Abstract:
According to the present invention, compounds selected from the thiozole class of organic molecules are employed as antagonists for receptor-operated ion channels (ROICs) useful in the treatment of a variety of disease states in which ROICs are implicated. ROICs of particular interest are receptor-operated calcium channels (ROCCs). Compounds of particular interest with respect to the subject invention are compounds of the thiazolidinedione class of organic molecules, more particularly ciglitazone. Disease states in which particular utility is expected include vasculoproliferative diseases such as atherosclerosis, restenosis following angioplasty, and other conditions in which PDGF is known to play a role. The utility of the present invention with respect to further studies on the operation of ROICs and ROCCs and their role in disease will be apparent to those of skill in the art.

Description:
This is a Continuation of application Ser. No. 08/206,909, filed Mar. 4, 1994 (now abandoned), which is a continuation of application Ser. No. 07/766,727, filed Sep. 26, 1991 (abandoned). 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates generally to the treatment of disease and, more particularly, to the treatment of diseases associated with receptor-operated calcium channels, including the treatment of hypertension, atherosclerosis, atherogenesis, thrombosis, vasospasm, and the consequences of these conditions. 
     Agonist-mediated increases in intracellular calcium in mammalian cells can occur via multiple mechanisms, most notably increased conductance of Ca 2+   ions through voltage-operated calcium channels (VOCCs) and receptor-operated calcium channels (ROCCs), both being located in the plasma membrane. 
     The VOCCs were the first identified in cardiac muscle and their characterization were greatly facilitated by the discovery of the highly sensitive and specific dihydropyridine antagonists. Fleckenstein et al., Am. J. Cardiol. (1987)59:177B-187B. These channels occur primarily in so-called electrically excitable cells and have been studied extensively in cardiac, smooth, and skeletal muscle. Channel opening, or more precisely, the probability that specified numbers of calcium channels are biophysically in the open state, is directly related to the potential difference across the plasma membrane. Depolarization increases the probability of the existence of VOCCs in the open state, whereas hyperpolarization results in the opposite, that VOCCs are likely to be closed. 
     The ROCCs, on the other hand are insensitive to changes in membrane potential and although sensitivity to dihydropyridine antagonists have been reported, the concentration required for significant blockade are orders of magnitude greater than those required for antagonism of VOCCs. ROCCs are activated by specific ligands and have been identified in all mammalian cell types examined including electrically excitable cells, e.g. cardiac and vascular smooth muscle. ROCCs have been identified in cells not ordinarily thought of being electrically excitable (Table 1). 
     The activity of ROCCs appear to be modulated primarily through agonist interaction with specific cell surface receptors with consequent phosphinositide breakdown and induction by inositol phosphates, e.g. inositol trisphosphate and inositol tetrakisphosphate, and activation of calcium entry via ROCCs. Ligands that transmit their biological signals by mechanisms involving calcium entry via ROCCs produce a transient increase in intracellular calcium concentration ( Ca 2+  !i) typified by a characteristic time-dependent kinetic form, regardless of the particular chemical agonist or the cell (tissue) type. 
     
                       TABLE I______________________________________Disease processes and potential sites of lesions where receptor-operatedcalcium channel antagonists are expected to function to offset theparticular lesion(s)Pathophysiologicprocesses,Disease states      Tissues        Neurohumoral agents______________________________________Atherogenesis,      VSM, endothelium                     growth factors, A IIAtherosclerosis,          endothelin, cytokines,Vasospasm, Angina         PDGF, thrombinPectorisRestenosis after      VSM, endothelium                     PDGF, growth factorsangioplastyThrombosis &amp;      VSM, platelets PDGF, thrombin, A II,infarction                endothelium, EGF,                     TGF-betaHypertension      VSM cell, endothelium,                     vasopressors, EDRF,      autononic innervation,                     neurotransmitters,      juxtaglomerular                     endothelinsNeoplasia and      various        transforming growth,oncogenesis               factors, endothelin,                     PDGFImmunodeficient      T cell, B cell,                     immune responseconditions mast cells, eosinophil                     modifiers, cytokines      neutrophil, macrohage                     chemotactic peptidesWound-healing      mesenchyme and other                     growth factors, cell      cells          adhesion moleculesReactive airway      bronchial VSM cell                     histamine, endothelin,disease, asthma      &amp; endothelium, mast                     beta-2 agonists,allergy    cells,         cromolynNeural dysfunction      glial cells, others                     neurotransmittersglioblastoma              nerve growth and other                     factors, endothelinSchizophrenia      CNS            endothelinEncephalopathy      CNS            neuropeptides, amino                     acids, nitric oxideMemory dysfuncton      CNS, other neural cells                     neurotransmittersOrganogenesis      various        colony-stimulating and                     chemotactic factors,                     neuropeptidesEndometriosis      endometrial cells                     PDGF, other growth                     factorsAlgesia    CNS            kinin/kininogen                     modulators______________________________________ VSM = vascular smooth muscle; EDRF = endotheliumderived relaxing factor; TGFbeta = transforming growth factor; A II = angiotensin II; EGF = epidermal growth factor 
    
     Subsequent to agonist-receptor binding, depending on the activator, there is a sharp initial spike in  Ca 2+  !i either immediately or after a delay followed by a slow decay in the calcium signal, which may be followed in some cases by oscillatory excursions in the calcium signal. Depending on cell-type or agonist, this general form has subtle but important variations, and may represent different subtypes of receptor-operated channels (see &#34;Experimental Findings&#34; below, and Ref: Rink, FEBS Lett, 1990). 
     A first aspect of the invention focuses on two physiological ligands that function via modulation of ROCCs and are implicated in the pathogenesis of hypertension, atherogenesis and atherosclerosis and other vasculoproliferative disease. 
     A second aspect of the invention includes the general implications of the role of ROCCs in the pathophysiological derangements in a variety of cellular functions, including those involving, neoplasia, immune response modification, endocrine and exocrine dysfunction, neuropsychiatric disease, wound-healing, and cell growth and proliferation in diverse organ systems. These vast implications will be disclosed in the context of small neurohumoral peptides, particularly platelet-derived growth factor (PDGF) and bradykinin. 
     A further aspect of the invention includes the utility of the subject use of thiazole compounds as antagonists for ROCCs in the further illucidation of ROCC operation. 
     A still further aspect of the invention includes the use of thiazole compounds in the treatment of disease in humans such as atherosclerosis. 
     SUMMARY OF THE INVENTION 
     According to the present invention, compounds selected from the thiazole class of organic molecules are employed as antagonists for receptor-operated ion channels (ROICs) useful in the treatment of a variety of disease states in which ROICs are implicated. ROICs of particular interest are receptor-operated calcium channels (ROCCs). Compounds of particular interest with respect to the subject invention are compounds of the thiazolidinedione class of organic molecules, more particularly ciglitazone. Disease states in which particular utility is expected include vasculoproliferative diseases such as atherosclerosis, restenosis following angioplasty, and other conditions in which PDGF is known to play a role. The utility of the present invention with respect to further studies on the operation of ROICs and ROCCs and their role in disease will be apparent to those of skill in the art. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the effect of PDGF on  Ca 2+  !i on A172 cells; 
     FIG. 2 shows the effect of PDGF on  Ca 2+  !i on A172 cells pretreated with ciglitazone (2 μg/ml). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The compounds employed according to the subject invention, the thiazole class of organic compounds, are the subject of extensive disclosure in the literature and methods of synthesis are, therefore, known to those of skill in the art. Of particular interest, however, are the disclosures of U.S. application Ser. No. 07/421,102, filed 13 Oct. 1989 and U.S. application Ser. No. 07/725,327, filed 8 Jul. 1991, the disclosures of which are hereby incorporated by reference. 
     The compounds of the subject invention are useful as antagonists of ROCCs which are implicated in a variety of tissues as indicated in Table II. The role of ROCCs in disease is only now being illucidated. For example, it is believed that the neurodegenerative effects of the human immunodeficiency virus (HIV) seen in many HIV infecteed patients is the result of the dysfunction of ROCCs in neurol tissue wherein the virus holds open ROCCs, causing the target cell to &#34;flood&#34; with calcium and ultimately die. The utility of ROCCs antagonists is obvious in such a condition. 
     According to the subject invention, ROCC antagonists are useful in modifying the effect of certain growth factors, particularly PDGF. PDGF is implicated in a variety of cardiovascular disease states including atherosclerosis, unstable angina, and restenosis following angioplasty. Monoclonal antibodies to PDGF have been shown to inhibit neointimal smooth muscle accumulation following angioplasty. Gordon, et al., Science(1991)253:1129-32. It is believed that efficacous doses of the compounds of the subject invention would have a similar effect on disease states wherein PDGF is implicated. An efficacous dose would be in the range of about 0.2 mg/kg to 100 mg/kg, particularly about 1 mg/kg to about 50 mg/kg, and more particularly about 2 mg/kg to 20 mg/kg. Efficacy is defined as the modification of calcium or other ion transport across the cell membrane of host cells from the level of ion transport prior to the administration of the therapeutic agent. 
     
                       TABLE II______________________________________Tissue Diversity of Ligand-Activated, Receptor-Operated CalciumChannels in Mammalian Cells (Partial List)Cell/Tissue        Agonist (examples)______________________________________Human T lymphocyte PHA, antibodies to              CD2, CD3 and T cell              receptor epitopesHuman B lymphocyte anti Ig antibodies, PHAHuman mast cell    histamineHuman platelet     thrombin, ADPVascular smooth muscle              norepinephrine, PDGF,              endothelin-1, histamine,              vasopressin, angiotensinIleum smooth muscle              acetylcholineHuman A172 glioma cell              bradykinin, PDGFVascular endothelium              thrombin, histamine              EDRF, nitric oxideHepatocyte         vasopressinLacrimal acinar cell              acetylcholineNeutrophil         fMLPJ774 macrophage    ATPNeural cells       neurotransmitters              endothelin-1,              endothelin-3Bronchial smooth muscle              endothelin-1Glomerular mesangial cells              endothelinParotid cell       muscarinic agonistsHuman glioma       endothelin______________________________________ PHA = phytohemaglutinin; Ig = immunoglobulin; fMLP = formylmethionyl-leucyl-phenylalanine; ATP = adenosine triphosphate; EDRF endotheliumderived relaxing factor; ADP = adenosine diphosphate 
    
     EXPERIMENTAL 
     MATERIALS AND METHODS 
     A. Effect of ciglitazone on the receptor-operated calcium channel agonist-induced  Ca 2+  !i signal. 
     The A172 human glioblastoma cell line was chosen to test the above-stated hypothesis for three reasons. First, the effects of PDGF on  Ca 2+  !i in A172 cells has been studied in detail. Second, the A172 cell does not contain voltage-sensitive calcium channels. Third, PDGF was chosen for its diverse cellular effects, including vasoconstriction, chemotaxis, and cell growth. Unless indicated otherwise, the methods employed in these studies are identical to those described in Szollosi et al., Cell Calcium (1991)12:477-91, the disclosures of which are hereby incorporated by reference. 
     A172 cells were grown to approximately 95% confluence and serum-starved for 4.5 hr prior to loading with the intracellular calcium indicator indo 1 by exposure to 3 μM of the acetoxymethyl ester (indo 1-AM). Ciglitazone, dissolved in dimethylsulfoxide, was added to a final concentration of 2 μg/ml immediately before loading with indo 1. The cells were incubated at 37 degrees for 45 min in an humidified incubator with 95% air plus 5% CO 2  as gas phase. The extracellular fluid was removed, the cells washed thrice with serum-free medium, and reincubated for a further 30 min to ensure complete hydrolysis of the indo 1-AM. The cells were then analyzed individually in a laser scanning microspectrophotofluorometer. 
     The effects of PDGF on  Ca 2+  !i on A172 cells are shown in FIG. 1. The data shown is the sum of the signal obtained by scanning 8 adjacent cells. The vertical bar signifies the time at which PDGF (20 ng/ml) was added to the well containing the cells. There is an initial delay followed by a sharp rise in  Ca 2+  !i. The peak value was sustained for approximately 3 min and is followed by a slow, sustained decay in the calcium signal. The proof that the initial spike in Al 72 cells is caused by the release of calcium from intracellular stores, and that the secondary sustained phase is the result of calcium entry via calcium channels located in the plasma membrane, is described in detail by Szollosi, et al. 
     The effect of PDGF on  Ca 2+  !i on A172 cells pretreated with ciglitazone (2 μg/ml) is shown in FIG. 2. Treatment with ciglitazone had no significant effect on the unstimulated (resting)  Ca 2+  !i, or peak value attained after addition of PDGF (20 ng/ml). However, the calcium signal was truncated by abolition of the secondary sustained portion of the calcium signal, the portion caused by opening of plasma membrane calcium channels. The data shown in FIG. 2 is the sum of the signal obtained by scanning 10 adjacent cells. The vertical bar shows the time at which PDGF was added. 
     The effects of PDGF and ciglitazone on various parameters relating to  Ca 2+  !i are summarized in Table III. The data was obtained by independently evaluating each of the 8 and 10 cells shown in FIGS. 1 and 2, respectively. The data in Table III confirms the lack of a significant effect by ciglitazone on the pre-peak delay or the initial calcium peak response. On the other hand, the prolonged post-peak elevation in  Ca 2+  !i was virtually eliminated at this concentration of ciglitazone (FIG. 1). These data indicate that the effect of ciglitazone was maximal at the concentration used (2 μg/ml). 
     
                       TABLE III______________________________________Effect of ciglitazone on the calcium signal induced by platelet-derivedgrowth factor        Control       Ciglitazone (2 μg/ml)        20 ng/ml PDGF + 20 ng/ml PDGFParameter    (n = 8)       (n = 10)______________________________________Baseline calcium ratio*        0.67 +/- 0.01 0.68 +/- 0.01Pre-peak dalay (sec)        57 +/- -6     54 +/- 5Peak response (sec)        1.0 +/- 0.1   1.1 +/- 0.1Response duration (sec)        701 +/- 37**  172 +/- 15**______________________________________ *Calcium concentration is expressed as the ratio of Ca2+:indo 1 chelate t free (umcomplexed) indo 1. Absolute concentration of free calcium is proportional to this ratio. The PDGF used in these studies is the purifei BB homodimer (G, Pierce, Amgen Biologicals, Thousand Oaks, CA). The vehicle for ciglitazone was used in control experiments. See text for further details. **Significance between control and ciglitazonetreated cells was p &lt; 0.001 as determined by Student&#39;s paried ttest. 
    
     The possibility that ciglitazone directly blocked PDGF-sensitive calcium channels was tested using the manganese maneuver in A172 cells loaded with indo- 1 (Szollosi et al., supra). Partial quenching of the second phase calcium response suggested partial direct blockade of the PDGF-operated calcium channel. Because, at the concentration of ciglitazone used, complete blockade of the second phase calcium response was achieved, the fact that a post-receptor effect of the drug is an inescapable conclusion. These seemingly disparate effects may simply be explained by a yet to be defmed structuraVfunctional relationship between the agonist receptor and the coordinate ROCC. Genetic uniqueness in either or both of agonist/receptor or coupled calcium channel response could, in a general sense confer the molecular organization required for the kind of diversity obviously inherent in biological signals conveyed via ROCCs. 
     Similar experiments on the A172 glioma cells were conducted with bradykinin, another hormone that transmits its signal via increased  Ca 2+  !i. The results were similar, namely, that ciglitazone blocked the second phase sustained plateau of  Ca 2+  !i without significant effect on the initial peak response. Interestingly, bradykinin has a vaso-relaxing effect on the vasculature, quite opposite to that of PDGF which is a vasoconstrictor. Furthermore, bradykinin has analgesic effects via effects on the central nervous system. Therefore, because of our findings with bradykinin and A172 cells, we also claim that thiazoles, appropriately designed, will prove a potential repository of therapeutically efficacious analgesic medicines. 
     The findings described herein demonstrate that ciglitazone blocks the portion of the PDGF-stimulated calcium transient which is mediated by receptor-operated calcium channels in the plasma membrane of A172 human glioblastoma cells. The fact that the other parameters (phases) of the calcium signal remained unaffected strongly suggests that the effect of ciglitizone is highly selective. 
     Identical experiments were carried out on cultured A10 rat vascular smooth muscle cells (VSMC). The results were virtually identical to those obtained with the A172 cells, namely that ciglitazone eliminated the secondary sustained phase of the calcium transient induced by PDGF in VSMC. Again, the effect of the ciglitazone appeared to be maximal at the concentration used (2 μg/ml). 
     B. Effect on DNA synthesis. 
     Ciglitazone inhibited PDGF-stimulated thymidine incorporation by approximately 55% as shown in Table IV. The inhibitory effects were similar at both drug concentrations, viz 0.5 and 2.0 μg/ml. These findings suggest that ciglitazone inhibits DNA synthesis induced by PDGF. This is believed to be a consequence of the antagonist effect of the drug on PDGF operated calcium channels and suggests that ciglitazone would be expected to inhibit cell growth, proliferation, and/or migration via this effect. 
     These data further suggest that blockade of PDGF receptor-operated calcium channels maybe ageneral property of the thiazoles, thus making them anovel class of calcium antagonists. While the precise mechanism is unknown the early data suggests a partial direct blockage of calcium entry across the cell membrane in cells having ROCCs. These findings imply that this novel class of calcium channel blockers may have therapeutic efficacy in a variety of cardiovascular pathologies, including hypertension, cardiac arrhythmia, coronary artery spasm, and atherosclerotic vascular disease. 
     
                       TABLE IV______________________________________Effect of ciglitazone on the incorporation of  .sup.3 H!-thymidine intohuman A172 glioma cellsSerum DMSO    Ciglitazone                   PDGF   .sup.3 H!-thymidine incorporation(%)   (%)     (μg/ml)                   (ng/ml)                         (counts per minute)______________________________________10.0  none    none      none  26700.1   none    none      none  8400.1   0.5     none      none  9100.1   none    0.5       none  8000.1   none    2.0       none  7800.1   none    none      10.0  15600.1   0.5     none      10.0  14900.1   none    0.5       10.0  10900.1   none    2.0       10.0  1180______________________________________ NB: The values shown represent the mean of triplicate determinations (internal error &lt;10.0%). See text for further details. 
    
     It will be apparent to those of skill in the art that the present invention adds to the state of the art the discovery of an entire new class of ROCC antagonists useful in the treatment of a variety of disease states and the consequences of such disease. 
     Although the present invention has been described in some detail for the purposes of clarity and understanding, it will be obvious to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims.