CA2332825A1 - Peripheral benzodiazepine receptor ligands - Google Patents

Abstract

Compounds which bind with high affinity to peripheral benzodiazepine recepto rs are useful as antiinflammatory agents. Such compounds include isoquinoline derivatives, such as PK 11195, and benzodiazepine derivatives such as Ro 5- 4864. A method of treating an inflammatory condition in a mammal with such compounds is provided. Pharmaceutical compositions comprising such compounds are described. A method is provided for identifying compounds that are therapeutically effective for treating inflammatory conditions.

Description

FIELDS OF THE INVENTION
This invention pertains to the use of compounds which bind with high affinity to peripheral benzodiazepine receptors as antiinflammatory agents. Such compounds :i include isoquinoline and benzodi~~zepine derivatives. These compounds may be used in the treatment of human diseases such as rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome, osteoarthritis, multiple sclerosis, Behcet's disease, temporal aretritis and dementia of the Alzheimer type.
ly BACKGROUND OF THE INVENTION
Historically, two broad classes of benzodiazepine receptors have been described: central and peripheral. Ligands for central benzodiazepine receptors, such as diazepam, flunitrazepan and clonazepam, produce an interaction with GABA"
receptors, enhancing the activity of GABA (gamma-aminobutyric acid). These 15 ligands possess anticonvulsant, muscle relaxant, sedative and anxiolytic properties.
They are widely used clinically. The receptors are highly concentrated in brain, but are also found peripherally.
Ligands that are selective for peripheral benzodiazepine receptors, such as 4' chlorodiazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-( 1-methylpropyl)-20 isoquinolinecarboxamide (PK 11.195), do not interact with GABA receptors and do not possess the same pharmacological properties as the central benzodiazepine receptor ligands. Instead they blind to peripheral benzodiazepine receptors which are widely distributed throughout the body, including the central nervous system.
The peripheral benzodiazepine receptor ligands have no well defined pharmacological properties, and so far there are no generally aceepted clinical applications for their use.
In this application, "peripheral benzodiazepeine receptors" means the class of peripheral benzodiazepine receptors as distinguished from the class of central benzodiazepine receptors, and "'peripheral benzodiazepine receptor ligands"
means :30 ligands that bind with high affuuty to peripheral benzodiazepine receptors.
PK 11195 has a particularly high affinity for peripheral benzodiazepine receptors. The equilibrium dissociation constant, or affinity (Kd) of PK 11195 for SUBSTITUTE SHEET {RULE 26) human brain tissue is estimated to be 4.3 nM (Doble et al., 1987). Its affinity for mouse peritoneal macrophages is~ reported to be 5 . 6 nM (Zavala and Lenfant, 1987b) .
A compound's ability to displace bound PK 11195 from peripheral benzodiazepine receptors (Ki) is a convenient measure of the relative strength of the compound's binding to these receptors. In brain, this has been determined to be 3.5 nM for PK / 1195 itself, 44 nlVt for 1-N,N-diethyl-a-methyl-2-phenylquinoline-propranamide (PK-14067), 178 nM for Ro 5-4864. By contrast, the value for the central benzodiazepine receptor ligand clonazepam is 46,000 nM (Doble et al., 1987) .
:l0 The peripheral benzodiazepine receptors are said to be highly associated, if not exclusively associated, with the outer mitochondria) membrane. The presumed association of these receptors with mitochondria (Snyder et al. 1990; Hirsch et al.
1989) has led to the proposal treat they affect mitochondria) respiration.
However, this view has been challenged on the basis of a poor correlation between receptor :l5 density and the ability of ligands to stimulate respiration (Zisterer et al. 1992).
The reported effects of peripheral benzodiazepine receptor ligands on immune functions are diverse and in many respects controversial. PK 11195 has been shown to inhibit in vitro mitogen-driven T- and B- cell stimulation, properties shared by a number of central benzodiazepine ligands (Ramseir et al., 1993; Bessler et al., 1992).
20 In contrast, PK 11195 has also been reported to stimulate antibody production in mice following immunization with sheep red blood cells (Lenfant and Zavala, 1986;
Zavala et al. , 1984; Zavala and Lenfa~nt, 1987b) which would enhance the inflammatory response. Peripheral benzodiazepine receptor ligands, including Ro 5-4864, have also been reported to enhance the rEapiratory burst system of macrophage-like :ZS cells stimulated with arachidonic: acid. PK 11195 in the same situation reportedly had little effect (Zavala and lenfant, 1987a).
In brain, peripheral benzodiazepine receptors are believed to be associated with glial cells. However, it is not clear what the physiological function of these receptors in brain might be. Various authors have reported increases in peripheral 30 benzodiazepine receptor levels in rat brain following kainic acid, ischemic, or neoplastic lesions. Similar findings have been reported for human brain tissue in SUBSTITUTE SHEET (RULE 26) patients with brain tumours, neoplasms, multiple sclerosis, cerebrovascular disorders and Alzheimer's disease. The teaching is that these increases in peripheral benzo-diazepine receptor levels reflect glial proliferation (Diorio et al., 1991;
Leong et al., 1994).
Following kainic acid lesioning to rat brain, binding of PK 11195 increased in both time and spatial localization with the appearance of macrophages, but PK
11195 binding apparently did not correlate with the appearance of astrocytes (Myers et al., 1991). In PET studiea of human gliomas, there was a large increase in binding of PK 11195, but not of Ro 5-4864 (Junck et al., 1989).
It has been suggested that inhibitory agonists of peripheral benzodiazepine receptors are useful for speeding the recovery of damaged central nervous system tissue (Gee, 1993) . The mechanism proposed to account for this effect is inhibition of glial cells and macrophages, and inhibition of cytokine production by macrophages at the site of acute injury. But Taupin et al. (1991, 1993) find that the inflammatory cytokines interleukin-1 and tumor necrosis factor are increased, rather than decreased by peripheral benzodiazepine receptor ligands.
In summary, it can be said that the art and teaching in the field is inconsistent, Until the United States patent application of McGeer et al. (serial 08/520, 211), there was no overall concept as to the function of peripheral benzodiazepine receptors, or the pharmacological properties of their ligands.
SUMMARY OF THE INVENTION
The invention discloses that ligands which bind with high affinity to peripheral benzodiazepine receptors act as antiinflammatory agents. A method of treating an inflammatory condition in a mammal is provided. The method comprises administer-ing to a mammal requiring such treatment a therapeutically effective amount of a compound which binds with high affinity to peripheral benzodiazepine receptors.
This may require dosages in the range of 0.1-100 mg/kilogram of body mass per day or roughly (0.3-320 micromoles/kilogram per day depending on the molecular weight of the agent), as determined by a medical practitioner or veterinarian.
Preferably, 4 PCT/CA9$/00502 the compound is selected from the group consisting of compounds which bind with micromolar or submicromolar affinity to peripheral benzodiazepine receptors, as, for example, PK 11195 or pharmacologically acceptable salts thereof.
The invention includes ~pharmaeutical compositions comprising a peripheral benzodiazepine receptor ligan~d in combination with one or more compatible pharmaceutically acceptable adjuvents or diluents which may be inert or physiologi-cally active. These compositions may be administered by the oral, parenteral or rectal route or locally. Compositions of the invention comprising a peripheral benzodiazepine receptor ligand may be packaged in packaging material that comprises l0 a label which indicates that the. composition can be used for treating inflammatory conditions.
The present invention provides a method of identifying a compound that is therapeutically effective for treating an inflammatory condition in a mammal.
The method comprises selecting a compound:
l5 (1) that binds with micromolar or submicromolar affinity to peripheral benzodiazepvie receptors; and {2) that is therapeutically effective in treating inflammatory symptoms in MRIrlpr rnice.
Additional steps that may be taken in the method of indentifying an :LO antiinflammatory compound include:
(1) selecting a compound that inhibits respiratory burst in cultured macrophages;;
(2) selecting a .compound that inhibits the neurotoxic effects of products secreted by cultured monocytic type cells, such as THP-1 :LS cells, following stimulation by inflammatory agents; and (3) selecting a compound that exhibits increased binding to human Alzheimer brain tissue compared to normal human brain tissue.
The compound utilized in the various aspects of the invention may be selected from the group consistung of: PK 11195, PK-14067, PK 14105, Ro 5-6993, :30 Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524, Ro 5-5122. In each case, -$-the compound is preferably 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195).
DETAILED Dl~?SCRIPTION OF THE INVENTION
This invention discloses the use of high affinity peripheral benzodiazepine receptor ligands as a new class of antiinflammatory compounds. This class of compounds includes isolquinoliine drivatives such as PK 11195 and benzodiazepine derivatives such as Ro 5-4864. The following examples illustrate various aspects of this invention, including four assays for defining the antiinflammatory pharmacologi-cal profile of such drugs. These assays establish the utility of high affinity peripheral benzodiazepine receptor ligands as antiinflammatory agents on the basis of the following results:
(1) Pronounced therapeutic activity in the MRL-lpr mouse autoimmune disease. This disorder is an accepted model of a variety of human diseases:
rheumatoid arthritis (Koopma,n and Gay, 1988), systemic lupus erythematosus (Bartlett et al., 1988), Sjogren"s syndrome (Hayashi et al., 1994), connective tissue disease (Rosenberg, 1988), behavioral and neurological disorders (Sakic et al., 1993) and CNS inflammation (Vogehweid et al. , 1991) . PK 11195 demonstrated a more powerful prevention of pathology in this autoimmune disorder than standard antiinflammatory agents.
(2) Down regulation of respiratory burst activity in cultured macrophages.
PK 11195 was a more potent down regulator than standard antiinflammatory agents.
(3) Protection from the toxic effects of products secreted from cultured THP-1 (monocyte type) cells, following stimulation by inflammatory agents, against cultured SYSY (neuronal type) cells. PK 11195 and Ro 5-4864 were more effective neuroprotective agents than some standard antiinflammatory drugs.
(4) Enhanced high affinity binding to Alzheimer disease brain tissue compared with neurologically normal brain tissue. Cells involved in the inflammatory response in the central nervous system, including T-cells, reactive microglia (macrophages) and reactive astrocytes (Itagaki et al., 1986), are associated with WO 99/61024 PCT/CA9~/00502 Alzheimer disease lesions. Therefore, Alzheimer disease tissue is a model for inflammatory disease of the central nervous system. Both PK 11195 and Ro 5-showed greater high affinity binding to Alzheimer brain tissue than to neurologically normal brain tissue, with the difference being greater for PK 11195.
Together, the foregoing assay results are predictive of the utility of PK
11195 and other peripheral benz;odiazepine receptor ligands in the treatment of animal and human disorders of an inflammatory nature. These include, but are not limited to, rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome, osteroarthritis, multiple sclerosis, inflammatory bowel disease, Behcet's disease, myasthenia gravis, temporal arteritis, Hashimoto's disease, dermatitis herpetiformis and other diseases, including Alzheimer disease, where chronic inflammation may exacerbate the fundamental pathology (as discvussed in more detail below).
EXAMPLE 1: Treating Inflammatory Conditions in MRL-lpr Mice MLR-lpr mice are a widely studied strain which spontaneously develop a particularly severe autoimmune: disorder. Pathologies that are found in a variety of human idiopathic inflammatory and autoimmune diseases are reproduced in these animals. They are therefore considered to be an outstanding model for such individual human diseases, although the pathology represents a combination of several of them. They are regarded as the best animal model for rheumatoid arthritis (Koopman and Gay, 1988), systemic lupus erythematosus (Bartlett et al., 1988), and Sjogren's syndrome (Hayashi Ert al. , 1994) . They display vascular connective tissue disease (Rosenberg, 1988), as seen in several human arthropathies. They develop behavioral and neurological disorders (Sakic et al., 1993), as well as evidence of CNS
inflammation (Vogelweid et al., 1991) which is seen in isolation in such diseases as Alzheimer type dementia and multiple sclerosis, but is also part of the syndrome in systemic lupus erythematosus and Sjogren's syndrome. The model is considered to be a particularly demanding one for screening antiinflammatory agents because of the severity and comprehensiveness of the disease pathology. The lesions are character-ized by infiltration of B- lymphocytes and T-lymphocytes, indicating that it is a typical autoimmune disorder. The lesions include synovial inflammation, synovial _ 'J _ cell proliferation, pannus fonnation, and articular cartilage erosion and bone destruction. Only powerful antiinflammatory agents will inhibit the devastating changes that accompany this genetic disorder. Mice of the MRL-lpr strain spontaneously develop a mild fomn of the disease at 4 to 5 months of age. The onset and severity can be accelerated by injection of complete Freund's adjuvant (CFA), supplemented to 10 mg/ml with heat-inactivated M. tuberculosis, at 13-14 weeks of age, with 67-82% of animals becoming affected within 1 month (Ratkay et al., 1994; Ratkay et al., 1993).
In the first set of experiments, mice of the MRL-lpr strain were injected at :LO 13-14 weeks of age with 0.05 ml of CFA supplemented to 10 mg/ml with heat-inactivated M. tuberculosis at each of two intradennal thoracic sites according to the standard procedure, and, following injection (day 0), were started on a daily subcutaneous dose of PK 11195 dissolved in alcohol. Daily injections were continued until day 14. The animals were sacrificed at day 30 and joint histopathology :LS assessed. Four dose levels of 1'K 11195 were employed: 0.01 mg/kg (n=15), 0.1 mg/kg (N = 10), 0.5 mg/kg (N=9) and 1 mg/kg (N=7). The results were compared with those in littennates receiving CFA only (n = 14) or CFA and treatment with ethanol alone (n=8). Followuig sacrifice, animals were evaluated for subsynovial inflammation, synovial hyperplasia, cartilage destruction and pannus formation, and :ZO bone destruction. Using a rating system described by Ratkay et al (1993), the results are shown in Table 1. PK 11195 significantly reduced the total histopathological score observed in control mice, even at the lowest dose of 0.01 mg/kg. In comparison with results obtained by Ratkay et al. (1994), PK 11195, at the 0.1 mg/kg dose, was more effective than ten times that dose of indomethacin, forty times that of :25 cyclosporin, and whole body irradiation at 3 Gy from a 60 cobalt source on day I of arthritis injection.

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Since alcohol was found to be an irritating vehicle to the mice, dimethyl sulfoxide (DMSO) was chosen a.s a non-irritating solvent for PK 11195 in subsequent experiments. The method for inducing the adjuvant arthritis in these experiments remained the same.
The second series of experiments tested the ability of PK 11195 to prevent MRL-lpr adjuvant arthritis when dissoved in DMSO, using the same criteria as in the first experiment. Starting at day 0 (the day of CFA injection), animals received no treatment (n = 10), 0.1 ml DrvISO alone (n = 14) or various doses of PK 11195 dissolved in 0.1 ml DMSO subcutaneously until sacrifice at day 30. PK 11195 was administered at daily doses of 0.01 mg/kg (n = 6), 0.1 mg/kg (n = 6), 0.5 mg/kg (n = 19) and 1 mg/kg (n = 6;1. About 80 % of the non-txeated animals developed visibly arthritic joints. There was a dramatically reduced incidence of visible swelling in animals receiving higher doses of PK 11195, as well as a delay in the onset of disease. This clinical evaluation was confirmed following sacrifice by histological analysis of the joints. There was a dose response effect (Table 2), with PK

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The third series of experiments tested the ability of PK 11195 to inhibit MRL-lpr arthritis once it had been established. Mice were primed at day 0 with CFA, as in the previous two series of experiments. Treatment with DMSO alone, or PK 11195 in DMSO, was not commenced until day 10, at which time significant swelling was apparent. PK 111'95 in 0.1 ml DMSO or 0.1 ml DMSO alone was then administered subcutaneously an a daily basis until sacrifice at day 30.
Erythema and swelling of the adjuvant injected control group (n = 10) was evident over the course of the experiment. DMSO alone (n = 13), and PK 11195 at doses of 0.03 mg/kg (n = 8). 0.3 mg/kg (n = 16) and 3.0 mg/kg (n = 16) were the treatment groups. PK
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This series of experunents established that PK 11195 has a pronounced therapeutic effect on already established arthritis in the MRL-lpr model.

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EXAMPLE 2: Inhibiting~Resniiratory Burst in Cultured Macrophages The respiratory burst system is an attack mechanism possessed by professional phagocytes such as peritoneal macrophages. Its main function is to protect the body from hostile: invaders by generating superoxide radicals, but inappropriate activation can damage host tissue. Oxidative stress is believed to be one of the more harmful concomitants of inflammation. The respiratory burst system can be activated in cultured peritoneal macrophages in multiple ways, including exposure to zymosan particles opsonized by complement proteins (Klegeris and McGeer, 1994). The ability of PK 11195 to inhibit respiratory burst by administra-tion before or after exposure of peritoneal macrophages to opsonized zymosan is shown in Table 5. At 10-', PK 11195 inhibited respiratory burst by more than 50 %
whether given before or after o;psonized zymosan. This indicates that the effect was downstream from, or independent of, the opsonized zymosan receptors. Table 5 also shows the comparative effects of three agents effective in inflammatory disorders:
indomethacin, prednisone and dapsone. Each of these agents was substantially less effective than PK 11195, and tended to show less inhibition when administered after opsonized zymosan than before. These data illustrate that PK 11195 inhibits respiratory burst more powerfully and by different mechanisms than these well known antiinflammatory agents. It its also more effective than 4'-chlorodiazepam, the prototype benzodiazepine ligand for peripheral benzodiazepine receptors.

Table 5: Inhibition of Respiratory Burst in Cultured Rat Peritoneal Macrophages by Various Substances Percent of Control ( t S. E. ) when Substance was ivg en Substance ConcentrationBefore OZ# After OZ#

PK 11195 10-~M 99.0 t 5.6 97.9 f 0.2 10-sM 87. 9 t 5 . 5 83 . 5 t 8 . 7 10~M 48 .9 t 10. 8 47.1 t 13 .6 Ro 5-4864 10~M 94. 6 t 12.2 not done 10-'M 85 .4 t 2.5 not done 10~'M 74.7 t 1. 9 not done Dapsone 10-SM 94.7 t 1. 3 96.4 t 1.

10~M 73.6 t4.6 90.3 t6.2 Indomethacin10-sM 101. 3 t 3 . 5 not done 10~M 86. 3 t 10. 9 100. 9 t 10-3M 36 t 12. 5 64. 8 t 9.4 Prednisone 10~'M 88 . 8 t 7 . 2 101. 8 t 4. 9 10-'M 62.5 t 8.1 67 t 11.6 OZ# = opsonized zymosan For methods see Klegeris and McGeer, 1994 EXAMPLE 3: Reduction of Cvtotoxicitv to Neuronal SYSY Cells Caused by Secretorv Products of Monocvtic THP-1 Cells Stimulated by Inflammatory Mediators Cultured cells of the monocytic type, such as THP-1 cells, when stimulated by inflammatory agents, secrete products which in their aggregate are toxic to cultured neurons.
Human monocytic THP-1 cells were plated in 24-well plates at a concentra tion of Sx105 cells per well in 1 ml of Dulbecco's Modified Eagle Medium Nutrient Mixture-F12 containing 5% festal bovine serum (FBS). THP-1 cells were incubated WO 99161024 PCTICA9~/00502 in the presence or absence of drugs for 30 minutes prior to the addition of an inflammatory stimulus consisting; of 1 Jug/ml lipopolysaccharide (LPS) with 333 U/ml of interferon-~y (IFN-~y) . After :24 hours incubation in a humidified 5 %
C02/95 % air atmosphere at 37°C, 0.5 ml aliquots of cell-free supernatants were transferred to the wells containing SYSY cells which had been plated 24 hours earlier. After 72 hours of culture, cell death was evaluated by the amount of lactate dehydrogenase (LDH) which had been released into the medium from lysed cells. Cell survival was measured by the ability of the cell culture to reduce the formazan dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
LDH activity in cell culture supernatants was measured by the enzymatic assay described by Decker and Lohmann-Matthes (1988) in which formation of the formazan product of the iodonit~rotetrazolium dye INT was followed colorimetxically.
Optical densities were measured, and the amount of LDH was expressed as specific cell lysis (in percent) calculated) by the following formula:
%iysed cells = 100 x [D,A(treated)-DA(untreated)]/DA(lysed) where DA is absorbance of supernatants at 490 nm from which background absorbance has been subtracted.. ~A(treated) was the measurement on supernatant from drug-treated cells, AA(u:ntreated) that from cells incubated only with fresh medium, and DA(lysed) that from cells where complete lysis was achieved with 1 Triton X-100.
The MTT assay was perfumed as described by Mosmann (1983) and by Hansen et al. (1989) . This method is based on the conversion of MTT to colored formazan by viable but not lby dead cells. The viability of SYSY cells was determined by adding MT'T to the SYSY cell cultures to reach the final concentration of 1 mg/ml. Plates were placf;d overnight at 37°C and optical densities at 570 nm were measured by transferring 100 ~cl aliquots to 96-well plates and using the plate reader with a corresponding filter. The percentage of viable cells was calculated by the following formula:
viable cells = 100 x [OD (treated)-OD(lysed)]/[OD(untreated)-OD(lysed)]

where OD is the optical density. As in the LDH experiments, treated refers to cells treated with drug, untreated to those incubated only with fresh medium, and lysed to those where complete lysis was achieved with 1 % Triton X-100.
The results are shown in Table 6. They demonstrate that both PK 11195 and Ro 5-4864 significantly prevented toxicity to neuronal cultures at doses ranging from 2x10~M to SxlO-SM. Agents which failed to have any effect in this assay at equivalent concentrations included such well known antiinflammatory agents as prednisone, dexamethasone and propentofylline.
Table 6: Inhibition of THP-1. Human Monocytic Cell Toxicity Towards SYSY
Human Neuroblastoma Cells A. LDH assa y after 72 hours incubation ~ Dead cellsSignificance Level Substance Concentration( + S E (randamized blocks ) ANOVA) PK 11195 SxlO-s 33.411.9 F=11.6, p = 0.0007 2x10-5 27.413.4 2x10 35.313.3 0 45.112.6 Ro 5-4864 5x10-5 28.714.9 F=6.1, p = 0.009 2x10-5 30.616.1 2x10- 38.512.2 0 43.112.9 WO 99/61024 PCT/CA9g/00502 - ig -B. MTT assay after 72 hours uicubation %~ Live cells Significance Level Substance Concentration (+ S E.) (randomized blocks ANOVA) PK 11195 5x10-5 ti1.7t15.6 F=9.7, p = 0.002 2xlo-s ti5.7 t 14.4 2x10- 47.9 t 8.0 0 35.4 t5.6 Ro 5-4864 5x10-5 Ei0.6f 1.2 F=7.8, p = 0.004 2x10-' '.>8.4t7.9 :l0 2x10- 44.5 t6.9 0 39.1 t6.4 These experiments establish that PK 11195 and Ro 5-4864 have a protective effect against neurotoxic products secreted by monocyte type cells following inflammatory induction by the well known inflammatory mediators lipopolysaccharide and gamma-interferon.
EXAMPLE 4~ Bindins to Human Alzheimer Brain Tissue To date, the only method by which the levels of putative peripheral benzodiazepine receptors in tis sue can be determined is by high affinity binding of specific ligands such as PK 11195. To compare the levels in Alzheimer and normal brain tissue, we assessed the high affinity binding of PK 11195 to Alzheimer and normal brain tissue. The standard method of Schoemaker et al. (1983) was employed.
The results are shown in Table 7 for Alzheimer and control brain tissue.
As shown in the table, Alzheimer cases had 2.7 fold higher binding of PK 11195 than control brain tissue. This is greater than the difference observed with Ro 5-(McGeer et al., 1988).

Table 7: Specific High Affinity Binding of PK 11195 (at 3 nM) to Alzheimer and Control Cortical Tissue (in fentamoles/mg protein) Alzheimer Control 4g0 201 Means+ S.E. 449~~25 165+25 (difference significant at p < 0.001 ) A criterion by which the potency of other isoquinoline or benzodiazepine derivatives can be compared with PK 11195 is their ability to displace PK
11195 in a competitive binding assay (Doble et al., 198.
EXAMPLE 5: A Method of Identifying_Novel Antiinflammatory Compounds The invention provides a method of identifying a compound that is therapeutically effective for treating an inflammatory condition in a mammal.
The method comprises selecting a compound:
(1) that binds with micromolar or submicromolar affinity to peripheral benzodiazepine receptors; and (2) that is therapeutically effective in treating inflammatory symptoms in MRL-lpr mice.
Additional steps may be taken in the method of indentifying anti-inflammatory compounds;
(1) selecting the compound that inhibits respiratory burst in cultured macrophages;

(2) selecting the compound which inhibits the secretion of neurotoxic products by cultured monocyte type cells following exposure to an inflammatory stimulus; or (3) selecting the compound that exhibits increased binding to human Alzheimer brain tissue compared to normal human brain tissue.
In the method of identifying antiinflammatory compounds, the compound utilized may be selected from vthe group consisting of: PK 11195, PK-14067, PK
14105, Ro 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524, Ro 5-5122, therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts.
EXAMPLE 6: Methods of Treatment A method of treating an inflammatory condition in a mammal is provided.
The method comprises administering to a mammal requiring such treatment a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt of the compound, the compound being selected from the group consisting of compounds which bind with micromolar or submicromolar affinity to peripheral benzodiazepine receptors. The: method may be practiced where the mammal is a human being and the compounf. binds with micromoiar or submicromolar affinity to human peripheral benzodiazepine receptors. The inflammatory condition may be rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome, osteoarthritis, multiple sclerosis, Behcet's disease, temporal arteritis, and, without being limited by the foregoing, any inflammatory disorder which calls for the use of antiinflammatory agents. Alternatively, the inflammatory condition may be dementia of the Alzheimer type and the peripheral benzodiazepine receptors may be those that are found in brain.
Dementia of the Alzheimer type is included in this category since it has been shown to be characterized by chronuc inflammation of the brain and to respond to antiinflammatory therapy (U. S. Patent 5,192, 753; European patent 0 642 336 BI) .
A preferred compound is PK 11195. However, the compound may be selected from the group consisting of: PK 11195, PK-14067, PK 14105, Ro 5-6993, WO 99/61024 PCT/CA9t3/00502 Ro 5-4864, Ro S-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro S-5115, Ro 5-4608, Ro S-6524, Ro 5-5122, therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds that bind with as high or higher affinity to peripheral benzo-diazepine receptors.
EXAMPLE 7: Formulations The invention includes phanmaeutical compositions comprising a peripheral benzodiazepine receptor ligand in combination with one or more compatible :l0 pharmaceutically acceptable adjuvents or diluents which may be inert or physiologi-cally active. These compositions may be administered by the oral, parenteral or rectal route or locally. The peripheral benzodiazepine receptor ligand may be PK
11195. However, the ligand may be selected from the group consisting of: PK
11195, PK-14067, PK 14105, F;o 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-:15 6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 4608, Ro 5-6524, Ro S-5122, therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds that bind with as high or higher affinity to peripheral benzodiazepine receptors.
Tablets, pills, powders (gelatin capsules or cachets) or granules, may be :ZO used as solid compositions for oral administration. In these compositions, the active ingredient according to the invention may be mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica. These compositions may also contain substances other than diluents, for example one or more lubricants such as magnesium stearate or talcum, a colorant, a coating (dragees) or a lacquer.
25 Pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents ouch as water, ethanol, glycerol, benzoic acid, benzyl alcohol, sodium benzoate, dimethyl sulfoxide, vegetable oils or liquid paraffin may be used as liquid composition> for oral administration. These compositions may contain substances other than diluents, for example wetting agents, sweeteners, 30 thickeners, flavoring agents or stabilizers.

WO 99/61024 PCT/CA9$/00502 Sterile compositions for parenteral administration may preferably be nan-aqueous solutions, suspensions or emulsions. Water, ethanol, propylene glycol, polyethylene glycol, benzoic acid, benzyl alcohol, sodium benzoate, dimethyl sulfoxide, vegetable oils, especially olive oil, injectable organic acids esters, for example ethyl oleate or other suitable organic solvents may be used as the solvent or the carrier.
These compositions may also contain adjuvants, especially wetting agents, tonicity regulating agents, emulsifiers, dispersants and stabilizers. The sterilization may be carried out in several ways, for example by aseptic filtration, incorporating DLO a sterilizing agent, by irradiation or by heating. They may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in a sterile medium suitable for injection.
Compositions for rectal administration are suppositories or rectal capsules, which contain, in addition to the active product, excipients such as cocoa butter, :l5 semi-synthetic glycerides or polyethylene glycols.
Compositions for local administration may be for example creams, ointments, lotions, eye lotions, mouth-washes, nasal drops or aerosols.
The dosage depends on the effect sought, the length of treatment and the administration route employed. In general, the medical practitioner {or veterinarian) a0 will determine the appropriate dosage depending on the age, weight and all other factors specific to the subject to be treated. The approximate dosage range may be chosen from the dosage range shown to be effective in resisting damage in the MRL-lpr murine rheumatoid arthritis model, i.e. 0.1 mg/kg to 100 mg/kg, with the most probable range being 1-10 mg/kg body weight per day.
:25 EXAMPLE 8: Articles of Manufacture Compositions of the invention comprising a peripheral benzodiazepine ligand may be packaged in packaging material that comprises a label which indicates that the composition can be used for treating inflammatory conditions. Such conditions 30 include rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome, osteoarthritis, multiple sclerosis,Behcet's disease, temporal arteritis and dementia of the Alzheimer type. The peripheral benzodiazepine receptor ligand of the composition preferably exhibits micromolar or submicromolar affinity to peripheral benzodiazepine receptors, for example PK 11195. The ligand may be selected from the group consisting of:
PK
11195, PK-14067, PK 14105, R.o 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-S 6669, Ro 5-6902, Ro 5-6531, R.o 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro S-6524, Ro 5-5122, W erapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds that bind with as high or higher affinity to peripheral benzodiazepine receptors.
As will be apparent to those skilled in the art of the invention in the light of l0 the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit and scope thereof.
Accordingly, the scope of the invention is to be considered in accordance with the substance defined by the claims.

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Claims

WHAT IS CLAIMED IS:

1. A method of alleviating a chronic inflammatory condition in a mammal, characterized by administering to a mammal suffering from a chronic inflammatory condition a compound, or a pharmaceutically acceptable salt or derivative of the compound, said compound being one or more compounds which bind with high micromolar or submicromolar affinity to peripheral benzodiazepine receptors in the tissue of the mammal, said compound, or a pharmaceutically acceptable salt ar derivative of the compound, being administered to said mammal at a dosage in the range of 0.1-100 mg/kilogram of body mass of the mammal per day.
2. The method of claim 1 wherein the mammal is a human being and the compound binds with micromolar or submicromolar affinity to the human peripheral benzodiazepine receptors.
3. The method of claim 2, wherein the inflammatory condition is dementia of the Alzheimer type and the peripheral benzodiazepine receptors are located in the brain.
4. The method of claim 2, wherein the inflammatory condition is rheumatoid arthritis.
5. The method of claim 2, wherein. the inflammatory condition is lupus erythematosus.
5. The method of claim 2 wherein the inflammatory condition is Sjogren's syndrome.
6. The method of claim 1, 2, 3, 4, 5 or 6 wherein the compound is 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide.

8. The method of claim 1 wherein the compound is one or more compounds of: PK 11195, PK-14067, PR:14105, Ro 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6659, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524, Ro 5-5122, therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds of similar static configuration that bind with as high or higher affinity to peripheral benzodiazepine receptors in the mammalian tissue.
9. The method of claim 1 wherein the compound, or a pharmaceutically acceptable salt of the compound, is administered to the mammal orally, parenterally, rectally or locally.
10. An article of manufacture characterized by packaging material and a pharmaceutical composition contained within the packaging material, wherein the pharmaceutical composition is therapeutically effective for treating an inflammatory condition, and wherein the packaging material comprises a label which indicates that the pharmaceutical composition can be used for treating the inflammatory condition, and wherein the pharmaceutical composition comprises a compound, or a pharmaceutically acceptable salt of the compound, or a mixture of compounds which bind with micromolar or submicromolar affinity to peripheral benzodiazepine receptors in mammalian tissue.
1l. The article of manufacture of claim 10 wherein the compound is 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide.
12. The article of manufacture of claim 11 wherein the inflammatory condition is rheumatoid arthritis.
13. The article of manufacture of claim 11 wherein the inflammatory condition is lupus erythematosus.

14. The article of manufacture of claim 11 wherein the inflammatory condition is dementia of fine Alzheimer type.
15. The article of manufacture of claim 11 wherein the inflammatory condition is Sjogren's syndrome.
16. The article of manufacture of claim 10 wherein the compound is one or more of: PK 11195, PK-14067, PB:14105, Ro 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524, or Ro 5-5122, or therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds of similar steric configuration that bind with as high or higher affinity to peripheral benzodiazepine receptors in mammalian tissue.
17. A method of identifying a compound that is therapeutically effective for treating an inflammatory condition in a mammal characterized by selecting a compound:
that binds with micromola or submicromolar affinity to peripheral benzodiazepine receptors; and that is therapeutically effective in treating inflammatory symptoms in MRL-Ipr mice.
18. The method of claim 17 further comprising the step of selecting the compound that inhibits the neurotoxic effects of products secreted by cells of the monocytic type following activation by inflammatory stimulators.
19. The use of one or more compounds which bind with micromolar or submicromolar affinity to peripheral benzodiazepine receptors in mammalian tissue in alleviating a chronic inflammatory condition in a mammal suffering from a chromic inflammatory condition characterized by administering to the mammal one or more compounds or a pharmaceutically acceptable salt of the one or more compounds, said compound or compounds, or a pharmaceutically acceptable salt of the compound or compounds being administered to said mammal at a dosage in the range of 0.1-mg/kilogram of body mass of the mammal per day.
20. The use as claimed in claim 19 wherein the mammal is a human being and the compound or compounds bind with micromolar or submicromolar affinity to the human peripheral benzodiazepine receptors.
21. The use of claim 20, wherein the inflammatory condition is dementia of the Alzheimer type and the peripheral benzodiazepine receptors are located in the brain.
22. The use of claim 20, wherein the inflammatory condition is rheumatoid arthritis.
23. The use of claim 20, wherein the inflammatory condition is lupus erythematosus.
24. The use of claim 20 wherein the inflammatory condition is Sjogren's syndrome.
25. The use of claims 19, 20, 21, 22, 23 or 24 wherein the compound is 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide.
26. The use of claims 23, 24, 25, 26, 27 or 28 wherein the compound is one or more compounds of: PK 11195, PK-14067, PK 14105, Ro 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 3-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6324, or Ro 5-5122, or therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds of similar steric configuration that bind with as high or higher affinity to peripheral benzodiazepine receptors in the mammalian tissue.

27. A compound which binds with micromolar or submicromolar affinity to peripheral benzodiazepine receptors in the tissue of the mammal, said compound, or a pharmaceutically acceptable salt or derivative of the compound used in, the alleviation of a chronic inflammatory condition in an animal, said compound being administered to said mammal at a dosage in the rake of 0.1 to 100 mg/kg of body mass per day.
28. A compound as claimed in claim 27 wherein the compound is one or more of PK 11195, PK 14067, PK 14105, Ro 5-6993, Ro 5-4864, Ro 5-4900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 5-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524 or Ro 5-5122, or therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds of similar steric configuration that bind with as high or higher affinity to peripheral benzodiazepine receptors.
29. The use of a compound which binds with micromolar or submicromolar affinity to peripheral benzodiazepine receptors in mammalian tissue, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for alleviating a chronic inflammatory condition in a mammal suffering from a chronic inflammatory condition, said medicament being administered to said mammal at a dosage in the range of 0,1 to 100 mg/kg of body mass per day.
30. The use of a compound as claimed in claim 29 wherein the compound is one or more of the compounds of the group PK 11195 , PK 14067, PK
14105, Ro 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524 or Ro 5-5122, or therapeutically acceptable salts of these compounds or mixtures of these compounds or their salts, or other compounds of similar steric configuration that bind with as high or higher affinity to peripheral benzodiazepine receptors.