Title:  Liver cell clones for use in extracorporeal liver-assist device

United States Patent:  6,294,380

Assignee:  JMS Co., Ltd. (JP)

Filed:  July 29, 1999

A blood perfusion device or apparatus that is used for bioartificial liver support. Human hepatocyte lines established from normal regenerating liver tissue and modulated in toxin-challenging conditions are provided. These functional hepatocytes exhibit extraordinarily enhanced detoxification functions, which are characterized by the elevated glutathione content and glutathione S-transferase activity. A bioreactor is constructed with the functional hepatocytes for bioartificial liver support system, which includes perfusion inlets and perfusion outlets, a containment vessel, a centrifugal pump and macroporous microcarriers where the functional hepatocytes are grown.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention relates to a liver-assist device and to liver cell clones, which may find application in such device.

An embodiment of the present invention is a novel detoxification system, referred to as a "specific Extracorporeal Liver Assist Device" ("sELAD"), which is based on a special functional hepatocyte cell line. By the term "specific Extracorporeal Liver Assist Device" it is meant an extracorporeal liver support system which provides augmentation of functional activities which are typically diminished in hepatic dysfunction, and which are considered important in the recovery from hepatic coma, frequently seen in fulminant hepatic failure patients. Since it is difficult to obtain an extracorporeal liver support system which displays all of the biochemical potential of hepatocytes in vivo, the system may possesses some of the main hepatic functions, preferentially detoxification. Preferably the extracorporeal liver support system possesses enhanced or elevated detoxification function by way of employment of hepatocytes, inoculated into the system, which have several times higher the content of detoxification enzymes and detoxificants than freshly isolated or transformed hepatocytes.

A bioartificial liver support system that applies the principles of the present invention provides an effective alternative to treat patients with fulminant hepatic failure. The device and method of the present invention can be used not only in bridging the patient to orthotopic transplantation, but also in preventing the patient from developing encephalopathy.

The immediate objective of any extracorporeal liver-support system is to maintain a patient with fulminant hepatic failure until the patient's own liver regenerates, or to bridge the patient to orthotropic transplantation. Most liver support systems used today are directed to blood detoxification because detoxification is considered an essential requirement for an extracorporeal liver support system. The metabolism of toxic substances by living cells in a hepatocyte bioreactor reduces toxicity, thus producing beneficial effects for the recovery of patients.

One embodiment of the present invention describes a method and apparatus to enhance detoxification activity in immortalized human hepatocytes by a cellular approach. The pathway to enhance the specific detoxification functions of the hepatocyte, which is described in this invention, is to modulate glutathione (GSH) and glutathione S-transferase (GST) levels in an immortalized human hepatocyte line (DYD). Using the present disclosure, one may produce such cells in a sufficient mass equivalent to the counterpart in vivo.

The glutathione and glutathione S-transferase system (GSH/GST) play an important role in removing toxic substances accumulating in the circulation of liver failure patients. GSH is a prevalent low molecular weight peptide that has multiple physiological and metabolic functions. GSH participates in reactions that destroy hydrogen peroxide, organic peroxides, free radicals and foreign compounds and drugs. GSH also participates in the metabolism of various endogenous compounds. Evidence suggests that GST is involved in protection against oxidative stresses (Lenehan P. F. et al., Cancer Chemother. Pharmacol. 35:377-86, 1995). The GSH/GST pathway represents part of an adaptive response mechanism to chemical stress caused by electrophiles, activated forms of most metabolites.

Eukaryotic cells have evolved several mechanisms to protect cellular constituents from highly reactive molecules entering or being biotransformed in the cells. The greater affinity of electrophiles for thiol groups, than for hydroxyl or amine groups, provides a chemical-physical basis for using high concentrations of substances containing thiol moieties as protectants. The formation of .a thioether bond between electrophiles and GSH typically yields a conjugate that is less reactive than the parental compound, therefore, the actions of GSH generally result in detoxification. GSH conjugation with harmful electrophilic moieties increases the solubility of hydrophobic xenobiotics, which then can more easily be transported out of the cells by the ATP-dependent glutathione S-conjugate efflux pump (GSH-Px).

It has been reported that induction of the GSH/GST pathway detoxifies some of the toxic carbonyl-, peroxide-, epoxide-containing metabolites produced within the cell by oxidative stress (Meister A. in The liver: Biology and Pathology, 1988 by Raven Press, Ltd., New York). As GSH functions in protecting cells from reactive oxygen intermediates, free radicals and toxic compounds, an increase in cellular GSH levels, and GST activity, may be beneficial. For example, some clinical studies have shown that liver damage can be prevented and minimized by the application of a glutathione precursor (Prescott L. F. et al., Lancet 2:10913, 1976; Rumack B H et al., Arch. Intem. Med. 141:3 80-5, 1981; Smilkstein M. J. et al., N. Eng. J. Med. 319:1557-62, 1988).

Cellular GSH levels may be partially increased by supplying substrates for enzymes in GSH synthesis. The constitutive upregulation of GSH synthesis and GST activity can also be achieved by modulating the cells under a progressively toxin-challenging culture (Hamilton T. C., in Glutathione S-Transferase Structure, Function and Clinical Implications, pp. 173-85, 1996) (so does the induction of cellular defense system to the oxidants).

GST activity is increased in many organisms following exposure to select foreign compounds. Induction of GST has been most thoroughly studied in rodents, and at least 100 different chemicals have been discerned to induce GST in rats and mice. The wide spectrum of xenobiotics that act as GST inducing agents, suggest that GST induction is part of an adaptive response mechanism to chemical stress that is widely distributed in nature. From studies of rodents, it may be inferred that the adaptive response to chemical stress is clearly pleiotropic in character and involves the induction of many drug metabolizing enzymes. Collectively, these detoxification enzymes provide protection against a diverse spectrum of harmful compounds.

The conjugation reaction between GSH and xenobiotics represents the first step in the synthesis of mercapturic acids, an important group of excretion products. Following conjugation with GSH, the subsequent steps in mercapturic acid biosynthesis require the serial actions of glutamyl transpeptidie, cysteinyl glycinase, and N-acetyl transferase. Since GSH functions in protecting cells from reactive oxygen intermediates, free radicals and toxic compounds, an increase in cellular GSH level and GST activity may be beneficial under certain clinical conditions, such as hepatic coma in which a significant amount of toxins from necrotic tissue flushes into the blood stream.

It is preferred in the present invention that modulation of intracellular GSH content and GST activity be carried out in an immortalized hepatic cell line. A preferred cell line is designated DYD, a human hepatocyte clone established from normal liver tissue. DYD is a highly differentiated cell line capable of growing at a high density. By endowing such cells with specific detoxification functions, the cells have the ability to transform toxins more specifically, rapidly and efficiently, thus elevating the efficiency of a bioreactor in a bioartificial liver-support system.

The quantity of hepatocytes inoculated into a bioreactor is another important factor that influences the efficiency of current bioartificial liver support systems. Although the exact number of hepatocytes in a convention bioreactor is not known, it is generally accepted that the cell mass is in the order of 100-300 g extracorporeal support.

The number of cells in a single bioreactor of the present invention is is preferably about 2.5-7.5 billion in magnitude, about 25-75 gram per single module. In order to accommodate such a huge number of cells, a special bioreactor configuration was designed. The cells were allowed to grow to confluence on inacroporous microcarriers. The cell-attached microcarriers where then moved into a bioreactor, where a continuous supply of nutrition and oxygen was provided to guarantee maintenance of steady functioning of the hepatocytes.

In modulation of the detoxification function, it was determined that highly differentiated human hepatocytes are optimal. An immortalized cell line with highly differentiated functions is preferred. Besides providing an unlimited cell division capacity, immortalized human hepatocytes obviate concerns about species specific metabolic differences. Further any infusion of proteins from the human hepatocytes is less likely to cause immune-mediated reactions than non-human proteins, especially after prolonged or repeated use. Methods for the establishment of immortalized cell lines are well known in the art, and are available using advanced cell and tissue culture technology. However, in order to obtain a clonal expansion of hepatocytes from normal liver tissue, special procedures in developing cell line are necessary, such as the use of new type matrix, growth factors or conditioned medium and induction of clonal expansion.

Enhancement of differentiated he patocyte functions, specifically those functions specific to detoxification, can be brought about by the induction and regulation of glutathione S-transferase, which is considered, as stated above, to represent a major group of detoxification enzymes. All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as non-catalytic binding properties (Hayes J. D. et al., Crit. Rev Biochem. Mol. Biol. 30:445-600, 1995). Through the concerted actions of several isoenzymes, the GST supergene family provides several tiers of defense against toxic chemicals. Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals.

It is known that some enhancers can regulate GST expression in the animal cells. Some enhancers interact with GST gene elements that respond to xenobiotics. They transcriptionally activate GST genes through different mechanisms according to their elemental structure (Hayes J. D. et al., Critical Reviews in Biochemistry and Molecular Biology 30: 445-600, 1995). The constitutive expression of GST up-regulation can be achieved by the effective modulations.

The constitutive up-regulation of GSH synthesis and GST activity can be achieved by modulating the cells under a toxin challenging culture condition. The chemicals that induce GST and GSH are extremely diverse. These include carcinogens, cytotoxins, chemotherapeutic drugs, heavy metals and metal-containing drugs. GST induction can also be performed using reactive oxygen species. Examples of chemicals inducing GST and GSH include: N-acetoxy-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine, aflatoxin B₁ -8,9-epoxide, benzo[a]pyrene-4,5-oxide, benz[a]anthracene-5,6-oxide, benz[a]anthracene-8,9-diol-10,11-oxide, butadiene monoepoxide, +anti chrysene-1,2-diol-3,4-oxide, 5-hydroxymethylchrysene sulfate, 7,12-dihydroxymethyl-benzo[a]anthracene sulfate, 7-hydroxymethyl-12methylbenz[a]anthracene sulfate, 1-methyl-2-nitro-1-nitrosoguanidine, 1-nitropyrene-4,5-oxide, 1-nitropyrene-9,10-oxide, 4-nitroquinoline 1-oxide, benzo[a]pyrene, dimethylaminoazobenzene, 7,12-dimethylbenz[a]anthracene, 5,9-dimethyl-7H-dibenzo[c,g]carbazole, 3-methylcholanthrene, acetochlor, acifluorfen, alachlor, aldrin, atrazine, azinphosmethyl, 1,4-benzoquinone, chlorimuron ethyl, cumen hydroperoxide, DDT, N,N-diallyl-2-chloroacetamide, diazinon, dichlobenil, dichlofluanid, 2,4-dichlorophenoxyacetic acid, S-ethyl N,N-dipropylthiocarbamate sulfoxide, ethylene oxide, ethylparathion, fenoxapropethyl, fluorodifen, lindane, malathion, methyl bromide, methyl chloride, methyl parathion, metolachlor, trans, trans-muconaldehyde, naphthalene 1,2-oxide, parathion, propachlor, propetamphos, styrene oxide, tetrachlorvinphos, trans-stilbene oxide, tridiphane, vinyl chloride, ampicillin, fosfomycin, penicillin, 1,3-bis(2-chloroethyl)-1-nitrosourea, chlorambucil, cyclophosphamide, ethacrynic acid, mechlorethamine, melphalan, mitozantrone, nitrogen mustard, thiotepa, acrolein, adenine propenal, cholesterol .alpha.-oxide, cytosine propenal, dilinoleoylphosphatidylcholine hydroperoxide, dilinoleoylphosphatidylethanolamine hydroperoxide, dilinoleoylphosphatidylglycerol hydroperoxide, epoxyeicosatrienoic acid, 4-hydroxynonenal, linoleic acid hydroperoxide, methyl linoleate ozonide, thymine propenal, uracil propenal, chlorotrifluoroethane, 1,4-dibromo-2,3-epoxybutane, dibromomethane, 1,3-dichloroacetone, dichloroacetylene, dichloromethane, 1,2,3,4-diepoxybutane, 1,2-epoxy-4-bromobutane, ethylenedibromide, hexachlorobutadiene, allobarbital, 1-.beta.-D-arabinofuranosyl cytosine, barbital, bisethylxanthogen, butylated hydroxyanisole, butylated hydroxytoluene, 3,5-di-tert-butylcatechol, tert-butylhydroquinone, 2-n-butylthiophene, cisplatin, clonazepam, cyclophosphamide, dexamethasone, diethyldithiocarbamate, diethyl maleate, diethylnitrosamine, 5,6-dihydro-2H-pyran-2one, dimethyl fitmarate, dimethyl maleate, dimethyl itaconate, disulfiram, 1,2-dithiole-3-thione, erucin, erysolin, ethanol, ethoxyquin, 5-ethyl-5-phenylhydantoin, 2-n-heptylftiran, .alpha.-hexachlorocyclohexane, .gamma.-hexachlorocyclohexane, hexachlorobenzene, 3,4,5,3',4',5'-hexachlorobiphenyl, 2,4,5,2',4',5'-hexachlorobiphenyl, 2,3,5,2',3',5-hexachlorobiphenyl, interferon-.alpha./.beta., iproplatin, isosafrole, lead acetate, p-methoxyphenol, methyl acrylate, 3-methylcholanthrene, 2-methylene-4-butyrolactcne, methyl selenocyanate, musk xylene, .beta.-naphthoflavone, oltipraz, phenobarbital, polychlorinated biphenyl, propylthiouracil, rifampicin, trans-stilbene oxide, steptozotocin, sudan, 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, tetrachlorodibenzo-p-dioxine, 2,3,5,6-tetrafluorophenol, 1-(2-thiazolylazo)-2-naphthol, vinylidene chloride, adriamycin, arsenic, bleomycin, 2-[3-(chloroethyl)-3-nitrosoureido]-D-dioxyglucopyiranose, ethacrynic acid, etoposide, hepsulfam, mitomycin C, mitoxantrone, novantione, oxazaphosphorine, TGF-.beta.1 and vincristine. It appears that the majority of GST substrates are either xenobiotics or products of oxidative stress in nature. The common feature for these compounds is that they are involved in the metabolizing process or the generation of metabolic cascade. Some endogenous compounds are also inducers of GST.

Modulation of intracellular GSH content and GST activity may be carried out in a replicating hepatic cell line DYD, a human hepatocyte clone established from normal liver tissue. A modulating procedure such as described in Example 1 below may be undertaken. By endowing the DYD hepatic cells with enhanced specific detoxification functions, the hepatocytes can transform toxins more specifically, rapidly and efficiently, thus elevating the efficiency of bioreactor for bioartificial liver support system.

An embodiment of the present invention comprises functional human liver cell clones (DYD-) derived from a normal human hepatocyte cell line (DYD) which exhibits enhanced detoxification activities as characterized by increased GSH content and elevated GST activity. The functional human liver cell clones preferably elicit increased GSH content and elevated GST activity as compared to immortalized human hepatocytes cell lines and a primary human hepatocyte culture isolated from human liver tissue. A particularly advantageous cell line, demonstrating unexpectedly superior properties, has a GSH content above 50 nmol/mg protein or 50 nmol/10⁶ cells; after 24 hours passage and a GST activity above 15 nmol/mg protein/min or 80 nmol/10⁶ cells /min with CDNB as substrate after 24 hours passage.

In another embodiment of the present invention, there is disclosed a method to obtain the functional human liver cell clones eliciting increased GSH content and elevated GST activity as compared to immortalized human hepatocytes cell lines and a primary human hepatocyte culture isolated from human liver tissue, which comprises a process of exposing a normal hepatocyte cell line in culture medium to to a toxin and modulating the expression of GSH and GST with inducers. The inducers may possess a carcinogenic or cytotoxic nature.

In another embodiment of the present invention, there is disclosed an extracorporeal liver-assist device, which has a bioreactor containing functional human liver cell clones eliciting increased GSH content and elevated GST activity as compared to immortalized human hepatocytes cell lines and a primary human hepatocyte culture isolated from human liver tissue. Preferably, the functional human liver cell clones are grown on macroporous microcarriers or other biocompatible support matrix system, for example, capillaries or ceramics. Preferably, the extracorporeal liver-assist device comprises plasma filtration circulation system and dialysis cirtridge circulation system. The plasma filtration circulation system may employ an adsorbent column.

In yet a further embodiment of the present invention, there is disclosed a method to obtain the functional human liver cell clones eliciting increased GSH content and elevated GST activity as compared to immortalized human hepatocytes cell lines and a primary human hepatocyte culture isolated from human liver tissue, which comprises a process of exposing a normal hepatocyte cell line in culture medium over a period of time to increasing concentrations of a compound that induces GSH and GST. The inducer compounds may possess a carcinogenic or cytotoxic nature.

Claim 1 of 12 Claims

What is claimed is:

1. An improved extracorporeal liver-assist device of the prior art of the type including a human liver-derived cell line, wherein the improvement comprises: a reactor containing a human liver-derived cell line artificially-modified to display at least about twice the GSH content as compared to the same human liver-derived cell line that is not artificially-modified.

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