Abstract:
The current invention provides methods for the up-regulation of trkA in a mammal, including humans, comprising the administration of an effective amount of a compound of formula I ##STR1## wherein R 1  and R 3  are, independently, --H, --CH 3 , --CO(C 1  -C 6  alkyl), or --COAr, where Ar is optionally substituted phenyl; 
     R 2  is selected from the group consisting of pyrrolidine, piperidine, and hexamethyleneimino; or 
     a pharmaceutically acceptable salt or solvate thereof. 
     In addition, the current invention provides method to increase the effect of NGF (Nerve Growth Factor), NGF-like activities or NGF agonists, whether such is from endogenous or exogenous sources. 
     Further, the current invention provides methods for maintaining the homeostasis and health of the hippocampus, hypothalamus, and cortex, thus maintaining their biological functions.

Description:
This Application claims the benefit of U.S. Provisional Application No. 60/059,617, filed Sep. 23, 1997. 
    
    
     FIELD OF THE INVENTION 
     The current invention deals with the areas of medicinal chemistry, molecular biology, and pharmacology related to the regulation of trkA, the receptor for the neurotrophin Nerve Growth Factor (NGF), by the administration of 2-aryl-3-aroylbenzo[b] thiophenes. 
     BACKGROUND OF THE INVENTION 
     The trk proteins are a family of receptor tyrosine kinases, whose function is the signal transduction of neurotrophins. The trk receptor family consists of three known members (A-C), each binding specific neurotrophins. Thus, the neurotrophins induce many of their effects through one of these trk receptors. Most germane to the current invention is the trkA receptor, which binds the neurotrophin Nerve Growth Factor (NGF). 
     NGF is an essential neurotrophin in maintaining homeostasis of nerve tissue in both the peripheral and central nervous system. The critical activity of NGF has been demonstrated in the development, maintenance, and repair of nerves. In addition to the homeostatic benefit of endogenous NGF, it has been suggested that exogenously administered NGF, molecules having NGF-agonist activity or molecules that can stimulate NGF or NGF-like activity, may be useful in treating damaged nerve tissue, e.g., nerve damage due to trauma, surgery, disease, old age, etc. It would be useful if an agent were available which would enhance the beneficial effects of endogenous or exogenous NGF, NGF-like activities or NGF agonists. Such an agent might be one which would increase the response of nerves to NGF via up-regulating the molecular target of NGF, i.e., its receptor, trkA. For further information, see: &#34;Severe Sensory and Sympathetic Neuropathies in Mice Carrying a Disrupted trk/NGF Receptor Gene&#34;, Smeyne, R. J., et al., Nature, 368, pp. 246-248, Mar. 17, 1994. 
     Recently, it has been shown that the hormone estrogen regulates the gene coding the NGF receptor, viz., trkA. It has been known that estrogen appears to have a beneficial effect on maintaining healthy nerve tissue in many animal species, including humans. For further information, see: &#34;Estrogen Differentially Regulates Estrogen and Nerve Growth Factor Receptor mRNAs in Adult Sensory Neurons&#34;, Sohrabji, F., et al., J. Neurosci., 14(2), pp.459-471 (1994) and &#34;The Effects of Ovariectomy and Estrogen Replacement on trkA and Choline Acetyltransferase mRNA Expression in the Basal Forebrain of the Adult Female Sprague-Dawley Rat&#34;, McMillan, P. J., et al., J. Neurosci., 16(5), pp. 1860-1865, 1996 and references cited therein. 
     Therefore, it would seem reasonable that estrogens, e.g., 17-β-estradiol, estrone, and the like, would be useful in the treatment of conditions where increasing the effect of molecules that directly or indirectly activate trkA receptor would be beneficial. However, estrogens are known to have many detrimental side-effects, e.g., cancer risk, uterine stimulation, and the like, vitiating their use in such cases. 
     It would be beneficial if an agent were available that would up-regulate the trkA/NGF system without estrogenic side-effects. 
     SUMMARY OF THE INVENTION 
     The current invention provides methods for the up-regulation of trkA in a mammal, including humans, comprising the administration of an effective amount of a compound of formula I ##STR2## wherein R 1  and R 3  are, independently, --H, --CH 3 , --CO(C 1  -C 6  alkyl), or --COAr, where Ar is optionally substituted phenyl; 
     R 2  is selected from the group consisting of pyrrolidine, piperidine, and hexamethyleneimino; or 
     a pharmaceutically acceptable salt or solvate thereof. 
     In addition, the current invention provides methods to increase the effect of NGF (Nerve Growth Factor), NGF-like activities or NGF agonists whether provided from endogenous of exogenous sources. 
     Further, the current invention provides methods for maintaining the homeostasis and health of the hippocampus, hypothalamus, and cortex, thus maintaining their biological functions. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The current invention is related to the discovery that a select group of 2-aryl-aroylbenzo[b] thiophenes i.e., the compounds of formula I, are useful for up-regulating trkA, and other methods of the current invention. 
     General terms used in the description of compounds herein described bear their usual meanings. For example, &#34;C 1  -C 6  alkyl&#34; refers to straight or branched aliphatic chains of 1 to 6 carbon atoms including methyl, ethyl, propyl, iso-propyl, n-butyl, pentyl, hexyl and the like. 
     The term &#34;substituted phenyl&#34; refers to a phenyl group alone or having one or more substituents selected from the group consisting of C 1  -C 4  alkyl, C 1  -C 4  alkoxy, hydroxy, nitro, chloro, fluoro, or tri (chloro or fluoro)methyl. &#34;OC 1  -C 4  alkyl&#34; refers a C 1  -C 4  alkyl group attached through an oxygen bridge such as methoxy, ethoxy, n-propoxy, iso-propoxy, and the like. 
     The term &#34;pharmaceutically acceptable salt&#34; refers to either acid or base addition salts which are known to be non-toxic and are commonly used in the pharmaceutical literature. Commonly used acid addition salts are inorganic salts formed by the addition of sulfuric acid, nitric acid, hydrochloric acid, hydrobromic acid phosphoric acid, phosphorous acid and the like; or organic salts formed by the addition of acetic acid, formic acid, benzoic acid, citric acid, methanesulfonic acid and the like. Commonly used basic addition salts are the salts formed by alkali or alkaline earth hydroxides, ammonium hydroxide, alkyl or aromatic amines and the like. A preferred salt of this invention is the hydrochloride salt. 
     The term &#34;solvate&#34; refers to a molecular complex of a compound of formula I with one or more solvent molecules. Such solvent molecules would be those commonly used in the pharmaceutical literature, which are known to be innocuous to the recipient, e.g., water, ethanol, and the like. 
     The compounds of this invention are derivatives of centrally located carbon, i.e., the &#34;--CO--&#34; moiety in formula I, thus derivatives are methanones, e.g., a compound of A--CO--B, would be named [A][B]methanone. Further the compounds of formula I are derivatives of benzo[b]thiophene which is named and numbered according to the Ring Index, The American Chemical Society, as follows: ##STR3## 
     Thus, Raloxifene hydrochloride, which is a preferred embodiment of this invention, is a compound of formula I, where R 1  and R 3  are hydrogen; R 2  is N-piperidinyl; and it is as its hydrochloride salt. Raloxifene hydrochloride is named: [2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thie-3-yl][4-[2-(1-piperidenyl)ethoxy]phenyl]methanone hydrochloride. 
     All of the compounds used in the methods and formulations of the current invention can be made according to procedures, such as those detailed in U.S. Pat. No. 4,133,814 and U.S. Pat. No. 4,418,068, each of which is included by reference, herein. In general, the process starts with a benzo[b]thiophene having a 6-hydroxyl group and a 2-(4-hydroxylphenyl) group. The starting compound is protected, alkylated, and de-protected to form the compounds of formula I. The formula I compounds which are carboxylic esters may be prepared by methods described in U.S. Pat. No. 5,393,763, which is included by reference, herein. 
     The compounds of formula I are members of a group of compounds known as &#34;anti-estrogen&#34; which have selective estrogenic agonist and antagonist pharmacologic activities. For example, formula I compounds act as estrogen agonists in treating pathologic sequelae caused by the cessation of menses in females (see: Draper et al., &#34;Effects of Raloxifene (LY139481 HCl) on Biochemical Markers of Bone and Lipid Metabolism in Healthy Postmenopausal Women&#34;, Hong Kong, Fourth Int&#39;l. Symp. on Osteoporosis, Mar. 29, 1993.; U.S. Pat. Nos. 5,393,763, 5,464,845, and 5,391,557). In addition, the compounds of formula I have been shown to have the potential to protect CNS nerve tissue by inhibiting the damaging effects initiated by the causal condition, hypoglycemia, (U.S. Pat. No. 5,512,296), and to help correct inappropriate and imperfect tissue repair (U.S. Pat. No. 5,574,047). 
     As used herein, the term &#34;effective amount&#34; means an amount of compound of the present invention which is capable of up-regulating the trkA receptor in neurons, thereby increasing the pharmacologic action of NGF, NGF-like activities or NGF agonists and maintaining the healthy homeostasis of nerve tissue. 
     Pharmaceutical formulations can be prepared by procedures known in the art. For example, the compounds of this invention can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as agar agar, calcium carbonate, and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate and solid polyethyl glycols. Final pharmaceutical forms include pills, tablets, powders, lozenges, syrups, aerosols, saches, cachets, elixirs, suspensions, emulsions, ointments, suppositories, sterile injectable solutions, or sterile packaged powders, depending on the type of excipient used. 
     Additionally, the compounds of this invention are well suited to formulation as sustained release dosage forms. The formulations can also be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. Such formulations would involve coatings, envelopes, or protective matrices which may be made from polymeric substances or waxes. 
     The particular dosage of a compound of formula I required to up-regulate the trkA receptor according to this invention will depend upon the particular circumstances of the conditions to be treated. Such considerations as dosage, route of administration, and frequency of dosing are best decided by the attending physician. Generally, accepted and effective doses for oral or parentral administration will be from 10 mg to 800 mg, and more typically between 20 mg and 100 mg, expressed as equivalents of the free base of a compound of formula I. Such dosages will be administered to a patient in need of treatment from once to three times each day or as often as needed to effectively up-regulate the trkA receptor. A preferred amount is 60 mg of Raloxifene hydrochloride (56 mg of free base) per day via an oral route of administration. 
     The formulations which follow are given for purposes of illustration and are not intended to be limiting in any way. The total active ingredients in such formulations comprises from 0.1% to 99.9% by weight of the formulation. The term &#34;active ingredient&#34; means a compound of formula I, preferably Raloxifene hydrochloride. 
     
         ______________________________________Formulation 1: Gelatin CapsulesIngredient        Quantity (mg/capsule)______________________________________Active Ingredient 50-600Starch NF         0-500Starch flowable powder             0-500Silicone fluid 350 centistrokes             0-15______________________________________ 
    
     The ingredients are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules. 
     
         ______________________________________Formulation 2: TabletsIngredient         Quantity (mg/tablet)______________________________________Active Ingredient   50-600Starch             10-50Cellulose, microcrystalline              10-20Polyvinylpyrrolidone              5(as 10% solution in water)Sodium carboxymethyl cellulose              5Magnesium stearate 1Talc               1-5______________________________________ 
    
     The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules thus produced are dried at 50-600° C. and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl cellulose, magnesium stearate, and talc, previously passed through a No. 60 mesh U.S. sieve, are added to the above granules and thoroughly mixed. The resultant material is compressed in a tablet forming machine to yield the tablets. 
     
         ______________________________________Formulation 3: AerosolIngredient        Weight %______________________________________Active Ingredient 0.50Ethanol           29.50Propellant 22     70.00(Chlorodifluoromethane)Total             100.00______________________________________ 
    
     The active ingredient is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to -30° C. and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container. 
     
         ______________________________________Formulation 4: SuspensionSuspensions each containing 100 mg of a compoundof formula I per 5 mL dose.Ingredient             Weight______________________________________Active Ingredient       100 mgSodium carboxymethyl     50 mgcelluloseSyrup                  1.25 mLBenzoic acid solution (0.1M)                  0.10 mLFlavor                 q.v.Color                  q.v.Purified water to total                  Total 5 mL______________________________________ 
    
     A compound of formula I is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor, and color diluted in water are added and the mixture is stirred thoroughly. Additional water is added to bring the entire mixture to the required volume. 
    
    
     ASSAY 
     The following demonstration of the methods of the current invention are presented for the purposes of illustration and are not intended to limit the scope of this invention in any way. 
     Forty female Sprague Dawley rats (weight range of 200 to 225 g, seventy-five days old) are obtained from Charles River Laboratories (Portage, Mich.). The animals are either bilaterally ovariectomized (OVX) or exposed to a sham surgical procedure at Charles River Laboratories, and then shipped after one week. Upon arrival, they are housed in metal hanging cages in groups of 3 or 4 per cage and have ad libitum access to food (calcium content approximately 0.5%) and water for one week. Room temperature is maintained at 22.2°±1.7° C. with a minimum relative humidity of 40%. The photoperiod in the room is 12 hours light and 12 hours dark. 
     The animals are dosed daily by oral gavage with either compound 1, [2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1-pyrolidinyl)ethoxy]phenyl]methanone hydrochloride, at 3 mg/kg, or with a placebo vehicle containing 10% cyclodextrin. There are twenty animals per each dosing regimen. Groups of four animals are taken off study at time periods of 0.5, 3, 12, 24, and 48 hours. The animals are sacrificed and brains dissected, the particular portions of the brains are pooled, homogenized, and RNA extracted. Twenty microgram samples of the pooled RNAs are applied to a gel and separated on a gel with electrophoresis and blotted to Hybond membranes for a Northern Analysis. The samples are analyzed by hybridization with a  33  P-labeled PCR fragment probe to either trkA or 18S rRNA. The samples are quantitated by exposure to a phosphorimager screen and the data processed by ImageQuant software. The 18S rRNA blots are a normalizing control, thus increases in trkA mRNA expression are in relation to the 18S rRNA level. 
     The results of this demonstration are given in Table 1 and increases in trkA mRNA expression are increases between drug treated and placebo animals. 
     
                       TABLE 1______________________________________           trkA mRNA Change in TreatedBrain Section   Animals______________________________________Hippocampus     2× Increase @ 24 hoursHypothalamus    3× Increase @ 24 hoursCerebellum      No changeCortex          2× Increase @ 48 hours______________________________________ 
    
     The receptor, trkA, and NGF are found not only in the CNS, but also in the peripheral nerves. NGF and the other neurotrophins exhibit much the same activity in the peripheral nerves as they do in the CNS. Thus, compounds of the current invention would have the same beneficial effect in peripheral nerves, i.e., enhancement of the effect of NGF, NGF-like activities or NGF agonists, as they would in the CNS. 
     Compounds of the current invention may be used both in treatment and prophylactic modalities. For example, in a treatment modality, patients suffering from a neuro-degenerative disease or trauma where neurons have suffered damage and are attempting to regenerate aided by the action of either endogenous or exogenous NGF, NGF-like activities or NGF agonists may be benefited. Particular circumstances illustrating a treatment modality include, but are not limited to neuronal healing and regeneration after cerebrovascular events, e.g., ischemic stroke, arterial occlusions, arterial insufficiency, embolism, aneurysm rupture, hemorrhage, and the like; trauma due to accident or surgery to either the CNS or peripheral nerves; or infection; inflammation; hypertension of cranial pressure; etc. 
     As mentioned, supra, the methods of the current invention would enhance the beneficial action of both endogenous and exogenous NGF, NGF-like activities or NGF agonists. Enhancement of the beneficial action of endogenous NGF or NGF-like activities would be in treatment of those conditions where the natural healing process is being allowed to proceed without external addition of NGF, e.g., a normal recovery from meningitis, encephalitis, an ischemic event, or the like. Enhancement of the beneficial action of exogenous NGF or NGF agonists would be in treatment of those conditions where the healing process is being accelerated with external addition of NGF or NGF agonists, e.g., administering such at the site of a surgical procedure, ischemic site, trauma, or the like. 
     The methods of the current invention when used in a prophylactic mode to enhance the beneficial action of endogenous NGF or NGF-like activities to prevent the pathogenic sequelae which would result from any condition or disease before such sequelae are manifested. Additionally, the methods of the current invention are useful for ameliorating the neuro-degenerative effects of aging. Thus, the current invention maintains good neuronal health. 
     This maintenance of good neuronal health is especially beneficial to the CNS and, in particular, the hippocampus, hypothalamic, and the cortex regions of the brain. Not only are the methods of the current invention especially beneficial to the particular portions of the brain, but also would be especially useful in maintaining the neurological functions of those portions, e.g., cognitive functions associated with the cortex, sensory perception associated with the hippocampus, etc.