharmaceutically acceptable counteranion and is preferably a halogen, and most preferably chloride or bromide.

It can be seen that the compound as represented in the formula shown above is a quaternary ammonium ion salt form. If R" is hydrogen, the compound represents a tertiary amine salt. Other biologically acceptable salt forms of the compounds represented by the general formula above may of course be

employed and are contemplated for use in this

invention, as long as they have the necessary organic structure to provide the ophthalmically active tear stimulant when topically administered, and are still in a form which is pharmaceutically acceptable for topical administration, i.e. generally soluble in acceptable pharmaceutical carriers.

The opioids that are useful for the present invention represent compounds such as morphine and its natural and synthetic derivatives. Examples include the benzomorphans and synthetic opiods such as cyclazocine, pentazocine and N-allylnormetazocine.

Examples of butyrophenones which can be used include the compound commercially sold as Haldol™, spiperone, droperidol, trifluperidol, and 1-[4-fluorophenyl]-4-[4-(5-fluoro-2-primidinyl)piperazinyl]-1-butanol. An additional class of sigma site binders that may be useful in the present invention include sigma site hallucinogens such as phencylidine. The fourth group includes sigma site binding anti-psychotics as illustrated by rimcazole.

The ophthalmically effective tear stimulant compositions containing the above described active compounds will generally contain a small but tear stimulating effective amount of the active in an ophthalmically acceptable carrier. On a weight/ volume basis it has been found that the amount of active may generally be within the range of about 0.1% to about 5%, and preferably from 0.2% to about 0.6% by weight/volume basis. The amounts of the active compound within these ranges, dissolved in suitable ophthalmically acceptable carriers have been demonstrated to effectively provide tear stimulation in the tests below described.

Suitable ophthalmically acceptable carriers are generally known and of course must be non-eyeirritating, non-toxic, and allow for safe, easy eye administration topically. Generally, for this invention, aqueous-base systems wherein the carrier includes a buffer system to provide eye safe pH, a viscolyzer to provide suitable viscosity for eye comfort, an antibacterial agent, and a chemical preservative are adequate. The ophthalmically acceptable buffer should provide a composition having a pH within the range of about 5.5 to about 7.8, preferably from about 6.8 to about 7.4.

Suitable ophthalmically acceptable buffers can be selected from the water soluble salt forms of citrate, borate, phosphate, carbonate, and acetate.

The viscolyzer suitable for use in this

invention should provide the composition with a viscosity within the range of from about 4

centipoises to about 100 centipoises, preferably from about 5 centipoises to about 35 centipoises.

Suitable viscolyzers can be selected from the group consisting of hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose and a

polyacrylamide sold under the trade name Gelamide™ 250 by American Cyanamide.

In addition, the ophthalmic composition ideally will include antibacterials to provide safety and efficacy for storage stability. The amount of antibacterial can be within the range of from about 0.005% to about 0.2% by weight/volume of the

composition. A suitable antibacterial would

include, for example, from about 0.005% to about 0.2% by weight/volume of benzalkonium chloride, from about 0.25% to about 0.5% of chlorobutanol, about 0.1% of thimerosal, about 0.05% methylparaben, about 0.01% propylparaben, and sodium chloride in an amount sufficient to make an isotonic solution.

Finally, chemical preservatives may also be used, for example sodium thiosulfate at about a 0.3% level and ethylenediaminetetraacetic acid at about 0.05%. It goes without saying that the precise ophthalmic carrier must be selected to provide pharmaceutical elegance, to provide eye comfort and to allow for effective topical administration.

Formulation of such is well within the skill of the ordinary artisan who prepares ophthalmic carrier compositions.

While the ophthalmic compositions of the present invention have been developed primarily to provide a method of topically administering to the eye a composition which treats dry eye syndrome by

stimulating the eye's own tear production, it is also contemplated that the compositions of this invention may also be useful in combination with tear

substitutes as well.

The following examples are offered to further illustrate but not limit the invention. The first series of examples shows preparation of compounds falling with the scope of the general formula for the active compounds of this invention, and the second series of examples, together with the drawing, illustrates application of these ophthalmically active compounds to the eye of rabbits to stimulate effective tear production. The rabbit eye is known to active investigators in the ophthalmic arts to closely parallel, and predict, and

correlate well with human eye activity.

Example 1

Preparation of N-cyclohexyl-N-methylbenzylamine The compound of N-cyclohexyl-N-methylbenzylamine was synthesized by treatment of N-methylcyclohexylamine (1) [1.98 ml, 1.70 g, 0.015 moles] with benzyl bromide (2) [1.78 ml, 2.56 g 0.015 moles] in the presence of potassium carbonate [2.28 g, 1.1 equivalents] in 4-methyl-2-pentaone (50 ml). The reaction mixture was heated under reflux overnight. After filtering the insoluble potassium salts, the filtrate was concentrated to dryness. The solid was dissolved in boiling 4-methyl-2-pentanone, treated with activated carbon, followed by hot filtration after which the purified compound precipitated upon cooling. It was collected by vacuum filtration, m.p. 173.5º-174.5°C. Proton NMR (CDCl₃) reported downfield from TMS: 1.11-1.92 ppm multiplet 10 H cyclic alkyl ring, 2.65 ppm singlet 3 H N-methyl, 3.01-3.11 ppm multiplet 1 H tertiary proton, 4.19 ppm singlet 2 H benzylic --CH₂--, 7.44-7.80 ppm 5 H aromatic; C-13 nmr 131.12, 130.19, 129,08, 128,40, 63.11, 55.12, 35.23, 26.37, 25.06, 24.33. El Mass Spec. M/e-(int) M+203 (28.4), M + + 1 204 (4.7), M + + 2 205 (0.2), 160(70.7), 146(10.6), 91(100),

92(7.1), 85(5.6), 82(5.2), 70(17.6), 65(24/7),

57(5.7), 56(5.3), 55(15.0), 43(3.6), 42(32.3),

41(20.1).

Example 2

N-cyclohexyl-N-methyl-2-phenylethylamine The preparation of N-cyclohexyl-N-methyl-2-phenylethylamine was accomplished in the following manner. This was prepared by treating a suspension of N-methylcyclohexylamine (1.98 ml, 0.015 moles, 1.70 g) and potassium carbonate (4.15 g, 0.03 moles, 2 eq.) in 4-methyl-2-pentanone (50 ml) with

phenethylbromide (2.05 ml, 0.015 moles, 2.78 g). The reaction was heated under reflux 16 hours. The insoluble material was removed by a hot gravity filtration and the filtrate was concentrated in vacuo on the rotary evaporator. The resulting oil was flash chromatographed on davisil 633 using 20% ethylacetate/hexane as the eluent. The appropriate fractions were pooled and concentrated. The oil was dissolved in ether and treated with HCl gas. The resulting precipitate was collected by vacuum filtration and recrystallized from absolute ethanol. m.p. 187°-188° C. El Mass Spec. M + 217(0.9),

126(92.2), 113(4.5), 112(1.2), 105(6.9), 91(32.2), 83(9.8), 77(11.6), 70(66.0), 65(15.9), 57(26.0), 55(39.8), 53(8.6), 45(2.8), 44(100), 43(8.2),

42(42.2), 41(34.9).

Example 3

Preparation of N-isopropyl-N-methylbenzylamine A flask containing N-methylbenzylamine (3.87 ml, 3.63 g, 0.03 moles), potassium carbonate (8.29 g, 0.06 moles) and 4-methyl-2-pentanone (100 ml) was treated with isopropyl bromide (2.69 ml, 0.03 moles) then heated under reflux for 18 hours. The reaction was checked by thin layer chromatography (tic) to ensure the starting materials had been consumed. The reaction mixture was gravity filtered hot to remove the potassium salts and the filtrate was

concentrated to an oil under reduced pressure on the rotovap. The oil was purified by flash chromatography (davisil 633, 20% ethyl acetate/hexane).

The appropriate fractions were pooled and

concentrated to an oil which was dissolved in

anhydrous diethyl ether. The ether solution was treated with gaseous HCl. The ether was removed under reduced pressure. The residue was dissolved in absolute ethanol, treated with activated carbon, filtered, and cooled. The liquid was concentrated with acetone to remove traces of water. The solid was collected by vacuum filtration, m.p. 127.5°- 128.5°C. Proton NMR (CDCl3 + TMS) 1.36 doublet J = 6.66 Hz 3 H ipr methyl, 1.51 doublet J = 6.66 Hz 3 H ipr methyl, 1.88 ppm NH + 2.62 doublet J = 5.04 Hz 3 H N-Methyl, 3.47 septette 1 H tertiary, 4.15 doublet J = 6.05 Hz 2 H benzylic CH_2-, 7.28-7.42 5 H

aromatic; C-13 (DMSO) ref:39.5, 131.16(11.88),

130.53(8.72), 129.12(5.74), 128.56(10.92), 55.99 (2.06), 55.11(7.14), 39.51(1.48), 34.36(6.93),

1845(3.11), 16.89(6.18), 15 19(5.20). El Mass spec. M/e-(int) M + 163(6.3), 148(40.7), 120(1.2), 92(8.0), 91(100), 90(1.5), 89(3.1), 78(2.2), 77(4.1),

65(18.8), 57(1.6), 56(8.1), 51(3.5), 44(6.0),

43(3.5), 42(11.8), 41(5.2).

Example 4

N,N-dimethyl-N-cyclohexylbenzylammonium Iodide A purified sample of the sample prepared in Example 1 as the free base (1.0 g, 4.0 mmoles) was dissolved in 10 g of absolute ethanol. Methyl iodide (0.82 ml, 1.86 g, 13.2 mmoles) was added to the reaction mixture via pressure equalizing addition funnel. When the addition was complete, the reaction was heated under reflux for 30 minutes then removed from the heat and transferred to a beaker to cool. A fluffy yellow solid precipitated out upon addition of anhydrous diethyl ether which was collected by vacuum filtration. The solid was washed with additional ether (2X25 ml) and air dried, m.p.

198.5°-200° C. Proton NMR (CDCl₃ + TMS) 1.333-2.36 multiplet 10 alkyl protons. 3.12 ppm singlet 6 H N- dimethyl, 3.80 multiplet 1 H tertiary proton, 4.87 ppm singlet benzylic CH₂-2 H, 7.42-7.76 5 H aromatic; Carbon 13 (ref: CDCl₃ 77.0 ppm) 132.21(9.7),

129.51(3.68), 127.97(10.0), 126.19(6.2), 72.33(3.47), 63.56(2.07), 46.53(2.82), 25.95(6.2), 24.09(5.8), 23.50(2.7).

Examples of Tear Stimulant Activity Two methods of evaluating increased tear

production in the normal rabbit eye have been

developed and used. One method involves the use of Schirmer test strips following instillation of a test product. Another method involves the use of a Periotron^®, an instrument originally designed to measure the amount of fluid on the gum surface of the mouth but adaptable to the surface of the eye. With these two methods, one can test many commercial tear substitute products. None showed an increase in the tear film. However, the tear stimulant, bromhexine (0.2 and 0.5%, 100 μL instillation), did not show a statistical increase in the fluid of the tear film in the normal rabbit eye at 30 minutes and 60 minutes after instillation from measurements made with the Periotron. This was used for comparison purposes (see Figure 1). In an earlier study, identical to the one shown in Figure 1 but measured at 15 and 60 minutes, bromhexine showed statistical significance at 60 minutes compared to baseline measurements.

In Figure 1, test results are shown for

bromhexine for comparative purposes and the compounds prepared in each of the Examples 1-4 as designed in Figure 1. The preferred compound of this invention, N-cyclohexyl-N-methyl-2-phenylethylamine, showed greater potency than the compound produced in

Example 1 on a weight basis, and showed greater potency than bromhexine in the normal rabbit eye at 0.2% w/v. The compounds of Examples 3 and 4 show less activity than the preferred compound of Example 2. Clearly the preferred compound of Example 2 showed a statistically significant increase in tear production (see Figure 1) over bromhexine.

In conducting these evaluation tests,

measurements following topical instillation were made up to one hour. Whenever rabbits were tested on two consecutive days, base line measurements were higher in both eyes. These measurements would decline to previous base line measurements if the rabbits were not used for testing for three or more days. The test protocol for the measurements shown in Figure 1 included the following for use of the

Periotron. Small filter strips were placed at the corneal scleral junction for ten seconds. They were then put into the Periotron well which measures how much fluid soaked into the filter strip. These were graded on a scale of from 0 to 100 to calibrate the instrument readings. The standard weight of the dry strip was taken, and water was added to get weight measurements which correlated to a known water content. The active compounds, prepared as earlier described, were dropped into the eye in an isotonic aqueous solution having a small amount of hydrochloric acid present to provide a pH of 4.5. The active drug readily dissolved. In each instance the active drug was used at 0.2% w/v. Prior to

administering the dose to the eye, a zero instrument reading was taken to determine the amount of fluid in the rabbit eye without drug. This is shown in Figure 1 as pretest (t=0). This was measured at the limbus in the eye. Thereafter, 100 microliters of the active in two drops was placed in the eye of the rabbit and readings were taken at ½ hour and 1 hour. The instrument readings are recorded on the bar graph illustrated in Figure 1. It can be seen that in each instance, there was initial tear stimulus, as measured at 30 minutes, and as well for the

compounds prepared in Examples 1, 2 and 4 at 60 minutes. The compound of Example 3 showed an initial stimulation at 30 minutes, but a decrease at 60 minutes in the tests reported.

The test results here shown, as well as others completed and still in progress, show the compounds of the present invention to be ophthalmically active for tear stimulation when topically applied.

Example 5

The compound N-cyclohexyl-N-ethylbenzylamine hydrochloride prepared in example 1 was tested for sigma binding site inhibition in comparison with Haldol, a known selective receptor for sigma binding sites. In particular, a radioligand of Haldol was bound to sigma binding sites to provide radio labeled ligands at sigma binding receptor sites of tissue. In the tests, the compound of Example 1 was then added to the cell system to see how successfully it displaced the radioactive ligands at the sigma binding site. The higher the percentage of radio ligand displaced, the greater the percentage of specific binding at the sigma sites. The following table shows the results of testing the above referred to compound at molar concentrations of 10 -9, 10-7 and

10^-5. T A B L E 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

CONCENTRATION OF PSYCHOACTIVE DRUG OF EXAMPLE 1 VERIFICATION

Displaced % Sigma % Displaced % Sigma

Concentration Haldol Site Binding Haldol Site Binding

10^-9M 27.5 72.5 22.5 77.5 10^-7 M 99.1 0.9 61.1 38.9 10^-5M 96.0 4.0 86.7 13.3

Any number for a psychoactive drug over 50% is considered highly significant. By way of example, the following Table 2 illustrates a percentage of specific binding for certain known psychoactives capable of binding at other binding sites which are responsible for the undesirable side effects often associated with psychoactive agents.

TABLE 2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Percent Inhibition

( Average : N = 2 )

- - - - - - - - - - - - - - - - - - - - - - -

Receptor/

Selectivity Radioligand 10^-9M 10^-7M 10^-5M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Biogenic Amines

Dopamine 1 [³H]-SCH 23390 3.2 5.9 10.7

Dopamine 2 [³H]-Sulpiride -1.4 -1.5 14.8

PCP [³H]-TCP 9.4 7.1 25.1 ([³H]-TCP is a PCP derivative which selectively labels PCP site: PCP is phencyclidine)

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Clearly, from the results of tables 1 and 2, the drug of example 1 selectively binds to the sigma binding site but is excluded from binding at

dopaminergic receptors which are responsible for undesirable side effects when drug acts as an agonist at these sites.

Example 6

N-cyclohexyl-N-ethyl-2-phenylethylamine

hydrochloride was tested in a similar fashion to that previously described in order to test comparison with Haldol or sigma binding site activity. For this compound the following table shows observed results.

TABLE 3

CONCENTRATION OF PSYCHOACTIVE DRUO OF EXAMPLE 1 VERIFICATION

% Displaced % Sigma % Displaced % Sigma Concentration Haldol Site Binding Haldol Site Binding

1 0 ^{- 9} M 4 3 . 2 56.8 33.9 66.1 1 0 ^{- 7} M 8 4 . 7 15.3 69.0 31.0

1 0 ^{- 5} M 1 0 7 . 6 -7.6 91.7 8.3

From tables 3 and 4, it is observed that the compound here tested binds selectively to the sigma and PCP binding sites but only very weakly binds to the dopaminergic site. Therefore, the compound here tested is expected to be a psychoactive drug with minimal undesirable side effects. TABLE 4

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Percent Inhibition

(Average : N = 2 )

Receptor/

Biogenic Amines

Dopamine 1 [³H]-SCH 23390 0.1 2.8 7.5 Dopamine 2 [³H]-Sulpiride 5.9 7.8 47.2

P C P [ ³H ] - T C P 1 1 . 1 2 1 . 0 87 . 7 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Example 7

The following table illustrates testing of sigma binding capability of bromhexine. Again, the test protocol and procedure were as previously described.

TABLE 5 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Percent Binding

- - - - - - - - - - - - - - - - - - - - - - -

Receptor/

Selectivity Radioligand 10^-9M 10^-7M 10^-5M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - sigma [³H]-DTG 24.8 45.1 90.8

TABLE 6

Biogenic Amines

Dopamine 1 [³H]-SCH 23390 3.9 10.8 17.5

Dopamine 2 [³H] -Sulpiride 5.6 4.5 11.9

PCP [³H]-TCP -13.6 0.2 -2.5 Again, binding to dopaminergic and PCP sites is negligible as illustrated in Table 6. The examples and the data presented in the tables all illustrate that the compounds there prepared and bromhexine are psychoactive and bind to the sigma site which would make them useful antipsychotics. In each instance for the active compounds illustrated in the examples, by nature of the chemical structures they are

expected to have few if any side affects in

comparison with currently available psychoactives such as Haldol.

Example 8

Data on Sigma Site Binding of Haloperidol

Lacrimocytes (acinar cells) were isolated from New Zealand male adult rabbit eyes. Rabbits weighing 2-3 Kg were anesthesized using intravenous

administration of Beuthanasia-D (0.5 mL/Kg). The lacrimal glands were removed carefully to avoid contamination by blood and injected interstitially with chilled oxygenated modified Eagle's media (MEM) at 4°C. Subsequently, small lobules of lacrimal gland tissue 2-3 mm in diameter were easily

identified and separated from external connective tissue, lymph nodes, and fat (J.D. Castle, et al., J. Cell Biol., 1972, 53, 290-311). Intact lobules were then enzymatically digested by incubation for 45 minutes at 37ºC with 95% 02/5% CO₂ in MEM which contained collagenase, hyaluronidase and trypsin. This treatment, which permitted the isolation of acinar cells, was followed by washing with ethylenediamine tetracetic acid (EDTA), further incubation with MEM, filtration to isolate acinar cells, and final storage as suspended acinar cells in Eagle's media at 4ºC. Cell viability was estimated by the trypan blue exclusion test (H.J. Phillips, 1973).

Dye Exclusion Test for Cell Viability, In "Tissue Culture, Methods and Applications". (P.F. Kruse and M.K. Patterson, eds.), Academic Press, New York, p. 406-408). [³H]-haloperidol binding (S.E. Wolfe, et al., Endocrinology, 1989, 124, 1160-1172):

Lacrimocytes ( approximately 3 mg ) , prepared from excised rabbit lacrimal glands and suspended in

Eagle's media, were transferred to six 2 ml tubes.

[³H]-Haloperidol (SA, about 10-20 Ci/mmole; New

England Nuclear) was added to each of the six tubes and incubated for 90 minutes at 37°C. Also added to each sample was 25 nM spiperone to block [³H]-haloperidol binding to D₂ dopamine and 5HT₂ serotonin receptors. In another set of three samples 30 μM 1-butaclamol was added to define specific binding since it competes for [³H]-Haloperidol-binding sites in endocrine and cerebellum tissues. After incubating for 90 minutes, membrane-bound [³H]-haloperidol was separated from free radiologand by rapid filtration through Whatman GF/C glass fiber filters. After filtration, the precipitate was washed with Tris-HCl buffer [pH 7.5]. The precipitate was transferred to a scintillation vial containing tissue solubilizer and after dissolution, scintillation fluid was added and the mixture counted in a scintillation counter (Beckman model LS 3801). The percent bound of [³H]-haloperidol was determined.

TABLE 7

3H-haloperidol Binding To Sigma-Receptor Of Rabbit

Lacrimocytes*

Sample No. CPM (Bound) Percent Binding

1 5337502 72.71

2 4430942 59.77

3 4668564 63.16

4** 4320506 58.82

5** 4683100 63.37

6** 5247340 71.42

* each sample contains approximately 1 million

lacrimocytes and 25 nM spiperone

** each sample contains 30 μM 1-butaclamol

The results, expressed as counts per minutes (CPM) and % of [³H]-haloperidol bound, indicate significant binding to lacrimocytes (acinar cells) The percent binding to washed cells is high and identical with or without the presence of spiperone and 1-butaclamol indicating that [³H]-haloperidol is not binding to D₂ dopamine receptors. Under these experimental conditions, [³H]-haloperidol is known to bind to sigma receptors in pituitary, adrenal, testis and ovary tissues (S.E. Wolfe, et al.,

Endocrinology, 1989, 124, 1160-1172) and therefore the presence of sigma receptors in the lacrimocytes of rabbit lacrimal gland tissue is established.

Example 9

Tear Secretion Measurements

The eye lashes of each rabbit were trimmed. A known binding agent to sigma sites was tested for its ability to promote tear secretion in comparison to buffered (pH = 7.4), isotonic vehicle. The "sigma binding butanol derivative" [also known as BMY 14802; reference: D.P. Taylor & J. Deklava, in Sigma &

Phencylidine-like Compounds as Molecular Probes in Biology, E.F. Dimino and J.-M. Kamenka, Ed., NPP Books, Ann Arbor, MI, 1988, p. 345.], is 1-[4-fluorophenyl]-4-[4-(5-fluoro-2-primidinyl)-piperazinyl]-1-butanol, and is structurally related to haloperidol. A group consisting of six rabbits was tested per day. At least 2 days were allowed before using the same group of rabbits again. On a treatment day one or two groups of rabbits were administered 50 μL of each test solution topically to the right eye; vehicle was always administered to the left eye. Drug solution was applied to the right eye and vehicle to the left eye. The known sigma binding butanol derivative was dosed at a

concentration of 1%. Tear secretion was measured by placing a test strip of filter paper (Schirmer Test Strips) under the lower eye lid of both eyes and allowing it to adsorb tears over a five minute period after which the strip was removed and the length of wetting measured in mm. Eight minutes before placing the Schirmer strip under each eye lid, one drop of a local anesthetic (proparacaine) was administered to the rabbit eye. Tearing over a five minute period was measured at t=0 (just prior to administering test agent), 10 and 60 minutes post-administration.

The results for the "known sigma binding butanol derivative" appear in Figure 2. The treated eye (open circles) shows an increase over the baseline measurement (17.5 mm) at 10 and at 60 minutes

indicating an effect from topical application. The nontreated eye (closed circles) shows lower tear measurements at both 10 and 60 minutes. However, the nontreated eye also shows a slight increase in tear response when compared to its baseline measurement which indicates systemic absorption and recirculation back to the nontreated eye. Regardless of the route of administration to the eye, the known sigma

binding butanol derivative appears to be active in promoting tear secretion in the normal rabbit eye. One can conclude that the drug is acting via a sigma site [receptor] interaction.

The above-identified evidence exhibits and demonstrates that the tear stimulants of U.S. Patent 4,820,737 function as sigma site binders. It has not been known before that sigma sites existed in ocular tissues. Now knowing that sigma sites exist in ocular tissues, it therefore follows that sigma site binders will have ophthalmic activity. This is demonstrated by Example 9 above, which shows tear stimulants are bound to sigma sites and demonstrates that a known sigma site binder (Example 8 ) does indeed stimulate tears. This necessarily leads to the conclusion that the tear stimulants of U.S.

Patent 4,820,737 act via a sigma site mechanism.

It can therefore be seen that all of the

objectives of the present invention are accomplished.

Claims

What is claimed is:

1.

A method of stimulating lacrimal secretion, comprising;

topically applying to the eye a small but

opthalmically effective amount of sigma site binder.

2.

The method of claim 1 wherein the sigma site binder is selected from the group consisting of natural and synthetic opioids, butyrophenones, arylamines, sigma site hallucinogens and sigma site binding antipsychotics.

3.

The method of claim 2 wherein the sigma site binder is an arylamine.

4.

The method of claim 2 wherein the sigma site binder is a butyrophenone.

5.

The method of claim 1 wherein the amount of said compound is from about 0.2% to about 0.6% on a weight/volume basis of a topical ophthalmic

preparation.

6.

An ophthalmically effective tear stimulant composition, comprising: a small but ophthalmically effective amount of a sigma site binder and;

an ophthalmically acceptable carrier for said sigma site binder.

7.

The composition of claim 6 wherein the sigma site binder is selected from the group consisting of natural and synthetic opioids, butyrophenones, arylamines, sigma site hallucinogens and sigma site binding anitpsychotics.

8.

The composition of claim 6 wherein the amount of said sigma site binder is from about 0.1% to about 5% on a weight volume basis of said composition.

9.

The composition of claim 8 wherein the carrier includes an ophthalmically acceptable buffer to provide a composition having a pH within the range of about 5.5 to 7.8.

10.

The composition of claim 6 wherein the pH is within the range from about 6.8 to 7.4.

11.

The composition of claim 9 wherein the

ophthalmically acceptable buffer selected from the group of water soluble salt forms of citrate, borate, phosphate, carbonate and acetate.

12.

The composition of claim 6 wherein the carrier includes an ophthalmically acceptable viscolyzer for eye comfort and a composition viscosity within the range of about centipoises to about 8 centipoises.

13.

The composition of claim 12 wherein the

viscolyzer is selected from the group consisting of hydroxyethylecellulose, hydroxypropyl methylcellulose, methylcellulose and polyacrylamide.

14.

The composition of claim 6 wherein the carrier includes an antibacterial agent.

15.

The composition of claim 6 wherein the carrier includes a chemical preservative.

16.

The composition of claim 6 wherein the

ophthalmic carrier includes a buffer, a viscolyzer, an antibacterial, and a chemical preservative.