WO1999039743A2

WO1999039743A2 - Method of promoting hepatic cell proliferation

Info

Publication number: WO1999039743A2
Application number: PCT/US1999/002618
Authority: WO
Inventors: Kathleen E. Rodgers; Gere Dizerega
Original assignee: University of Southern California USC
Current assignee: University of Southern California USC
Priority date: 1998-02-09
Filing date: 1999-02-08
Publication date: 1999-08-12
Anticipated expiration: 2000-08-09
Also published as: WO1999039743A3; AU2590399A

Abstract

The present invention provides methods, improved cell culture medium and kits for promoting hepatic cell proliferation and differentiation by growth in the presence of angiotensinogen, AI, AI anoalogues, and/or AI fragments and analogues thereof, AII analogues, AII fragments or analogues thereof, and/or AII AT2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.

Description

METHOD OF PROMOTING HEPATIC CELL PROLIFERATION

Cross Reference

This application is a continuation in part of U.S. Application Serial No. 60/074,104 filed February 9, 1998 and a continuation of U.S. Application Serial No. 60/108,412 filed November 13, 1998.

Field of the Invention

This present invention relates to methods, cell culture media, and kits for use in accelerating the proliferation of hepatic cells.

Background of the Invention The liver is an epithelial organ that contains two major differentiated cell types: the hepatocyte and the bile ductule cell, which are thought to arise from a common hepatic stem cell. (Ponder, FASEB J. 10:673-684 (1996); hereby incorporated by reference in its entirety.) It is unclear what role the stem cell plays in normal postnatal liver growth, in the chronically regenerating liver, or in liver carcinogenesis. Hepatic cells are typically quiescent in adults. (Thorgeirsson, FASEB J.

10:1249-1256 (1996); hereby incorporated by reference in its entirety.) However, both hepatocytes and bile ductule cells have the capacity to meet replacement demands caused by cellular loss, as exemplified by their ability to proliferate and restore liver mass after partial hepatectomy (PH). (Id.) The ability of liver tissue to regenerate has been known for some time and is controlled by cytokines of paracrine and/or autocrine origin which either stimulate or inhibit hepatic cell growth. Hepatic stem cells, whose immediate progeny are referred to as oval cells or small epithelial cells (hereinafter referred to as 'SEC'), assume the burden of regenerative liver growth only after significant hepatocyte loss. In contrast to liver regeneration after PH, hepatic regeneration in acute liver failure (also known as fulminant hepatic failure) occurs by the proliferation and differentiation of hepatic stem cells. (Kay and Fausto, Mol. Med. Today 3:108-115 (1997); hereby incorporated by reference in its entirety.) Acute liver failure can be caused by several distinct diseases, including viral hepatitis, chemical injury (for example, caused by acetaminophen/paracetamol), Wilson's disease, and Reye's syndrome (Id.) Oval cells or SEC arise initially in the bile epithelium after activation of hepatic stem cells following physiologically- or experimentally-induced liver damage (Alison et al., 1996; Thorgeirsson, 1996; Kay and Faust, 1997; and references therein). They are capable of differentiating into both bile epithelial cells and hepatocytes when given appropriate stimuli (Fausto, Prog. Clin. Biol. Res. 331:325 (1990); Sell, Cancer Res. 50:3811 (1990); Sigal et al., Am. J Physiol. 263:G139 (1992); Thorgeirsson, Am. J. Pathol. 142:1331 (1993)).

Thus, the liver cannot be classified as a classical stem cell-fed tissue, like the epidermis, the intestinal epithelium, the hematopoietic system, or the bone marrow. Unlike other tissues capable of regeneration (e.g., skin and bone marrow), regeneration of the liver is not dependent upon small populations of stem cells or progenitor cells. However, large numbers of hepatic stem cells do appear when mature hepatocytes are inhibited from proliferation (Thorgeirsson, 1996; Sell, Mod. Pathol. 7:105 (1994); Fausto, et al, Proc. Soc. Exp. Biol. Med. 204:237 (1993)). A number of acute and chronic conditions can lead to the damage of liver tissue. These include, but are not limited to, hepatocarcinoma, hepatitis infection, cirrhosis of the liver, partial hepatectomy, fulminant hepatic failure, hepatocyte transplantation, and liver transplantation. In most instances, the survival of patients with these disorders depends upon successful liver regeneration, which occurs through the proliferation of hepatocytes and/or hepatic stem cells.

Thus, there is a need in the art for methods that increase the proliferation of hepatic stem cells and hepatocytes. Such methods will provide clinical benefits in liver regeneration following resection of hepatocarcinomas, hepatitis infection, cirrhosis of the liver, partial hepatectomy, fulminant hepatic failure, hepatocyte transplantation, liver transplantation, and other hepatic disorders where rapid regeneration of the liver is desirable. Such methods are also useful in rapidly providing a large population of hepatic cells for use in cell therapy and for providing a large population of transfected hepatic cells for use in gene therapy.

Summary of the Invention

In one aspect, the present invention provides methods that promote hepatic cell proliferation by contacting the cells with angiotensinogen, angiotensin I (hereinafter referred to as 'Al'), Al analogues, and/or Al fragments and analogues thereof, angiotensin II (hereinafter referred to as 'All') analogues, All fragments or analogues thereof, and/or All AT₂ type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.

In another aspect of the present invention, an improved cell culture medium is provided for the proliferation of hepatic cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and/or All AT type 2 receptor agonists.

In a further aspect, the present invention provides kits for the propagation of hepatic cells, wherein the kits comprise an effective amount of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and or All AT type 2 receptor agonists, and instructions for culturing the cells. Preferred embodiments of the kit further comprise cell culture growth medium, a sterile container, and an antibiotic supplement.

Brief Description of the Figures

Figure 1. Effect of All and All analogues on hepatocyte proliferation in vitro.

Detailed Description of the Preferred Embodiments As defined herein, the term "hepatic cells" refers to hepatic stem cells that are poorly differentiated, demonstrate extensive proliferation, and have multiple differentiation options (Thorgeirsson, FASEB J. 10:1249 (1996)), as well as hepatocytes and bile ductule cells derived therefrom. Examples of hepatic stem cells include, but are not limited to, SEC cells, oval cells, as well as cultured WB-F344 cells

and RLEΦ13 cells (Coleman et al., Am. J. Pathol. 151:353 (1997); Omori et al.,

Hepatology 26:720 (1997)). As defined herein, "proliferation" encompasses both cell self renewal and cellular proliferation with accompanying differentiation.

Unless otherwise indicated, the term "active agents" as used herein refers to the group of compounds comprising angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II analogues, All fragments or analogues thereof and All AT₂ type 2 receptor agonists.

U.S. Patent No. 5,015,629 to DiZerega (the entire disclosure of which is hereby incorporated by reference) describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of All in an amount which is sufficient for said increase. The application of All to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair. The term All refers to an octapeptide present in humans and other species having the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:l]. The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen (Circulation Research 60:786-790 (1987); Clouston et al., Genomics 2:240-248 (1988); Kageyama et al, Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200 (1983); all references hereby incorporated in their entirety). The substance so formed is a decapeptide called angiotensin I (Al) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from Al, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID NO:37]. All is a known pressor agent and is commercially available.

Studies have shown that All increases mitogenesis and chemotaxis in cultured cells that are involved in wound repair, and also increases their release of growth factors and extracellular matrices (diZerega, U.S. Patent No. 5,015,629; Dzau et. al., J. Mol. Cell. Cardiol. 21:S7 (Supp III) (1989); Berk et. al, Hypertension 13:305-14 (1989); Kawahara, et al, BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest. 83:1419-23 (1989); Taubman et al., J. Biol. Chem 264:526-530 (1989); Nakahara, et al, BBRC 184:811-8 (1992); Stouffer and Owens, Circ. Res. 70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107 (1992); Bell and Madri, Am. J. Pathol. 137:7-12 (1990)). In addition, All was shown to be angiogenic in rabbit corneal eye and chick chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6 (1991)). Therefore, All may accelerate wound repair through increased neovascularization, growth factor release, reepithelialization and/or production of extracellular matrix. The use of All analogues and fragments, AT2 agonists, as well as AIII and AIII analogues and fragments in wound healing has also been described. (U.S. Patent No. 5,629,292; U.S. Patent No. 5,716,935; WO 96/39164; all references herein incorporated by reference in their entirety.)

Although All has been shown to increase the proliferation of a number of cell types in vitro, it does not necessarily increase the proliferation of all cell types. The effect of All on a given cell type has been hypothesized to be dependent, in part, upon the All receptor subtypes the cell expresses (Shanugam et al., Am. J. Physiol. 268:F922-F930 (1995); Helin et al., Annals of Medicine 29:23-29 (1997); Bedecs et al., Biochem J. 325:449-454 (1997)). These studies have shown that All receptor subtype expression is a dynamic process that changes during development, at least in some cell types (Id.)

Recently, All has been identified as a co-mitogen for hepatocytes both in vitro and in vivo. (Huerta-Bahena et al., Biochim. Biophys. Acta 763:120 (1983); Booz et al, Endocrinology 130:3641 (1992); Hasewaga et al., J. Cell Physiol. 158:215 (1994); Kar et al., J. Cell Physiol. 168:141 (1992); Dajani et al., J. Cell. Physiol. 168:608 (1996)). However, it is not known whether angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and/or All AT₂ type 2 receptor agonists accelerate the proliferation of hepatic stem cells, hepatocytes, or bile ductule cells. For example, data suggests that the All fragment AII(l-7) acts through a receptor(s) that is distinct from the ATI and AT2 receptors which modulate All activity. (Ferrario et al., J. Am. Soc. Nephrol. 9:1716- 1722 (1998); Iyer et al, Hypertension 31:699-705 (1998); Freeman et al., Hypertension 28:104 (1996); Ambuhl et al., Brain Res. Bull. 35:289 (1994). Thus, the stimulatory effect of All through activation of the ATI or AT2 receptor does not shed any light on the potential stimulatory effect of AII(l-7).

A peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) is p-aminophenylalanine6-AII ["(p-NH₂-Phe)6-AII)"], Asp- Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH₂-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al, Eur. J. Pharmacol. 225:119-127 (1992).

The effects of All receptor and All receptor antagonists have been examined in two experimental models of vascular injury and repair which suggest that both All receptor subtypes (ATI and AT2) play a role in wound healing (Janiak et al., Hypertension 20:737-45 (1992); Prescott, et al, Am. J. Pathol. 139:1291-1296 (1991); Kauffman, et al, Life Sci. 49:223-228 (1991); Viswanathan, et al, Peptides 13:783-786

(1992); Kimura, et al., BBRC 187:1083-1090 (1992)).

Many studies have focused upon AII(l-7) (All residues 1-7) or other fragments of All to evaluate their activity. AII(l-7) elicits some, but not the full range of effects elicited by All (Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19 (Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265:664- 673 (1991); Jaiswal, et al, Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654 (1994)). As hereinafter defined, a preferred class of AT2 agonists for use in accordance with the present invention comprises Al, All, and Al or All analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AIL In addition to peptide agents, various nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.

The active All analogues, fragments of All and analogues thereof of particular interest in accordance with the present invention are characterized as comprising a sequence consisting of at least three contiguous amino acids of groups R -R in the sequence of general formula I

R¹ -R²-R³-R⁴-R⁵-R⁶-R^7"R⁸ in which R¹ and R² together form a group of formula

X-R^A-R^B-, wherein X is H or a one to three peptide group, R^A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asρ(NH₂) and Sue,

R^B is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys, R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer and azaTyr; R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;

R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and

R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group, wherein the active agent is not AIL Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AH analogues set forth above subject to the restriction that R is p-NH₂-Phe. Particularly preferred combinations for R^A and R^B are Asp- Arg, Asp-Lys, Glu-

Arg and Glu-Lys. Particularly preferred embodiments of this class include the

following: AIII, Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AH(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(l-7), Asp-Arg-Val- Tyr-Ile-His-Pro {SEQ ID NO:4]; AII(2-7). Arg- Val-Tyr-Ile-His-Pro [SEQ ID NO:5]; AH(3-7), Val-Tyr-Ile-His-Pro [SEQ ID NO:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID NO:7]; AII(l-6), Asp-Arg-Val-Tyr-Ile-His [SEQ ID NO:8]; AII(l-5), Asp-Arg-Val- Tyr-Ile [SEQ ID NO:9]; AII(l-4), Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(l-3), Asp- Arg- Val [SEQ ID NO: 11]. Other preferred embodiments include: Arg-norLeu- Tyr-Ile-His-Pro-Phe [SEQ ID NO: 12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO: 15] were also tested and found not to be effective. A class of particularly preferred compounds in accordance with the present invention consists of those with the following general structure:

Rl-Arg-R2-R3-R4-His-Pro-R5 wherein Rl is selected from the group consisting of H and Asp;

R2 is selected from the group consisting of Val and Pro; R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂;

R4 is selected from the group consisting of Ala, He, Leu, and norLeu; and

R5 is Phe, He, or is absent, and wherein the active agent is not AIL

Particularly preferred embodiment of this class are selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38

Another class of compounds of particular interest in accordance with the present invention are those of the general formula II

R²-R³-R⁴-R⁵-R⁶-R⁷-R⁸

in which R² is selected from the group consisting of H, Arg, Lys, Ala,

Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr; R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer and azaTyr;

R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly; R⁶ is His, Arg or 6-NH₂-Phe;

R⁷ is Pro or Ala; and

R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr.

A particularly preferred subclass of the compounds of general formula II has the formula

R²-R³-Tyr-R⁵-His-Pro-Phe [SEQ ID NO: 16]

wherein R², R³ and R⁵ are as previously defined. Particularly preferred is a compound of the formula Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:38]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro- Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18]. The

fragment AII(4-8) was ineffective in repeated tests; this is believed to be due to the

exposed tyrosine on the N-terminus.

In the above formulas, the standard three-letter abbreviations for amino acid residues are employed. In the absence of an indication to the contrary, the L-form of the amino acid is intended. Other residues are abbreviated as follows:

TABLE 1

Abbreviation for Amino Acids

It has been suggested that All and its analogues adopt either a gamma or a beta turn (Regoli, et al, Pharmacological Reviews 26:69 (1974)). In general, it is believed that neutral side chains in position R³, R⁵ and R⁷ may be involved in maintaining the appropriate distance between active groups in positions R⁴, R⁶ and R⁸ which are primarily responsible for binding to receptors and/or intrinsic activity. Hydrophobic side chains in positions R³, R⁵ and R⁸ may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.

Appropriate side chains on the amino acid in position R² may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R².

For purposes of the present invention, it is believed that R³ may be involved in the formation of linear or nonlinear hydrogen bonds with R⁵ (in the gamma turn model) or R⁶ (in the beta turn model). R³ would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr. With respect to R⁴, conformational analyses have suggested that the side chain in this position (as well as in R³ and R⁵) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R⁴ is preferably selected from Tyr, Thr, Tyr (PO₃)₂, homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).

In position R⁵, an amino acid with a β aliphatic or alicyclic chain is particularly

desirable. Therefore, while Gly is suitable in position R⁵, it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.

In the All analogues, fragments and analogues of fragments of particular interest in accordance with the present invention, R⁶ is His, Arg or 6-NH₂-Phe. The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R⁶. For example, conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R⁷. Similarly, it is presently considered that R⁷ should be Pro in order to provide the most desirable orientation of

R . In position R , both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention. Analogues of particular interest include the following: TABLE 2: Angiotensin II Analogues

The polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis. The disclosures of the foregoing treatises are incorporated by reference herein.

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.

In one aspect of the present invention, a method of increasing in vitro and ex vivo hepatic cell proliferation by exposure to angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, AH analogues, AH fragments or analogues thereof, and/or All AT type 2 receptor agonists ("active agents"), is disclosed. Experimental conditions for the isolation, purification, ex vivo growth and in vivo mobilization of hepatic stem cells and hepatocytes have been reported (Drakes et al., J. Immunol. 159:4268 (1997); Omori et al. Hepatology 26:720, (1997); U.S. Patent No. 4,914,032; U.S. Patent No. 5,227,158; all references incorporated by reference herein in their entirety) .

In one embodiment of the invention, hepatocytes are isolated using procedures known in the art. (for example, see Free Radical Biology and Medicine 18:303-310). The isolated hepatocytes are resuspended in appropriate tissue culture medium to induce cell adherence. After 5-7 days in culture, the cells are treated with the active agents at various concentrations, preferably ranging from 0.1 ng/ml to 10 mg/ml. Cell proliferation is assessed at various time points during culture using methods well known in the art, including, but not limited to, measuring the rate of DNA synthesis according to the method of Nakamura and coworkers (Nakamura et al., J. Biochem. (Tokyo) 94:1029 (1982); Nakamura et al., Biochem. Biophys. Res. Comm. 122:1450 (1984)) Trypan blue dye exclusion hemacytometer counting (Omori et al, Hepatology 26:720 (1997)), or flow cytometry (Drakes, J. Immunol. 159:4268 (1997); incorporated by reference herein in its entirety) to determine if the active agents have induced the proliferation of hepatocytes at a higher rate than control samples. In another embodiment of the invention, hepatic stem cells are isolated from non-transplantable adult mammalian livers, particularly from the bile duct epithelium (Drakes et al, J. Immunol. 159:4268 (1997); Omori et al. Hepatology 26:720, (1997)). Hepatic stem cells are then selected for in these samples and cultured under appropriate growth conditions, in the presence of the active agents of the invention. Hepatic stem cell proliferation is assessed at various time points during culture as described above.

In a preferred embodiment, hepatic stem cells are isolated from adult mammalian livers by centrifugal elutriation of dispersed liver tissue (Hayner et al., Cancer Res. 44:332 (1984); Germain et al., Cancer Res. 45:673 (1985) Germain et al., Cancer Res. 48:368 (1988); Drakes et al., J. Immunol. 159:4268 (1997); Omori et al., Hepatology 26:720 (1997)). Briefly, adult mammalian liver is perfused in situ via the portal vein in two steps, first with Mg ⁺- and Ca²⁺-free Hank's balanced salt solution immediately followed by 0.1% pronase and 0.1% collagenase in buffered William's solution. Dispersed liver cells are then successively filtered on 240mm-pore and 70mm-pore nylon screens. The parenchymal cell fraction is obtained after multiple washings by centrifugation at 50 x g for 3 minutes. The nonparenchymal cell fraction is obtained by centrifugation at 200 x g for 10 minutes of the supernatant from which hepatocytes had been repeatedly eliminated by centrifugation. Following isolation, hepatic stem cells are suspended in standard culture medium, including but not limited to, RPMI-1640 (Gibco BRL, MD) and incubated in the presence of, preferably, between about 0.1 ng/ml and about 10 mg/ml of the active agents of the invention. The cells are expanded for a period of between 8 and 21 days, then cellular proliferation is monitored by measuring the rate of DNA synthesis (Nakamura et al., 1982; Nakamura et al., 1984). Differentiation of hepatic stem cells to hepatocytes or bile ductule cells is detected by measuring the expression of typical differentiated hepatocyte markers including, but not limited to, albumin, transferrin, and alpha- 1-antitrypsin, using standard methods well known in the art. These methods can include, but are not limited to, Northern blot analyses or reverse transcriptase-polymerase chain reaction (RT-PCR) with marker-specific DNA primers (Omori et al., Hepatology 25:1115 (1997); Drakes et al., J. Immunol. 159:4268 (1997); Omori et al., Hepatology 26:720 (1997)). Similarly, "self-renewal" of hepatic stem cells is assessed periodically by phase-contrast light microscopy and or immunohistochemistry (Overturf et al., Am. J. Pathol. 151 :5 (1997)).

In a further preferred embodiment, hepatic stem cells, bile ductule cells, or hepatocytes that have been cultured in the presence of the active agents are used for ex vivo liver-directed gene therapy (Alt and Cushman, J. Hepatol. 23:746 (1995)). Prior to ex vivo liver-directed gene therapy, the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10⁶ and 50 x 10⁶ cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Following reinfusion, hepatic cell proliferation is assessed by the methods discussed above.

In one embodiment, assessment of the in vivo proliferative effect of the active agents of the invention on hepatic cells is performed by histochemical evaluations of the liver including, but not limited to, hepatocytic parenchyma, periportal parenchyma, biliary epithelium, hyperplastic ducts within the periportal zone, arborizing ductules, and the portal spaces. In a preferred embodiment, in vivo proliferation of hepatic cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA or cyclin; Zymed Laboratories) (Rodgers et al., J. Burn Care Rehabil. 18:381-388 (1997); incorporated herein by reference in its entirety).

In another aspect of the present invention the active agents are used to increase in vivo hepatic cell proliferation. For use in increasing the proliferation of hepatic cells, the active agents may be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, transdermally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intraarterial, intramuscular, intrastemal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.

The active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions) and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.

While the active agents of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compounds. When administered as a combination, the active agents and other compounds can be formulated as separate compositions that are given at the same time or different times, or the active agents and other compounds can be given as a single composition.

For administration, the active agents are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The active agents may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, the active agents of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions,

ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.

The dosage regimen for increasing in vivo proliferation of hepatic cells with the active agents of the invention is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg of the active agents per body weight are useful for all methods of use disclosed herein. The treatment regime will vary depending on the condition being treated, based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed.

In a preferred embodiment of the present invention, the active agents are administered parenterally. A suitable parenteral dose of the active agents is preferably between about 0.1 ng/kg and about 10 mg/kg administered twice daily. For parenteral administration, the active agent may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. In another aspect of the present invention, an improved cell culture medium is provided for the proliferation of hepatic cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of the active agents of the invention. Any cell culture media that can support the growth of hepatic cells can be used with the present invention. Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEM® Reduced-Serum Medium, and RPMI Medium, or combinations thereof.

The improved cell culture medium can be supplied in either a concentrated (ie: 10X) or non-concentrated form, and may be supplied as a liquid, a powder, or a lyophilizate. The cell culture may be either chemically defined, or may contain a serum supplement. Culture media and serum supplements are commercially available from many sources, such as GIBCO BRL (Gaithersburg, MD) and Sigma (St. Louis, MO)

In a further aspect, the present invention provides kits for the propagation of hepatic cells, wherein the kits an effective amount of the active agents of the invention, and a set of instructions for culturing the cells.

In a prefened embodiment, the kit further comprises cell culture media. Any cell culture media that can support the growth of hepatic cells can be used with the present invention. Examples of such cell culture media are described above. The improved cell culture medium can be supplied in either a concentrated (ie:

10X) or non-concentrated form, and may be supplied as a liquid, a powder, or a lyophilizate. The cell culture may be either chemically defined, or may contain a serum supplement.

In another preferred embodiment, the kit of the present invention further comprises a sterile container. The sterile container can comprise either a sealed container, such as a cell culture flask, a roller bottle, or a centrifuge tube, or a non- sealed container, such as a cell culture plate or microtiter plate (Nunc; Naperville, IL).

In a further preferred embodiment, the kit further comprises an antibiotic supplement for inclusion in the reconstituted cell growth medium. Examples of appropriate antibiotic supplements include, but are not limited to actimonycin D,

Fungizone®, kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO).

The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

Example 1. All and All analogues and fragments increase hepatocyte proliferation Hepatocytes were isolated from rabbits using the procedure of Morel et al.

(Free Radical Biology and Medicine 18:303-310). Briefly, the liver was minced and placed in culture with 0.02% Type VIII collagenase (Sigma, St. Louis, MO) in RPM 1640 overnight at 37° C. Thereafter, the cells were collected in RPMI 1640 medium containing 1 mg/ml bovine serum albumin and 5 μg/ml bovine insulin. The cell suspension was filtered through sterile gauze and allowed to sediment for 20 minutes to remove cellular debris and blood. The cells were then washed three times by centrifugation at 1200 rpm. Viability was assessed by Trypan blue exclusion. The hepatocytes were resuspended in 75%) Eagle's minimal essential medium/25%) Medium 199 (Gibco BRL) buffered with sodium bicarbonate and supplemented with 10%> fetal calf serum containing 50 μg/ml streptomycin, 7.5 IU/ml penicillin, 5 μg/ml insulin, and 1 mg/ml bovine serum albumin at 1 x 10⁶ cells/ml. The medium was changed after 3-4 hours of adherence and was changed again after 24 hours. The cells were cultured for 5-7 days prior to use in the following studies.

After 5-7 days in culture, the cells were detached from the tissue culture plastic with 0.05%) trypsin-EDTA (Gibco-BRL Products) at 37°C for 10 minutes. The cells were washed one time with sterile phosphate buffered saline (pH 7.2) and adjusted to a concentration of 200 cells/ml. Two hundred μl of this suspension was aliquoted into wells of 96 well plates and allowed to attach to the tissue culture plastic. After adherence, AH, All analogues and AH fragments were added to the wells in duplicate to a final concentration of 10 μg/ml. The identity of the AH analogues and fragments is shown in Table 1.

Table 1. Designation for Analogues

Name Abbreviation Sequence SEQ ID NO:

GSD 28 Ile⁸-AH DRVYIHPI SEQ ID NO: 38

GSD 24B Pro³-AII DRPYIHPF SEQ ID NO.31

GSD 22A Ala⁴-AIII RVYAHPF SEQ ID NO: 18

AH(l-7) DRVYIHP SEQ ID NO:4

AH DRVYIHPF SEQ ID NO. 1

On days 1, 2, or 3 after addition of the peptides, the medium was removed, the cells were stained with Geimsa stain to identify nuclei, and the number of nucleated cells present in each well was assessed microscopically. The results are shown in Figure 1. These data demonstrate that AH, AII(l-7) and the All analogues and fragments significantly increased the number of hepatocytes per well relative to a control.

The present invention, by providing methods for enhanced proliferation of hepatic cells, will greatly increase the clinical uses of hepatic cells for liver regeneration after resection of hepatocarcinomas and in other hepatic disorders where a more rapid regeneration of the liver is desirable. This is true both for increased "self- renewal", which will provide a larger supply of hepatic stem cells capable of generating additional hepatocytes, epithelial cells, and/or exocrine pancreatic cells and for proliferation with differentiation, which will provide a larger supply of epithelial cells, hepatocytes, or exocrine pancreatic cells, for use in liver regeneration.

The method of the present invention also increases the potential utility of hepatic cells as vehicles for gene therapy in hepatic system disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected hepatic cells.

The present invention is not limited by the aforementioned particular preferred embodiments. It will occur to those ordinarily skilled in the art that various modifications may be made to the disclosed preferred embodiments without diverting from the concept of the invention. All such modifications are intended to be within the scope of the present invention.

Claims

We claim:

1. A method for promoting hepatic cell proliferation comprising contacting hepatic cells with an amount effective to promote proliferation of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R - R⁸ in the sequence of general formula I

X-R^A-R^B-, wherein X is H or a one to three peptide group, R^A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asp(NH₂) and Sue,

R^B is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R³ is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser, homoSer and azaTyr;

R⁷ is Pro or Ala; and

R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group, and wherein the active agent is not AIL

2. The method of claim 1 wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36; SEQ ID NO:37, and SEQ ID NO:38.

3. The method of claim 1 wherein the concentration of active agent is between about 0.1 ng/kg and about 10.0 mg/kg.

4. A method for promoting hepatic cell proliferation comprising contacting hepatic cells with an amount effective to promote proliferation of an active agent comprising a

sequence of the following general formula: Rl-Arg-R2-R3-R4-His-Pro-R5 wherein Rl is selected from the group consisting of H and Asp;

R2 is selected from the group consisting of Val and Pro;

R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂;

R4 is selected from the group consisting of Ala, He, Leu, and norLeu; and R5 is Phe, He, or is absent, and wherein the active agent is not AIL

5. The method of claim 4 wherein the active agent is selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.

6. The method of claim 4 wherein the concentration of active agent is between about 0.1 ng/kg and about 10.0 mg/kg.

7. An improved cell culture medium for promotion of hepatic cell proliferation, wherein the improvement comprises addition to the cell culture medium an amount effective to increase proliferation of hepatic cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R - R in the sequence of general formula I

R¹ -R²-R³-R⁴-R⁵-R⁶-R^7"R⁸ in which R¹ and R² together form a group of formula X-R^A-R^B-, wherein X is H or a one to three peptide group,

R^A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me²Gly, Pro, Bet, Glu(NH₂), Gly, Asρ(NH₂) and Sue,

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃)₂, Thr, Ser,

homoSer and azaTyr; R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;

R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group, and wherein the active agent is not AIL

8. The improved cell culture medium of claim 7 wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO:2, SEQ ID NO:3,

SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36; SEQ ID NO:37, and SEQ ID NO:38.

9. The improved cell culture medium of claim 7 wherein the concentration of active agent is between about 0.1 ng/ml and about 10.0 mg/ml.

10. An improved cell culture medium for promotion of hepatic cell proliferation, wherein the improvement comprises addition to the cell culture medium an amount effective to increase proliferation of hepatic cells of at least one active agent comprising a sequence of the following general formula:

Rl -Arg-R2-R3-R4-His-Pro-R5 wherein Rl is selected from the group consisting of H and Asp;

R2 is selected from the group consisting of Val and Pro;

R3 is selected from the group consisting of Tyr and Tyr(PO₃)₂;

R4 is selected from the group consisting of Ala, He, Leu, and norLeu; and

R5 is Phe, He, or is absent, and wherein the active agent is not AH.

11. The improved cell culture medium of claim 10 wherein the active agent is selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.

12. The improved cell culture medium of claim 10 wherein the concentration of active agent is between about 0.1 ng/ml and about 10.0 mg/ml.

13. A kit for promoting hepatic cell proliferation comprising:

(a) an amount effective to promote hepatic cell proliferation of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R -R in the sequence of general formula I

R⁴ is selected from the group consisting of Tyr, Tyr(PO₃) , Thr, Ser, homoSer and azaTyr;

R⁵ is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly; R⁶ is His, Arg or 6-NH₂-Phe; R⁷ is Pro or Ala; and R⁸ is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R⁴ as a terminal Tyr group, wherein the active agent is not AH; and

(b) instructions for using the amount effective of active agent to promote hepatic cell proliferation.

14. The kit of claim 13 wherein the active agent is selected from the group consisting of angiotensinogen, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36; SEQ ID NO:37, and SEQ ID NO.38.

15. The kit of claim 13 wherein the concentration of active agent is between about 0.1 ng/ml and about 10.0 mg/ml.

16. A kit for promoting hepatic cell proliferation comprising an amount effective to promote hepatic cell proliferation of an active agent comprising a sequence of the following general formula:

R2 is selected from the group consisting of Val and Pro; R3 is selected from the group consisting of Tyr and Tyr(PO₃) ;

R4 is selected from the group consisting of Ala, He, Leu, and norLeu; and

R5 is Phe, He, or is absent, and wherein the active agent is not AH; and (b) instructions for using the amount effective of active agent to promote hepatic cell proliferation.

17. The kit of claim 16 wherein the active agent is selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38.

18. The kit of claim 16 wherein the concentration of active agent is between about 0.1 ng/ml and about 10.0 mg/ml.