USE OF CYTOPROTECTANTS

Abstract: It is described that flavonoids can be used to produce pharmaceutical preparations for protection of specific groups of living cells against cell death occurring spontaneously, under pathological conditions or as the result of a toxic insult. It is demonstrated that certain flavonoids protect groups of neuronal and liver cells during toxic insults produced by okadaic acid.

Description: Use of cytoprotectants The invention relates to use of cytoprotective fiavonoids, alone or in combination with cytotoxic drugs, to produce medicaments to prevent the death of normal cells under pathological or therapeutic conditions, preparations and pharmaceutical compositions and kit containing the same, and method to protect specific cells. A major problem in cancer chemotherapy is the side effects of cytotoxic drugs on the normal cells and tissues of the body, which often limit the effectiveness of the treatment. Thus an important goal in therapy-related cancer research is to develop cytotoxic drugs which act specifically on cancer cells. An alternative strategy would be to develop agents which specifically protect normal cells against cytotoxic drugs, and therefore could be used in combination with cytotoxic drugs to achieve selective killing of cancer cells. Furthermore, such cytoprotectants could conceivably be used to protect against cell death occurring under certain pathological conditions, e.g. in Alzheimer's disease. In the present invention, the algal toxin okadaic acid has been used both as a model cytotoxin, and as a disease model, by virtue of its ability to elicit an Alzheimer-like condition in neural cells. Induction of cell death by okadaic acid and related toxins Okadaic acid is a toxin, produced by marine dinoflagellates [Tachibana K et al (1981) J Am Chem Soc 103, 2469-2471 ], that can induce hyperphosphorylation of cellular proteins through inhibition of protein phosphatases 1 and 2A [Bialojan C & Takai A (1988) Biochem J 256, 283- 290; Haystead TAJ et al (1989) Nature 337, 78-81 ; Cohen P et al (1990) Trends Biochem Sci 15, 98-102]. Biogenic toxins with a similar mechanism of action include dinophysistoxin-1 and acanthifolicin [Cohen P et al (1990) Trends Biochem Sci 15, 98- 102], calyculin A [Kato Y et al (1986) J Am Chem Soc 108, 2780-2781 ; Ishihara H et al (1989) Biochem Biophys Res Commun 159, 871-877], microcystin-LR, microcystin-YR, nodularin and other microcystins [Nishiwaki-Matsushima R et al (1992) J Cancer Res Clin Oncol 118, 420-424; Yoshizawa S et al (1990) J Cancer Res Clin Oncol 116, 609-614], fostriecin [Ho DT & Roberge M (1996) Carcinogenesis 17, 967- 972], tautomycin [Hori M (1991) FEBS Lett 285, 145-148], cantharidin [Li Y-M & Casida JE (l 992) Proc Natl Acad Sci USA 89, 11867-1 1870; Honkanen RE (1993) FEBS Lett 330, 283-286], and endothall [Li Y-M et al (1993) Biochem Pharmacol 46, 1435-1443]. Okadaic acid is a polyether fatty acid, the structure of which is shown in Fig. 1 along with the structures of some of the other protein phosphatase-inhibitory toxins (from [Fujiki H & Suganuma M (1993) Adv Cancer Res 61, 143-194]). The effect of okadaic acid on animal cells seems to depend on cell type and treatment conditions. Okadaic acid has frequently been reported to behave as a growth inhibitor, causing cell cycle arrest in late Gl [Kim T-A et al (1993) Proc Natl Acad Sci USA 90, 5460-5463; Simm A et al ( 1994) Exp Cell Res 210, 160-165; Zhang W et al (1995) Cancer Res 55, 668-674] and/or G2/M [Van Dolah FM & Ramsdell JS (1992) J Cell Physiol 152, 190-198; Giese G et al (1995) J Cell Physiol 163, 145-154], depending somewhat on when in the cell cycle it is added [Schδnthal A & Feramisco JR (1993) Oncogene 8, 433-441 ]. Growth inhibition has been associated with a reduction in cyclin- dependent protein kinase (Cdk) activity [Schδnthal A & Feramisco JR ( 1993) Oncogene 8, 433-441 ], induction of the Cdk inhibitor Cipl [Zhang W et al (1995) Cancer Res 55, 668-674], and interruption of mitosis [Ghosh S et al (1992) J Cell Sci 103, 1 17-124]. However, okadaic acid is an effective skin tumour promoter [Suganuma M et al (1988) Proc Natl Acad Sci USA 85, 1768-1771], and it has also been claimed that it can stimulate DNA replication at extremely low doses (<1 nM) [Afshari CA & Barrett JC (1994) Cancer Res 54, 2317-2321 ]. Okadaic acid may elicit various responses generally associated with cell proliferation, such as phosphorylation of retinoblastoma protein and p53 [Van Dolah FM & Ramsdell JS (1992) J Cell Physiol 152, 190-198; Yatsunami J et al (1993) Cancer Res 53, 239-241 ; McClure JE et al (1993) Biochem Biophys Res Commun 197, 1578- 1584], Cdk activation [Yamashita K et al (1990) EMBO J 9, 433 1-4338; Devault A et al (1992) J Cell Biol 118, 1 109- 1 120; Meikrantz W et al (1994) Proc Natl Acad Sci USA 91, 3754-3758] and MAP kinase activation [Casillas AM et al (1993) Biochem J 290, 545-550], but these effects are not always accompanied by actual cell growth [Van Dolah FM & Ramsdell JS (1992) J Cell Physiol 152, 190-198; Casillas AM et al (1993) Biochem J 290, 545- 550], and may in fact trigger apoptosis [Meikrantz W et al (1994) Proc Natl Acad Sci USA 91, 3754-3758]. Okadaic acid can cause extensive cell death in many cell types, e.g., lymphoid and leukemic cells [Sakurada K et al (1992) Biochem Biophys Res Commun 187, 488-492; Zheng B et al (1994) J Biol Chem 269, 12332-12338; Haldar S et al (1995) Proc Natl Acad Sci USA 92, 4507-451 1], fϊbroblasts [Yan Y et al (1997) J Biol Chem 111, 15220-15226], osteoblasts [Morimoto Y et al (1997) Exp Cell Res 230, 181-186], pituitary cells [Ritz V et al (1997) Biochem Pharmacol 54, 967-971 ], hepatocytes [Bøe R et al ( 1991 ) Exp Cell Res 195, 237-246], nerve cells [Fernandez MT et al (1991) Life Sci 49, PL157-PL162], mammary carcinoma cells [Bøe R et al (1995) Br J Cancer 72, 1151-1159; Sheikh MS et al (1996) Cell Growth Differentiation 7, 1599-1607], and HeLa cells [Meikrantz W et al (1994) Proc Natl Acad Sci USA 91, 3754-3758]. In vivo, okadaic acid (administered intragastrically) elicits diarrhea, with accumulation of fluid in the stomach, small intestine and colon, and damage to the intestinal epithelium [Edebo L et al (1988) APMIS 96, 1029- 1035; Ito E & Terao K (1994) Nat Toxins 2, 371-377]. Of particular relevance to the present invention are the toxic effects of okadaic acid on liver and nerve cells, which are discussed in more detail below. Effects of okadaic acid and related toxins on liver cells In isolated rat hepatocytes, okadaic acid has been shown to induce widespread protein hyperphosphorylation as a result of the shift in the kinase/phosphatase activity balance [Haystead TAJ et al. (1989) Nature 337, 78-81 ]. Okadaic acid inhibits various hepatocellular metabolic pathways, like glycogen synthesis [Agius L & Peak M (1991) Biochim Biophys Acta 1095, 243-248; Pugazhenthi S et al (1993) Biochim Biophys Acta 1179, 271- 276], phospholipid synthesis [Hatch GM et al (1992) J Biol Chem 267, 15751- 15758; Tijburg LBM et al (1992) Biochem Biophys Res Commun 182, 1226-1231], hormone signalling [Garcia-Sainz JA et al (1991) Biochem Biophys Res Commun 179, 852-858; Colombatto S & Grillo MA (1993) Int J Biochem 25; 697-700], autophagy [Holen I et al (1992) Biochem J 284, 633- 636; Holen I et al (1993) Eur J Biochem 215, 1 13-122] and endocytosis [Holen I et al (1995) Biochem J 311, 317-326]. Furthermore, okadaic acid causes hyperphosphorylation and disruption of the cytokeratin cytoskeleton [Holen I et al (1992) Biochem J284, 633-636; Ohta T et al (1992) 13, 2443- 2447; Blankson H et al (1995) 218, 522-530], inhibits growth, and induces apoptotic cell death [Bøe R et al (1991) 195, 237-246; Mellgren G (1993) Exp Cell Res 205, 293-301 ]. Calyculin A and microcystins likewise inhibit metabolic processes in isolated rat hepatocytes [Claeyssens S et al (1995) Biochem J 306, 693-696], and induce cytoskeletal alterations and hepatocellular cell death [Runnegar MTC et al (1987) Toxicon 25, 1235- 1239; Ericsson JE et al (1990) Biochem Biophys Res Commun 173, 1347- 1353; Hooser SB et al (1991) Vet Pathol 28, 259-266; Falconer IR & Yeung DSK (1992) Chem-Biol Interact 81, 181-196; Mellgren G (1993) Exp Cell Res 205, 293-301]. In vivo effects of okadaic acid on the liver appear not to have been reported, but microcystins are potently and specifically hepatotoxic when administered orally or i.p. to mice [Adams WH et al (1985) Toxicon 23, 441-447; Runnegar MTC et al (1986) Toxicon 24, 506-509; Falconer IR et al (1988) J Toxicol Environ Health 24, 291-305] or rats [Miura GA et al (1991) Toxicon 29, 337-346]. Toxic effects of okadaic acid on nerve cells, and the role of protein overphosphorylation in neuronal damage Overphosphorylation of proteins has been implicated in several types of nerve cell damage in the central nervous system. For example, ischemic brain injury has been associated with tyrosine-phosphorylation and activation of protein kinase C [Cardell M & Wieloch T (1993) J Neurochem 61, 1308- 1314] and MAP kinases (MAPKs) [Campos-Gonzalez R & Kindy MS (1992) J Neurochem 59, 1955-1958; Hu, BR & Wieloch T (1994) J Neurochem 62, 1357-1367]. Kindy [Kindy MS (1993), J Cereb Blood Flow Metab 13, 372- 377] showed that protein-tyrosine-kinase inhibitors like genistein and lavendustin A could block MAPK phosphorylation and prevent neuronal death induced by ischemia/reperfusion in gerbil hippocampus. In Alzheimer's disease, hyperphosphorylation of the cytoskeletal cross- binding protein tau causes formation of neurofibrillary tangles implicated in the etiology of the disease [Mandelkow EM et al (1996) Ann NY Acad Sci 111, 96-106]. Several protein kinases kan phosphorylate tau, including MAPKs [Trojanowski JQ & Lee VMY (1996) FASEB J 9, 1570-1576; Roder HM et al (1995) Biochem Biophys Res Commun 193, 639-647; Goedert M et al (1992) FEBS Lett 312, 95-99]. In PC 12 rat pheochromocytoma cells, often used as neuronal models because of their neuroectodermal origin, the Alzheimer amyloid beta protein (Abeta) was shown to induce MAPK activation [Sato N et al (1997) Biochem Biophys Res Commun 232, 637-642]. Injection of okadaic acid directly into sheep brains has been reported to precipitate an Alzheimer-like neuronal dystrophy [Nelson PT & Saper CB (1996) Neurosci Lett 208, 77-80]., 65-76]. Okadaic acid induces both MAPK activation and overphosphorylation of tau protein in cultured neurons [Arias C et al (1993) J Neurochem 61, 673-682; Garver TD et al (1995) Mol Pharmacol 47, 745-756; Baum L et al (1995) Brain Res Mol Brain Res 34, 1- 17; Saito T et al (1995) FEBS Lett 376, 238-242; Burack MA & Halpain S (1997) Neuroscience 72, 167-184; Malchiodi-Albedi F et al (1997) J Neurosci Res 48, 425-438; Ho DT et al (1997) J Neurochem 68, 106-111], and has been shown to induce cytoskeletal disruption [Sacher MG et al (1992) Biochem Biophys Res Commun 186, 524-530] and the death of cultured neurons [Fernandez MT et al (1991) Life Sci 49, PL157-PL162; Fernandez MT et al (1993) Ann NY Acad Sci 679, 260-269; Fernandez- Sanchez MT et al (1996) FEBS Lett 398, 106-112; Li YP et al (1996) Brain Res 738, 196-204] and neuroblastoma cells [Bøe et al (1991) Exp. Cell Res 195, 237-246; Uberti D et al (1997) Neurosci Lett 235, 149-153; Nuydens R et al (1998) J Neurochem 70, 1124-1133]. However, although isoquinolinesulfonamides in general have been found to protect neurons against okadaic acid-induced cell death, no protective effects have been obtained with more specific inhibitors of serine/threonine protein kinases [Candeo P et al (1992) J Neurochem 59, 1558-1561 ; Cagnoli CM et al (1996) J Mol Neurosci 1, 65-76]. Effects of fiavonoids on living cells A particularly versatile class of protein kinase inhibitors are the fiavonoids, which have been shown to inhibit a variety of serine/threonine protein kinases [Ferriola PC et al (1989) Biochem Pharmacol 38, 1617-1624; Jinsart W et al (1992) Hoppe-Seyler's Z Physiol Chem 373, 205-211; Worland PJ et al (1993) Biochem Pharmacol 46, 1831-1840; Higashi K & Ogawara H (1994) Biochim Biophys Acta 1221, 29-35], tyrosine-protein kinases [Akiyama T et al (1987) J Biol Chem 262, 5592-5595; Ogawara H et al (1989) J Antibiotics 42, 340-343] and histidine-protein kinase [Huang J et al (1992) JBiol Chem 267, 15511-15515]. In addition, many fiavonoids are reactive substances which display a variety of chemical and biological effects that may be unrelated to protein kinase inhibition. The effects of fiavonoids on cell growth are mostly inhibitory [Ogawara H et al (1989) J Antibiotics 42, 340-343; Yoshida M et al (1992) Cancer Res 52, 6676-6681 ; Worland PJ et al ( 1993) Biochem Pharmacol 46, 1831-1840; Higashi K & Ogawara H (1994) Biochim Biophys Acta 1221, 29-35]. Genistein has thus been shown to inhibit DNA synthesis and growth in various cell types [Okura A et al (1988) Biochem Biophys Res Commun 157, 183-189; Traganos F et al (1992) Cancer Res 52, 6200-6208; Yanagihara K et al (1993) Cancer Res 53, 5815-5821 ; Matsukawa Y et al (1993) Cancer Res 53, 1328-1331 ; Kuo S-M (1996) Cancer Lett 110, 41-48], including hepatocytes [Arakaki N et al (1992) Biochem Biophys Res Commun 185, 22- 28]. Several other fiavonoids have also been reported to inhibit the growth of cells or tumours. [Suolinna E-M et al (1975) Cancer Res 35, 1865-1872; Ogawara H et al (1989) J Antibiotics 42, 340-343; Post JFM & Varma RS (1992) Cancer Lett 67, 207-213; Yanagihara K et al (1993) Cancer Res 53, 5815-5821; Higashi K & Ogawara H (1994) Biochim Biophys Acta 1221, 29- 35; Hirano T et al (1994) Life Sci 55, 1061-1069; Namgoong SY et al (1994) Life Sci 54, 313-320; Zi et al. (1997) Biochem. Biophys. Res. Commun. 239, 334-339] Fiavonoids have frequently been reported to induce cell death. For example, quercetin, apigenin and luteolin induced p53 -dependent apoptotic cell death in fibroblastoid cells [Plaumann B et al. (1996) Oncogene 13, 1605-1614]. Tangeretin induced apoptotic DNA fragmentation in human HL60 leukemia cells [Hirano T et al. (1995) Br J Cancer 72, 1380-1388]. The antitumour effect of flavone acetic acid (FAA) seems to be due to an interruption of blood flow to the tumour [Zwi LJ et al. (1989) J Natl Cancer Inst 89, 1005- 1013]. A large number of fiavonoids, including genistein, were found to induce growth arrest and cell death in human mammary carcinoma cells [Hirano T et al. (1989) J Natl Cancer Inst 81, 1005-1013 ; Pagliacci MC et al. (1994) Eur J Cancer 30A, 1675-1682]; genistein has been shown to induce apoptotic death in several other cell types as well [Tallet A et al (1996) Cancer Res 56, 4255-4263; Carlo-Stella C et al (1996) Blood 88, 3091-3100; Wijesekera DS et al (1997) Kidney Int 52, 1511-1520]. Baicalein induced both apoptotic and necrotic cell death in various human hepatocarcinoma cell lines [Matsuzaki Y et al. (1996) Jpn J Cancer Res 87, 170-177]. Quercetin, dihydroquercetin and rutin could apparently protect human skin fibroblasts, keratinocytes, endothelial cells and sensory ganglion cells against cell death induced by oxidative stress (GSH depletion) [Skaper SD et al. (1997) Free Radic Biol Med 22, 669-678]. Genistein has been reported to prevent neuronal death induced by ischemia/reperfusion in gerbil hippocampus [Kindy MS (1993), J Cere b Blood Flow Metab 13, 372-377], and purpurogallin protected against ischemic injury in the rabbit heart [Wu TW (1994) Life Sci. 54, PL23-PL28]. PD98059 can apparently suppress the apoptosis related to oxidative stress in rat pleural mesothelial cells [Jimenez LA et al (1997) Am J Physiol 273, L 1029-L 1035 ] and to atherosclerotic ischemia (using a smooth muscle cell system) [Bono F et al (1997) FEBS Lett 416, 243-246]. Naringin has been reported to protect rodents against experimental peritonitis and lung oedema [Lambev I et al (1980) Eksp Med Morfol 19, 207-212]. All the protective effects of fiavonoids referred to above relate to nonspecific ischaemic, anoxic or oxidative tissue damage and/or cell death. No study has demonstrated or suggested cytoprotective effects of fiavonoids against okadaic acid-induced cell death, and no fiavonoids have been reported to prevent the death of liver cells. Apart from a described protection against cisplatin-induced nephrotoxicity by the white thistle flavonoid silibinin [Bokemeyer C et al (1996) Br J Cancer 74, 2036-2041], no therapeutic use of fiavonoids in combination with specific cytotoxic agents appears to have been demonstrated or suggested. Biological effects of naringin Naringin is the flavonoid responsible for the bitter taste of grapefruit [Hoffmann E (1879) Arch Pharm 14, 139-145]. It is a flavanone glycoside, the structure of which is shown in Fig. 2. Naringin is known to have free radical-scavenging [Maridonneau-Parini I et al (1986) Pharmacol Res Commun 18, 61-72; Chen YT et al (1990) Free Radic Biol Med 9, 19-21 ] and antimutagenic [Francis AR et al (1989) Carcinogenesis 10, 1953-1955] properties. However, naringin is non-inhibitory in most biological test systems [Kaul TN et al (1985) J Med Virol 15, 71-79; Moroney M-A et al (1988) J Pharm Pharmacol 40, 787-792; Nguyen TD & Canada AT et al (1993) JNutr 123, 259-268; Walle T et al (1995) Biochem Pharmacol 50, 731-734], whereas its aglycone, naringenin, is frequently inhibitory [Kimmich GA & Randies J (1978) Membrane Biochem 1, 221-237; Buening MK et al (1981) Cancer Res 41, 67-72; Moroney M-A et al (1988) J Pharm Pharmacol 40, 787-792; Guengerich FP et al (1991) Carcinogenesis 11, 2275-2279; Fuhr U et al (1993) Br J Clin Pharmacol 35, 431-436], suggesting that the sugar moiety of naringin, neohesperidose, helps to restrict its reactivity. Naringin does not seem to inhibit cell growth [Schwartz A & Middleton E et al (1984) Immunopharmacology 7, 115-126; Namgoong SY et al (1994) Life Sci 54, 313-320]. The inventor has previously shown that naringin antagonizes the effects of okadaic acid on hepatocytic intermediate filament organization [Blankson H et al (1995) 218, 522-530], autophagy, and endocytosis [Gordon PB et al (1995) J Biol Chem 270, 5830-5838], i.e. processes unrelated to cell death. Naringin was the most potent among 55 different fiavonoids tested, but okadaic acid-antagonistic effects were also observed with prunin, neohesperidin, neoeriocitrin, apiin, rhoifolin, and kaempferol 3-rutinoside, whereas genistein had a biphasic effect and other fiavonoids were inhibitory or inactive [Gordon PB et al (1995) J Biol Chem 270, 5830-5838]. In vivo, naringin has been reported to protect rodents against experimental peritonitis and lung oedema [Lambev I et al (1980) Eksp Med Morfol 19, 207-212]. No protective effects of naringin against cell death in any cell type or tissue have been reported, nor have any fiavonoids been reported to prevent the death of liver cells. Biological effects of PD98059 PD98059 is a flavone compound developed by the Parke-Davis company, with the structure shown in Fig. 2 [Dudley RT et al (1995) Proc Natl Acad Sci USA 92, 7686-7689]. It is an effective antagonist of MAPK activity due to its ability to prevent activation of the MAPK-activating enzymes MEK1 and MEK2 [Alessi DR et al (1995) J Biol Chem 270, 27489-27494]. Since MAPKs are essential mediators of the effects of many growth factors, PD98059 is usually found to act as a growth inhibitor, preventing the mitogenic effects of growth factors e.g. on fibroblasts [Weber JD et al (1997) Biochem J 326, 61-68], myoblasts [Milasincic DJ et al (1996) Mol Cell Biol 16, 5964-5973; Coolican SA et al (1997) J Biol Chem 272, 36653-6662], endothelial cells [Kroll J & Waltenberger J (1997) J Biol Chem 272, 32521- 32527; Xi XP et al (1997) FEBS Lett 417, 283-286], hepatocytes [Spector MS et al (1997) Mol Cell Biol 17, 3556-3565], smooth muscle cells [Servant MJ et al (1996) J Biol Chem 111, 16047-16052; Sachinidis A et al (1997) Mol Pharmacol 52, 389-397; Nelson PR et al (1998) J Vase Surg 27, 1 17- 125; Wilkie N et al (1996) J Biol Chem 271, 32447-32453; Karpova AY et al (1997) Am J Physiol 272, L558-L565], glomerular epithelial cells [Cybulski AV & McTavish AJ (1997) Biochem Biophys Res Commun 231, 160-166], glioma cells [Van Brocklyn et al (1997) J Neurochem 69, 116-125], pancreatic cancer cells [Kinoshita E et al (1997) FEBS Lett 407, 343-346], small cell lung cancer cells [Seufferlein T & Rozengurt E (1996) Cancer Res 56, 5758-5764], astrocytoma cells [Luo W et al (1996) Cancer Res 56, 4983- 4991], and various cell lines [Takahashi Y et al (1997) J Biol Chem 272, 12897-12900; Mcllroy J et al (1997) Mol Cell Biol 17, 248-255]. However, anti-cytostatic effects of PD98059 have been observed in cases where the MAPK pathway transmits a growth-inhibitory or apoptotic signal, such as in normal and malignant cells of pulmonary origin [Ravi RK et al (1998) J Clin Invest 101, 153-159], smooth muscle cells [Bornfeldt KE et al (1997) J Clin Invest 100, 875-885], mammary carcinoma cells [Alblas J et al (1998) Oncogene 16, 131-139], and fibroblasts [Pumiglia KM & Decker SJ (1997) Proc Natl Acad Sci USA 94, 448-452]. If the MAPK-induced growth arrest is accompanied by apoptosis, PD98059 can also be anti-apoptotic, e.g. in pulmonary [Jimenez LA et al (1997) Am J Physiol 273, L1029-L1035] or smooth muscle cells [Bono F et al (1997) FEBS Lett 416, 243-246]. PD98059 has been shown to promote cell death e.g. in hepatoma cells [Jarvis WD et al (1997) FEBS Lett 412, 9-14], cardiocytes [Aikawa R et al (1997) J Clin Invest 100, 1813-1821; Sheng Z et al (1997) J Biol Chem 272, 5783- 5791], PC 12 pheochromocytoma cells [Parrizas et al (1997) J Biol Chem 272, 154-161], and in the BaF3 cell line [Suzuki et al (1997) Oncogene 15, 1689-1697]. PD98059 has been shown to prevent MAPK activation in the brain [Bhat RV et al (1998) J Neurochem 70, 558-571], and to suppress the neuronal differentiation of neuroblastoma or phaeochromocytoma cells [Pang L et al (1995) JBiol Chem 270, 13585-13588; Kim B et al (1997) J Biol Chem 272, 21268-21273; Barrie AP et al (1997) JBiol Chem 272, 19666-19671 ]. PD98059 may prevent new long-term potentiation (LTP), but does not interfere with established LTP [English JD & Sweatt JD (1997) J Biol Chem 272, 19103-19106]. PD98059 has been shown to prevent the MAPK activation induced in PC 12 rat pheochromocytoma cells by the Alzheimer amyloid beta protein (Abeta) [Sato N et al (1997) Biochem Biophys Res Commun 232, 637-642]. However, PD98059 did not prevent tau hyperphosphorylation [Ho DT et al (1997) J Neurochem 68, 106-1 1 1], implicating other protein kinases in the latter phosphorylation. No protective effects of PD98059 against cell death have been reported in neurons or other cells of neurectodermal origin [Virdee K & Tolkovsky AM (1996) J Neurochem 67, 1801-1805; Creedon DJ et al (1996) JBiol Chem 111, 20713-20718; Parrizas M et al (1997) JBiol Chem 272, 154-161 ; Gunn- Moore FJ et al (1997) Biochem J 324, 365-369; Miller TM et al (1997) JBiol Chem 272, 9847-9853]. On the contrary, PD98059 was found to promote cell death in cerebellar granule cells [Villalba JN et al (1997) J Neurosci 17, 83-90]. The object of the present invention is to use fiavonoids for therapeutic protection of living cells against cell death. This object is obtained by the present invention as presented in the enclosed claims. In the present invention it is established that combinations of certain compounds surprisingly protect normal cells while pathological cells are killed. This represent a novel treatment strategy in which compounds selectively protect normal cells against cytotoxic agents and in combination with cytotoxic agents, protect normal cells when the cancer cells are destroyed. The present invention demonstrates that certain fiavonoids give selective protection of normal cells against a cytotoxic agent, while the corresponding cancer cells are not protected. Thus a combination of fiavonoids and cytotoxic agents surprisingly represents a novel preparation in the treatment of cancer. These cell-protecting fiavonoids can further be used to produce preparations active as therapeutica against degenerative diseases or disorders and other pathological conditions characterized by cell death. The present invention relates to the use of fiavonoids or derivatives thereof to produce a pharmaceutical preparation for therapeutic protection of specific groups of living cells against cell death occuring spontaneously, under pathological conditions, or as the result of a cytotoxin treatment. Further objects of the present invention comprise uses wherein the living cells are selected from the group containing neuronal cells, such as brain cells, except neuroblastoma cells and other cancer cells of neuronal origin, and liver cells, except hepatocellular cancer cells and other cancer cells of hepatic origin. Still further objects of the present invention comprise uses wherein the fiavonoids are selected from the group containing PD 98059 or a derivative thereof, and naringin or a derivative thereof. Further objects of the present invention comprise uses wherein the cell death occurs spontaneously, under pathological conditions, or as the result of cytotoxin treatment, e.g. with okadaic acid, microcystin or other protein phosphatase inhibitors or algal toxins, or antineoplastic agents, or other therapeutica, pharmaceutically acceptable vehicles and possibly further therapeutic and/or prophylactic compounds. Still further objects of the present invention comprise uses wherein fiavonoids and toxic agents are combined in one preparation or formulated in separate preparations so that the active ingredients are combined. In the following the present invention is described in more detail by examples and figures. Fig. 1. Chemical structures of okadaic acid and related protein phosphatase-inhibitory toxic agents. From [Fujiki H & Suganuma M (1993) Adv Cancer Res 61, 143-194]. Fig. 2. Chemical structures of naringin and PD98059. Fig. 3. Okadaic acid-induced cell death in rat brain (hippocampus) cultures. White areas indicate dead cells with propidium iodide-stained, fluorescent nuclei. Fig. 4. Okadaic acid-induced cell death in various regions of the hippocampus. The fluorescence from propidium iodide-stained, dead cells in various regions of the hippocampus (DG, CA1, CA3) was quantified from digitalized video images. Fig. 5. Okadaic acid-induced overphosphorylation of MAP kinases. Rat brain (hippocampus) cultures were incubated for up to 24h with up to 100 nM okadaic acid. At the time points and doses indicated, measurements were made of the extent of tyrosine-phosphorylation in protein bands of 42 and 44kDa size, corresponding to the MAP kinases (MAPKs) ERK2 and ERK1, respectively, using either a phosphotyrosine antibody (A) or a phospho- MAPK antibody (B). The total amounts of MAPK protein were also measured (C). Quantitative values from B and C are given in D. Fig. 6. Suppression of okadaic acid-induced MAP kinase phosphorylation by PD98059. Rat brain (hippocampus) cultures were incubated for Oh or 8h with up to 100 nM okadaic acid, in the absence or presence of 50 μM PD98059, and the extent of MAPK phosphorylation was measured with a phospho-MAPK antibody. The okadaic acid-antagonistic effect of PD98059 is evident both in the Western blot (upper part of figure) and in the quantitative representations of p42 (ERK2) and p44 (ERK1) phosphorylation (lower part of figure). Fig. 7. Prevention of okadaic acid-induced cell death by PD98059. Rat brain (hippocampus) cultures were incubated for up to 48h with 30 nM okadaic acid in the absence or presence of 10 μM PD98059. The okadaic acid-antagonistic effect of PD98059 is seen as a reduction in the area and intensity of propidium iodide fluorescence, indicative of cell death. Fig. 8. Okadaic-acid-antagonistic effects of PD98059 in various regions of the hippocampus. Rat brain (hippocampus) slices were incubated for 24 or 48h without or with 30 nM okadaic acid at the concentration of PD98059 indicated. The fluorescence from propidium iodide-stained, dead cells in various regions of the hippocampus (DG, CA1 , CA3) was quantified from digitalized video images. Fig. 9. Death of cultured rat hepatocytes treated with okadaic acid. Normal rat hepatocytes were cultured for 22h, then okadaic acid was added at the concentration indicated. At 48h, the cultures were stained with trypan blue or propidium iodide, and the number of intact (dye-excluding) cells was counted and expressed as per cent of the number in control (okadaic acid- free) cultures. Each value is the mean ± S.E. of 2-4 independent experiments. Fig. 10. Okadaic acid-induced alterations in cellular and nuclear morphology of cultured hepatocytes: protection by naringin. Normal rat hepatocytes were cultured for 24h, (A), without additions; (B), with 30 nM okadaic acid (added at 3.5h); (C), with 100 μM naringin (added at 3h); (D), with both okadaic acid and naringin. The nuclei were stained supravitally with Hoechst-33258. Left, phase contrast; right, fluorescence. Notice fragmented nuclei in (B). Magnification, 540x. Fig. 11. Induction of DNA fragmentation by okadaic acid. Normal rat hepatocytes (A) and Morris 7800C J hepatoma cells (B) were cultured for 22h; then various concentrations of okadaic acid were added (lane 1 , 0 nM; lane 2, 1 nM; lane 3, 10 nM; lane 4, 30 nM (A) or 15 nM (B); lane 5, 100 nM (A) or 30 nM (B); lane 6, 100 nM (B). At 48h, DNA was extracted and subjected to electrophoresis in 1.4% agarose gel as according to [Herrmann M et al (1994) Nucleic Acids Res 22, 5506-5507]. The gels were stained with SYBRTM(} reen an ^ photographed under ultraviolet illumination. Fig. 12. Flow-cytometric distribution of normal and apoptotic nuclei from cultured rat hepatocytes, labelled by the ISEL method. Normal rat hepatocytes were cultured for 24h, (A), without additions; (B), with 100 μM naringin; (C), with 30 nM okadaic acid; (D), with both naringin and okadaic acid. Nuclei were isolated and labelled with propidium iodide to measure the DNA content (ordinate) and with in situ end-labelling (ISEL) to detect nuclei with DNA strand breaks (abscissa). Nuclei with high fluorescence or a subdiploid DNA content were recorded as apoptotic (right-hand -shaped boxed area). Notice that normal hepatocyte nuclei can be diploid (2N), tetraploid (4N) or octoploid (8N). Fig. 13. Induction of apoptosis in cultured rat hepatocytes by okadaic acid, calyculin A, microcystin-LR and tautomycin. Normal rat hepatocytes were cultured for 24h in the presence of okadaic acid (0A,O), calyculin A (CA, •), microcystin-LR (MC, Δ), or tautomycin (TA, Δ ) at the concentration indicated. The nuclei were processed by the ISEL method and counted by flow cytometry, and the percentage of apoptotic cells was calculated. Each value represents a single experiment, or the mean ± range of two independent experiments. The background apoptosis (5- 10%) has been subtracted to facilitate comparison. Fig. 14. Effect of okadaic acid on cultured human hepatocytes. Normal human hepatocytes, prepared by collagenase perfusion of a fresh human biopsy specimen, were cultured for 24h under standard conditions [Mossin L et al (1994) Exp Cell Res 214, 551-560] in the absence (A,B) or presence (C,D) of 30 nM okadaic acid. The nuclei were stained supravitally with Hoechst-33258. Left, phase contrast; right, fluorescence. Notice fragmented nuclei in (D). Magnification, 540x. Fig. 15. Okadaic acid-antagonistic effect of naringin on cellular viability of normal hepatocytes, and lack of effect on hepatoma cells. Normal rat hepatocytes (A) and Morris 7800Cι hepatoma cells (B) were cultured for 22h; then okadaic acid (30 nM in A; 15 nM in B) was added, without (O) or with (•) naringin (added 30 min before) at the concentration indicated. At 48h, the number of morphologically intact (dye-excluding) cells was scored, and expressed as % of the control value (without added drugs). Each value is the mean ± S.E. of 3-4 independent experiments. Fig. 16. Protection, by naringin, of normal hepatocytes against apoptosis induced by microcystin or okadaic acid. Normal rat hepatocytes were cultured in the presence of microcystin (A) or okadaic acid (B) (added at 3h at the concentration indicated) with (•) or without (O) 100 μM naringin (added 30 min before). After 24h in culture, nuclei were isolated, in situ end- labelled and analysed by flow cytometry. The apoptotic index is given as per cent of the total numder of gated particles (10,000). Each value us the mean ± S.E or range of 2-5 experiments. Fig. 17. Lack of okadaic acid-antagonistic effect of naringin in rat and human hepatoma cells. Morris 7800C j rat hepatoma cells (A) or Hep3B human hepatoma cells (B) were cultured in the presence of okadaic acid (added at 3h at the concentration indicated) with (•) or without (O) 100 μM naringin (added 30 min before). After 24h in culture, the cells were harvested and analysed by flow cytometry after in situ end-labelling. The number of apoptotic cells is expressed as pre cent of the total number of gated particles (10,000). Each value is the mean ± S.E or range of 3-5 experiments. Similar results were obtained with the human C3A hepatoma cell line. The examples demonstrate that fiavonoids can have tissue-specific cytoprotective effects: PD98059 specifically protects normal neuronal cells against okadaic acid cytotoxicity, but we have found no cytoprotective effect of this flavonoid on hepatocytes. Conversely, naringin specifically protects normal hepatocytes against okadaic acid cytotoxicity, but we have found no cytoprotective effect of naringin on neuronal cells. The examples indicate, furthermore, that the cytoprotective effects of fiavonoids may be restricted to normal cells, since no cancer cells yet examined have been found to be protected. This suggests the possibility of using cytoprotective fiavonoids in combination with cytotoxic agents as a general strategy for the treatment of malignant disease, achieving a more selective killing of cancer cells by protecting their normal counterparts. It may also be possible to use tissue-specific fiavonoids to protect tissues other than the target tissue for the toxic chemotherapeutic treatment. Regarding PD 98059, two novel observations are of particular relevance for the present invention: (a) PD98059 is able to protect normal neurons against toxin-induced damage and cell death. This suggests the possibility of using PD98059 as a neuroprotective drug in the therapeutic treatment of neurodegenerative disorders. (b) PD98059 selectively protects normal neurons, but not malignant cells of neuronal origin, against toxic damage and cell death. This suggests the possibility of a combination chemotherapy against human cancers of neurectodermal origin, wherein PD98059 is combined with a nonspecific toxin like okadaic acid to achieve selective killing of cancer cells while protecting normal nerve cells. Regarding naringin, two novel observations are of particular relevance for the present invention: (a) Naringin is able to protect normal hepatocytes against toxin-induced damage and cell death. This suggests the possibility of using naringin as a hepatoprotective drug in the therapeutic treatment of hepatic disorders. (b) Naringin selectively protects normal hepatocytes, but not malignant cells of hepatocellular origin, against toxic damage and cell death. This suggests the possibility of a combination chemotherapy against human cancers of hepatocellular origin, wherein naringin is combined with a nonspecific toxin like okadaic acid to achieve selective killing of cancer cells while protecting normal hepatocytes. In this connection it is of interest that we have found okadaic acid to specifically induce hepatic damage, and death, when injected intravenously in mice. In contrast, we found intragastric administration of okadaic acid to elicit diarrhea, with no detectable effect on the liver. For use in combination chemotherapy, PD98059, naringin, or other cytoprotective fiavonoids can be combined with any antineoplastic or cytotoxic drug, selected from the group consisting of Shionogi 254-S, acanthifolicin, acetaminophen, acitretin, aclarubicin, actinomycin D, actinoplanone, aeroplysinin derivative, aldophosphamide analogues, alpha- carotene, alpha-difluoromethylarginine, alstonine, altretamine, aminothiodiazole, amonafide, amphetinile, amsacrine, anaxirone, Angiostat, anisomycins, ankinomycin, anthracycline, antineoplastons, aphidicolin, aspariginase, azinomycin-A, baccharin, batracyclin, benfluron, benzotript, bisantrene, bisucaberin, Boehringer BBR-2207, BCG-vaccine, bestrabucil, bleomycin, brequinar, bromofosfamide, bryostatin- 1 , budotitane, Wakunaga CA-102, calichemycin, calyculin A, cantharidin, caracemide, carboplatin, carmethizole, carmofur, carmustine, Ciba-Geigy CGP-30694, Chinoin-139, Chinoin- 153, chlorambucil, chlormethine, chlorsulfaquinoxalone, chromoximycin, cisplatin, clanfenur, claviridenone, Contracan, crisnatol, curaderm, cyclopentylcytosine, cyclophosphamide, cyplatate, cytarabine, cytochalasins, cytocytin, dacarbazine, dactinomycin, datelliptinium, Lilly DATHF, daunorubicin, Merrel Dow DDFC, deazaguanine, delta- aminolevulinic acid, 2-deoxycoformycin, didemnin-B, dideoxyadenosine, dideoxycytidine, dideoxyguanosine, didox, dihaematoporphyrin, dihydrolenperone, dinaline, diphenylspiromustine, diplatinum, dinophysistoxin-1, distamycin, ditrisarubicin B, Yoshitomi DMDC, doxifluridine, doxorubicin, Wellcome EHNA, elliprabin, elliptinium acetate, elmustine, elsamicin-A, endothall, epirubicin, erbstatin, ergotamine, esorubicin, esperamicin-Al , esperamicin-Alb, estramustine, etoposide, etretinate, Merck EX-015, fazarabine, fenretinide, fiavonoids, floxuridine, fludarabine, 5-fluorouracil, 5-FU-fιbrinogen, Daiichi Seiyaku FO-152, fostriecin, fotemustine, gallium nitrate, genkwadaphnin, glidobactin, gregatin-A, grifolan, grincamycin, haematoporphyrin derivatives, hepsulfam, herbimycin, hexadecylphosphocholine, homocysteine, homoharringtonine, hydroxyurea, idarubicin, ifosfamide, illudins, ilmofosine, 5-iodotubercidin, iproplatin, isoglutamine, isopropylpyrrolizine, isotretinoin, kazusamycin, leukoregulin, lomustine, lonidamine, Lilly LY-18801 1, Lilly LY-264618, mafosfamide, marycin, menogaril, merbarone, merocyanine derivatives, methionine, methobenzaprim, methotrexate, metylanilinoacridine, microcystin-LR, microcystin- YR, minactivin, mitoguazone, mitolactol, mitonafide, mitomycin, mitoquidone, mitoxanthrone, mopidamol, motretinide, Wellcome MZPES, nafazatrom, nocodazole derivatives, nodularin, norspermidine, NCI NSC- 127716, NCI NSC-264880, NCI NSC- 39661 , NCI NSC-612567, octreotide, oquizanocine, oxaliplatin, oxalysine, oxaunomycin, paclitaxel, Warner-Lambert PALA, pancratistatin, pazelliptine, pentostatin, peplomycin, pilatin, pirarubicin, piritrexim, piroxantrone, Asahi PL-AC, plicamycin, polyhaematoporphyrin, polypreic acid, porothramycin, porphyrins, prednimustine, probimane, procarbazine, proglumide, protease nexin I, protein phosphatase inhibitors, protein tyrosine phosphatase inhibitors, protein kinase inhibitors, protein tyrosine kinase inhibitors, pyrindamycin, ranimustine, rapamycin, razoxane, restrictin-P, retelliptine, retinoids, retinoic acid, rhizoxin, rodorubicin, semustine, sibanomicin, siwenmycin, sorangicin-A, sparsomycin, spatol, spirocyclopropane derivatives, spirogermanium, spiromustine, steffimycin B, strypoldinone, superoxide dismutase, Takeda TAC-788, talisomycin, tauromustine, taxol, temozolomide, teniposide, teroxirone, terpentecin, tetraplatin, thaliblastine, thioguanine, thiotepa, thrazine, tiazofurin, Erbamont TIF, tocotrienol, Topostin, tricrozarin A, trimelamol, trimetrexate, Taiho UFT, ukrain, uricytin, vinblastine, vincristine, vindesine, vinestramide, vinorelbine, vintriptol, withanolides, and zorubicin. Use of the active compounds according to the invention may be as the chemical compound, or the active ingredients may be included in a pharmaceutical preparation. The pharmaceutical preparation may comprise the active compound or a derivative thereof, and/or more pharmaceutically acceptable vehicles and possible further therapeutic and/or prophylactic compounds. The pharmaceutical preparations are suitable for oral, rectal, nasal, topical, vaginal or parenteral (included intra-muscular, subcutareous or intravenous) administration. The preparations are also suitable for inhalation or insuflation. The pharmaceutical preparation can be in the form of solids, such as tablets, capsules, powders, suppositories, or granules, or liquids such as solutions, suspensions, emulsions or elixirs, or sterile injectable solutions for parenteral use. The pharmaceutical preparation of the present invention may contain the fiavonoids or its derivates combined with one or several antineoplastic agent, ocadaic acid or other phosphatase-inhibitory toxic agents, or the fiavonoids may be contained in a separate preparation. Furthermore, the pharmaceutical preparation of the present invention may contain the active ingredients, one or more pharmaceutically acceptable vehicles and possibly other therapeutic and/or prophylactic compounds. Example 1. Okadaic acid induces MAP kinase activation and extensive neuronal cell death in brain slice cultures from rat hippocampus Brain slice cultures were prepared as previously described [Gahwiler BH (1988) Trends Neurosci 11, 484-489; Laake JH et al (1995) J Neurochem 65, 871-881 ]. Hippocampal slices, 400 μm thick, were cut from male rat pups (P4-P7) and fixed onto glass coverslips with a chicken plasma/thrombin clot. The coverslips were incubated at 35-36°C in rotating tubes, in 50% Eagle's basal medium with 25% Hanks' balanced salts and 25% inactivated horse serum, with weekly medium change. After 13-14 days, the cultures were used for experiments. Cell death was recorded by labelling the nuclei of dead cells with propidium iodide, which gave a red nuclear fluorescence that could be quantitated in the Olympus IMT2 fluorescence microscope with the aid of a Hamamatsu C4880-96 cooled CCD camera and associated image processing software. Pictures of whole cultures were converted to black-and-white and analysed for gray-level intensity within outlined regions by means of AnalySIS software (Soft Imaging Software GmbH). For MAP kinase immunoblotting, five cultures were pooled and homogenized in the presence of proteinase inhibitors and a protein-tyrosine- phosphatase inhibitor (vanadate). Samples containing 10 μg protein were separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes (Bio-Rad), incubated at 4°C overnight with primary antibody, and then for lh with an HRP-coupled secondary antibody. Labelling was detected by the enhanced chemiluminescense technique (ECL, Amersham). Antibodies to ERK1/ERK2 and phospho-MAP kinase were purchased from New England Biolabs Inc, and phosphotyrosine antibodies from Transduction Labs Inc. Okadaic acid, in the concentration range 1-300 nM, induced extensive neuronal death as seen in the fluorescence microscope (Fig. 3) and from the quantitative data (Fig. 4). All the major hippocampal regions (DG, CAl and CA3) were affected. Electron microscopic examination indicated that the neuronal cell death was predominantly of the necrotic rather than the apoptotic type, whereas glial cells seemed to die by apoptosis. Okadaic acid induced rapid hyperphosphorylation of the MAP kinases ERK1 (44 kDa) and ERK2 (42 kDa) as assessed with either a specific phosphoMAPK antibody (Fig. 5B), or a general anti-phosphotyrosine antibody (Fig. 5A). Only the phosphorylation (and, by inference, the activity) was stimulated, while the overall protein concentrations remained unaltered, as shown with a general MAPK antibody (Fig. 5C). Example 2. PD98059 protects normal neurons against okadaic acid- induced MAP kinase activation and cell death The flavonoid PD98059 strongly antagonized the okadaic acid-induced MAPK phosphorylation (Fig. 6), and the okadaic acid-induced neuronal death, as seen in the fluorescence microscope (Fig. 7) and from the quantitative data (Fig. 8). Neuronal death in all hippocampal regions was prevented by PD98059, even at a dose as low as 10 μM. PD98059 is thus an effective protectant of normal brain neurons. Example 3. Okadaic acid induces extensive cell death of cultured NB-1 human neuroblastoma cells The human neuroblastoma cell line NB- 1 [Ishikawa S (1977) Acta Pathol Jpn 27, 697-711 ] was cultured in RPMI medium with 10% foetal calf serum. After the seeding of new subcultures, the cells were allowed 4h to attach to the culture dish. Treatment with up to 50 nM okadaic acid was then initiated, and already 6h later, toxic effects were indicated by the detachment of some of the cells. After 20h in culture, the majority of the cells had detached at the highest okadaic acid concentrations (30 and 50 nM). Most of these were dead as indicated by propidium iodide staining; in addition, 5- 10% of the attached cells had died, giving an overall death rate of 50-90%) at the highest okadaic acid concentrations (Table 1). Cell death was apparently of the necrotic type, since Hoechst-stained nuclei did not show typical apoptotic morphological changes at any time point, and flow-cytometric analysis of DNA strand breaks by the TUNEL method did not show the typical apoptotic response seen with many other cell types. Example 4. PD98059 does not protect neuroblastoma cells against okadaic acid-induced cell death Addition of PD98059 (10-50 μM) to NB-1 neuroblastoma cell cultures 3h after seeding had no detectable effect on its own, and failed to affect the okadaic acid-induced cell detachment or cell death (Table 1). In contrast to its ability to antagonize okadaic acid in normal neurons, PD98059 would thus seem to have no protective effect on these malignant cells of neuronal origin. Table 1. Okadaic acid-induced cell detachment and cell death in NB-1 neuroblastoma cell cultures: lack of protection by PD98059. NB- 1 human neuroblastoma cells were cultured for up to 20h in the absence or presence of 50 μM PD98059, added at 3h after seeding, with up to 50 nM okadaic acid, added at 4h after seeding. The percentage of dead (propidium iodide- staining) cells was recorded after 10 or 20h in culture. Data from a single experiment, representative of 5 performed (the other 4 without quantification). Percentage of dead cells in the culture lO h in culture 20 h in culture Okadaic acid -PD98059 +PD98059 -PD98059 +PD98059 (nM) 0 7 6 13 12 10 9 6 12 22 30 10 8 59 46 50 22 25 62 87 Example 5. No detectable in vivo toxicity of PD98059 Intravenous injection of PD98059 in mice at 10 mg/kg body weight caused no detectable change in the behaviour or health status of the animals. Example 6. Okadaic acid and related protein phosphatase-inhibitory toxins induce apoptotic cell death in cultured rat and human hepatocytes Normal rat liver cells (hepatocytes) were isolated from adult male rats by the two-step collagenase perfusion method [Seglen PO (1976) Meth Cell Biol 13, 29-83], and cultured in a serum- free medium supplemented with EGF, insulin and dexamethasone as previously described [Mossin L et al (1994) Exp Cell Res 214, 551-560]. Addition of okadaic acid after 22h in culture caused a dose-dependent detachment and cell death as measured by staining with trypan blue or propidium iodide at 48h (Fig. 9). At the higher okadaic acid concentrations, most of the attached cells also had an abnormal morphology, indicative of cell damage. Supravital staining with the Hoechst 33258 dye revealed that many of these cells had fragmented nuclei (Fig. 10B), a typical feature of ongoing apoptosis, or programmed cell death [Bøe R et al (1991) Exp Cell Res 195, 237-246]. Apoptosis was confirmed by the ability of okadaic acid to induce extensive internucleosomal DNA cleavage detectable as a "DNA ladder" in agarose gels (Fig. 1 IA), another hallmark of this cell death mechanism [Gerschenson LE et al ( 1992) FASEB J 6, 2450-2455]. As a quantitative measure of apoptosis in the adherent cells, DNA damage (strand breakage) was measured in hepatocytic nuclei by in situ end-labelling (ISEL) of free DNA ends [Cain K et al (1996) Cytometry 23, 312-321 ]. The method is based on the incorporation of digoxigenin-UTP into formaldehyde- fixed nuclei by means of a terminal transferase enzyme, subsequent immunocytochemical staining with anti-digoxigenin-FITC, and flow- cytometric enumeration of the highly fluorescent apoptotic nuclei along with fragmented nuclei of a subdiploid DNA content (Fig. 12). As shown in Fig. 13, the fraction of apoptotic cells among the adherent hepatocytes after 24h of okadaic acid treatment increased as a function of toxin dose to almost 30% apoptosis at 50 nM. The related toxins, calyculin A, microcystin-LR and tautomycin, were less potent than okadaic acid (no effect below 50 nM), but they all induced pronounced apoptosis; calyculin A even to higher levels (70% at 100 nM) than okadaic acid (Fig. 13). Okadaic acid is also an effective inducer of apoptosis in normal human hepatocytes: in cultures established from a clinical liver biopsy (the only biopsy we have obtained so far at the Norwegian Radium Hospital), there was, after 24h, 7% apoptotic cells in control cultures, and 29% in cultures treated with 30 nM okadaic acid for 24h (morphology, see Fig. 14). Example 7. Naringin protects normal rat hepatocytes against okadaic acid-induced cell death The flavonoid naringin, added to cultured rat hepatocytes just before the addition of okadaic acid, completely prevented the formation of fragmented, apoptotic nuclei as seen in the fluorescence microscope (Fig. 10D). The death of okadaic acid-treated hepatocytes was also strongly suppressed by naringin at concentrations up to 100 μM (Fig. 15 A). At higher concentrations, the flavonoid was less effective, perhaps indicating the onset of nonspecific toxicity. The ISEL method confirmed the effectiveness of naringin as an antagonist of okadaic acid-induced apoptosis in normal rat hepatocytes (Fig. 16). No tests have so far been done with normal human hepatocytes, since further biopsy material has not yet been available. Example 8. Okadaic acid induces apoptotic cell death in various rat and human cancer cell lines of hepatocellular and colonic origin, but naringin has no protective effect on these malignant cells The 7800Cj Morris hepatoma cell line, derived from a rat hepatocellular carcinoma [Richardson Ul et al (1913) J Cell. Physiol 83, 141-149], was susceptible to okadaic acid-induced apoptosis, as indicated by the DNA fragmentation assay (Fig. 1 IB). Furthermore, the percentage of cells which were dead after 24h of okadaic acid treatment (Fig. 15B) was comparable to that of the normal rat hepatocytes, as was the percentage of apoptotic nuclei detected by the ISEL method (Fig. 17A). However, in contrast to the normal hepatocytes, naringin had no protective effect on the hepatoma cells (Figs. 15B and 17). A human hepatoma cell line, Hep3B, exhibited massive apoptosis after okadaic acid treatment, but failed to be protected by naringin (Fig. 17B). Similar results were obtained with two other human hepatoma cell lines (HepC3A and HepG2), and with three human colon carcinoma cell lines (Colo 155, Colo 205 and CaCo2). Example 9. No detectable in vivo toxicity of naringin Naringin, administered by intragastric intubation to mice or rats at doses of up to 8 g/kg body weight, or intraperitoneally to rats at up to 2 g/kg, did not detectably impair the health status of the animals.

Claims: CLAIMS 1. Use of fiavonoids, except the white thistle fiavonoids, silymarin and silibinin, or derivatives thereof to produce a pharmaceutical preparation for therapeutic protection of specific groups of living cells against cell death occurring spontaneously, under pathological conditions, or as the result of treatment with a toxic agent, except agents that cause death by ischaemic, anoxic or oxidative tissue damage. 2. Use according to claim 1 wherein the living cells are neural cells, including brain cells, except neuroblastoma cells and other cancer cells of neural origin. 3. Use according to claim 1 or 2 wherein the flavonoid is PD98059, or a derivative of PD98059. 4. Use according to claims 1, 2 or 3 wherein the cell death is induced by a therapeutic agent. 5. Use according to claims 1, 2 or 3 wherein the cell death is induced by an antineoplastic agent. 6. Use according to claims 1 , 2 or 3 wherein the cell death is induced by okadaic acid, microcystin or other algal toxins or protein phosphatase- inhibitory toxic agents. 7. Use according to claim 1 wherein the cells are liver cells (hepatocytes) except hepatocellular carcinoma cells and other cancer cells of hepatic origin. 8. Use according to claims 1 or 7 wherein the flavonoid is naringin, or a derivative of naringin. 9. Use according to claims 7 or 8 wherein the cell death is induced by a therapeutic agent. 10. Use according to claims 7 or 8 wherein the cell death is induced by an antineoplastic agent. 1 1. Use according to claims 7 or 8 wherein the cell death is induced by okadaic acid, microcystin or other algal toxins or other protein phosphatase- inhibitory toxic agents. 12. Use according to one of the preceding claims wherein the fiavonoids and the toxic agents are combined in one preparation. 13. Use according to one of the preceding claims wherein the fiavonoids and the toxic agents are formulated in separate preparations. 14. Use according to claims 12 and 13, wherein the pharmaceutical preparations comprise the active ingredients, one or more pharmaceutically acceptable vehicles and possibly further therapeutic and/or prophylactic compounds. 15. Preparation characterized in that it comprises the compounds as defined in claims 1, 3 alone or together with the compounds as defined in claims 4, 5 and 6. 16. Preparation characterized in that it comprises the compounds as defined in claims 1, 8, alone or together with the compounds as defined in claims 9, 10 and 11. 17. Pharmaceutical composition characterized in that it comprises fiavonoids as defined in claims 3 or 8, alone or together with compounds as defined in claims 4 - 6 or 9 - 1 1 , and one or more pharmaceutically acceptable adjuvant or filler. 18. Method to protect specific groups of living cells agains cell death, characterized in administering a flavonoid or derivative thereof alone or together with a cell death inducing agent such as a therapeutic agent, an antineoplastic agent, okadaic acid or other protein phosphatase inhibitory toxic agents. 19. Kit characterized in that it contains the fiavonoids as defined in claims 1 , 3, 8 and the compounds as defined in claims 4 - 6 and 9 - 1 1.

Inventors: SEGLEN PER, SEGLEN, PER
Applicants: SEGLEN PER, SEGLEN, PER
Application year: 1999
Main classification: A61K31
IPC codes: A61K31, A61K38