WO1994016685A1

WO1994016685A1 - Ophthalmic uses of signa agonists

Info

Publication number: WO1994016685A1
Application number: PCT/US1994/000591
Authority: WO
Inventors: Ronald D. Schoenwald; Charles F. Barfknecht; Roger E. Newton
Original assignee: University Of Iowa Research Foundation
Priority date: 1993-01-28
Filing date: 1994-01-18
Publication date: 1994-08-04
Also published as: AU6124494A

Abstract

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

Description

Title: OPHTHALMIC USES OF SIGMA AGONISTS

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 accom¬ plishments 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 urn), wets the lipophilic epithelial surface of the cornea with the middle aqueous layer. The aqueous layer (7 um) contains dissolved proteins, carbohy¬ drates, glycoproteins, oxygen, and inorganic salts. The outer lipid layer (0.1 um) retards evaporation of the aqueous component.

Dry eye syndrome or eratoconjuctivitis 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 admini¬ stration. 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 pharmaco¬ logical 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. - A -

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 characterization 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 buty- rophenone antipsychotic, exhibits high affinity for sigma binding sites and several psychotomimetics, including PCP and benzomorphan-type 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 but not intentionally as a result of sigma site binding. 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. Sigma agonists cause the release of proteins from the lacrimocytes.

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 agonist 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 here named three joint inventors, 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 protein release in tears. The method comprises topically applying to the eye an ophthalmically effective amount of a sigma site binder. Because the treatment involves stimulation of protein release 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. In addition the molecules may also have the ability to stimulate tears.

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 Ophthalmologlca 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. Figure 3 shows identification of agonist activity for N,N-dimethyl-2-phenylethylamine using protein stimulation of lacrimocytes.

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, C. to C₅ alkyl, C., to C-. hydroxyalkoxy and C.. to C-. alkoxy; Z is selected from the group consisting of C. to C_g alkylene, C. to C_β oxyalkylene, and C. to C_fi aminoalkylene; R is selected from the group consisting of C.. to C_β alkyl and C₃ to C_β cycloalkyl; R* is selected from the group consisting of hydrogen, C₁ to C_g alkyl and C„ to C_β cycloalkyl; and X is a pharmaceutically acceptable counteranion.

Y can be in either an ortho-, meta- or para- position and is preferably hydrogen. Z likewise represents a moiety which can be ortho-, meta- or para- with respect to the Y, but is preferably para- positioned with respect to Y, and is preferably a C. to C_fi alkylene, and most preferably C, to C . alkyl, R is preferably a C₁ to C₃ alkyl, and most preferably methyl. R* is preferably cycloalkyl, most preferably cyclohexyl. 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 protonated tertiary amine salt form, except where R' is hydrogen and it is then a secondary amine. 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 admini¬ stration, i.e. generally soluble in acceptable pharmaceutical carriers. 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 0.05% to about 5%, and preferably from 0.1% 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-eye- irritating, 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 hydroxyethyl- cellulose, hydroxypropyl methylcellulose, methyl- cellulose and a polyacrylamide sold under the trade name Gelamide-jj- 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-methylcyclohexyl- amine (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-pentanone (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. 173.5°-174.5°C. Proton NMR (CDC1₃) 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 —CH_—, 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-ethyl-2-phenylethylamine

The preparation of N-cyclohexyl-N-ethyl-2-phenyl- ethylamine was accomplished in the following manner. This was prepared by treating a suspension of N- ethylcyclohexylamine (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 chromato- graphed 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 HC1 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,N-dimethyl -2-phenylethylamine hydrochloride

2-Phenylethyl bromide [0.01 mol.] and dimethylamine hydrochloride [0.02 mol.] are stirred at room temperature [24 hrs] and then refluxed [1 hr.] with potassium carbonate [0.06 mol.] in methanol. The insoluble materials are filtered and the solution evaporated and distilled to form N,N-dimethjyl-2- phenylethylamine. The hydrochloride salt [m.p. 163-4 ^βC] is formed and recrystallized from ethanol. The structure was confirmed by its proton nmr spectrum, by its chemical ionization mass spectrum, and its correct elemental analyses.

Example 4

Preparation of N-methyl-N-n- hexyl-2-phenylethylamine hydrochloride

A mixture of N-methyl-2-phenylethylamine [0.03 moles], potassium carbonate [0.06 moles], and 1- bromohexane [0.03 mol.] were refluxed in 100 ml. of 4- methyl-2-pentanone for 12 hrs. The mixture was filtered and the filtrate reduced to dryness which in turn was chromatographed on silica gel and eluted with a solvent system containing ethyl acetate, hexane, and chloroform [8:11:1], The product, N-methyl-N-n-hexyl- 2-phenylethylamine, was collected and evaporated to yield a yellow oil which was converted to the hydrochloride salt [m.p. 104.5-6 °C] . The structure was confirmed by its proton nmr spectrum, by its chemical ionization mass spectrum, and its correct elemental analyses. Example 5

Preparation of N-methyl-N- cvclohexyl-3-phenylpropylamine hydrochloride

A mixture of N-methylcylohexylamine [0.03 moles], potassium carbonate [0.06 moles], and l-bromo-3- phenylpropane [0.03 mol.] were refluxed in 100 ml. of 4-methyl-2-pentanone for 12 hrs. The mixture was filtered and the filtrate reduced to dryness which in turn was chromotographed on silica gel and eluted with a solvent system containing ethyl acetate and chloroform [7:3]. The product, N-methyl-N-cyclohexyl- 3-phenylpropylamine, was collected and evaporated to yield an oil which was converted to the hydrochloride salt [m.p. 102-4 °C] . The structure was confirmed by its proton nmr spectrum, by its chemical ionization mass spectrum, and its correct elemental analyses.

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 uL 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, the compound of Example 3, 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 compound of Example 3 shows slightly less sigma agonist activity than the compound of Example 2, but it is clearly the preferred compound for therapeutic purposes.

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 measure-ments 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 with a pH of 7.0. 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 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 6

The compound N-cyclohexyl-N-methylbenzylamine 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

TABLE 1

CONCENTRATION OF PSYCHOACTIVE DRUG OF EXAMPLE 1 VERIFICATION concen- % Displaced % sigma site % Displaced % Sigma tration Haldol Binding Haldol Site Binding

10^{" 9}M 27.5 72.5 22.5 77.5 10^-7M 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 7

N-cyclohexyl-N-ethyl-2-phenylethylamine hydrochloride prepared as in Example 2 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 DRUG OF EXAMPLE 1 VERIFICATION

Concen- % Displaced % sicjrna Site % Displaced % Sigma tration Haldol Binding Haldol Site Binding

10^"9M 43.2 56 . . 8 33.9 66.1 10~⁷M 84.7 15 .. 3 69.0 31.0 10^"5M 107.6 -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 nhibition (Average; N=2)

Receptor/Selectivity Radioligand 10^{" 9}M 10^{" 7}M 10^{" 5}M

Biogenic Amines

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

PCP [³H]-TCP 11.11 21.0 87.7 Example 8

The following tables illustrate 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

Percent Binding

Receptor/Selectivity Radioligand 10^"9M 10^"7M 10^~5M

Bioqenic Amines

Dopamine 1 [³H]-SCH 23390 3.9 10.8 17.5 Dopamine 2 [³H]-Sulpiride 5.6 4.5 11.9

PCP [³H1-TCP -13.6 0.2 -2.5

Again, binding to dopaminergic and PCP sites is negligible for bromhexine 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 9

Data on Sigma Site Binding of Haloperidol in the Lacrimal Gland

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 37^βC with 95% 02/5% C0₂ 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 4°C. 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). Radioligand Binding Procedure

The procedure by Wolfe et al. (Wolfe, Jr. S.A., Culp, S.G. and De Souza, E.B. s-Receptors in Endocine Organs: Identification, Characterization, and Autoradiographic Localization in Rat Pituitary, Adrenal, Testis, and Ovary. Endocrinology. 124: 1160- 1172, 1989) was used to determine binding to s-receptors.

New Zealand White rabbits of either sex and weighing 1.8-2.2 Kg were sacrificed and whole main lacrimal glands, free of blood, were dissected and frozen in cold oxygenated Modified Eagle's Medium

(MEM). The glands were distended by injection of chilled oxygenated MEM into interstitial tissue in order to locate and remove small lobules (2-3 mm).

Intact lobules were first incubated over a 15-minute period in 95% 0₂/5% CO., media containing collagenase

(100 U/mL), hyaluronidase (400 U/mL) and trypsin (9000

U/m ). The enzymatic digestion was interupted with the use of 2 mM EDTA followed by a wash of MEM containing 1.8 mM Ca++, 0.8 mM Mg++, and 10 mM glucose. The lobules were again placed in freshly prepared enzymatic media for an additional 45 minutes.

After disintegration of the lobules, the lacrimocytes were dispersed in MEM without enzymes, filtered through a 25-mm mesh siliconized steel gauze (Whatman

GF/C filters), and finally resuspended in cold L-15 medium containing 0.1 mg/ml trypsin inhibitor. ₃ H-Haloperidol (16.75 nM, New England Nuclear

Research Products, Boston, MA) was incubated over a wide concentration range in media containing the intact, but isolated lacrimocytes. Spiperone (25 nM) was added to prevent binding to D₂ dopamine, or 5HT₂ serotonin receptors. Incubation was stopped with the addition of 5 mL cold 100 mM Tris-HCl (pH 7.4) followed by vacuum filtration through glass microfiber filters that had been pretreated with 0.5% poly- ethyleneimine for 15 min. After filtration, the precipitate (radioligand bound to sigma receptor) was washed twice with 50 mM Tris-HCl buffer (pH 7.4) at room temperature. The filter, containing the precipitate, was transferred to a scintillation vial containing 1 mL of tissue solubilizer (Soluene 350,

Packard Inst. ) and 4 mL of scintillation fluid (Budget

Solve-| , Fisher Scientific, Fair Lawn, NJ) and counted

(Beckman LS 3801). Nonspecific binding (NSB) was determined by saturating the system with nonradio-

3 active haloperidol (10 μM) for H-haloperidol binding.

Specific binding was determined by substracting NSB

3 from total binding. The affinity of H-haloperidol for s-receptors (K.) and receptor density in tissue ( (BB_mm_aa_χx)) wweerree ccaallccuu;lated using nonlinear computer curve- fitting programs.

Results

3 Saturation binding of H-haloperidol to isolated, intact lacrimocytes was determined over the concentration range tested (0.5-3000 nM). At 4 °C equilibrium was reached in 45 minutes, whereas, at 25 and 37 °C equilibrium was reached in 45 minutes, whereas, at 25 and 37 °C, it took 30 and 15 minutes, respectively. Specific binding at saturation (4 °C) was approximately 60-65% of total binding, the remainder representing NSB. A Scatchard analysis of the saturation data was nonlinear and statistically significant from a single site model, yielding K, values of 28.8 and 161.9 nM for each site and Bmax values of 123 and 188 fmol/mg of protein, respectively.

Example 10

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 l-[4- fluorophenyl]-4-[4-(5-fluoro-2-pyrimidinyl)- piperazinyl]-l-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 uL 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 (pro- paracaine) 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.

Example 11

Data on Agonist/Antagonist Activity

Intact lacrimocytes were incubated (250 mg wet wt/mL) in 1 mL of L-l5 medium (Sigma) containing 0.1 mg/mL trypsin inhibitor (Sigma) either alone, with N,N dimethyl-2-phenylethylamine (10- 4 M), haloperidol (10

4

M) or in combination at 37 °C for 15 minutes. The mixture was gassed continuously with 95% 0₂/5% CO,,.

After 15 min. the culture tubes were immediately placed in ice, followed by centrifugation at 3000 rpm for 10 min. The supernatant was analyzed for protein content (Bradford, M.M. Anal. Biochem., vol 72: 248- 254, 1976). A volume of 0.1 ml of each supernatant was reacted with 2.5 ml of Coomassie reagent in a stoppered quartz cell. After 1 minute the absorbance was measured at 595 nm. The protein content in each experiment was compared by using a Student's t test. Figure 3 shows the results of incubating intact lacrimocytes alone (control) and with N,N-dimethyl-2- phenylethylamine (7A: 10 -4 M) as well as with and without haloperidol (HP: 10^"4 M), all statistically significant from the control (p<0.05). The baseline protein secretion rate was increased by 28 ± 2.3% in the presence of N,N-dimethyl-2-phenylethylamine, decreased by 20 ± 7.6% in the presence of N,N- dimethyl-2-phenylethylamine (7A) and haloperidol (HP) and decreased by 11.1 ± 0.70% when in the presence of haloperidol only. These results suggest that N,N- dimethyl-2-phenylethylamine is behaving as an agonist and haloperidol an antagonist.

The above-identified evidence exhibits and demonstrates that the tear stimulants of U.S. Patent

4,820,737 function as sigma site binders and agonists.

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. It has not been known what compounds are agonists (i.e., stimulate protein secretion) and which are antagonists. Example 11 establishes that N,N-dimethyl-2-phenylethylamine is an agonist and haloperidol is an antagonist. 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 agonist.

2. The method of claim 1 wherein the sigma agonist is selected from the group consisting of non-analgesic isomers of natural and synthetic opioids and arylamines.

3. The method of claim 2 wherein the sigma agonist is an arylamine.

4. The method of claim 1 wherein the amount of said compound is from about 0.1% to about 0.6% on a weight/volume basis of a topical ophthalmic preparation.

5. An ophthalmically effective tear stimulant composition, comprising a small but ophthalmically effective amount of a sigma agonist binder and an ophthalmically acceptable carrier for said sigma site binder.

6. The composition of claim 5 wherein the sigma agonist is selected from the group consisting of non- analgesic isomers of natural and synthetic opioids and arylamines.

7. The composition of claim 5 wherein the amount of said sigma site binder is from about 0.1% to about 5% on a weight volume basis of said composition. 8. The composition of claim 7 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.

9. The composition of claim 5 wherein the pH is within the range from about 6.8 to 7.4.

10. The composition of claim 8 wherein the ophthalmically acceptable buffer selected from the group of water soluble salt forms of citrate, borate, phosphate, carbonate and acetate.

11. The composition of claim 5 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.

12. The composition of claim 11 wherein the viscolyzer is selected from the group consisting of hydroxyethylocellulose, hydroxypropyl methylcellulose, methylcellulose and polyacrylamide.

13. The composition of claim 5 wherein the carrier includes an antibacterial agent.

14. The composition of claim 5 wherein the carrier includes a chemical preservative.

15. The composition of claim 5 wherein the ophthalmic carrier includes a buffer, a viscolyzer, an antibacterial, and a chemical preservative.