HK1060050A - Use of strains of the parapox ovis virus against organ fibrosis - Google Patents

Description

Use of strains of parapoxvirus ovis against organ fibrosis

The present invention relates to the use of inactivated parapoxviruses in humans for the prophylaxis and treatment of diseases which are accompanied by an increased collagen deposition associated with an affected internal organ, such as the liver and the skin, and their additional structures. The invention relates in particular to liver fibrosis and cirrhosis following viral hepatitis, or ethanol-induced liver disease, and also cystic fibrosis.

The invention particularly relates to isolates of Parapoxvirus ovis (Parapoxvirus ovis) such as the D1701 strain, the orf-11 strain, the Greek orf strain 176, the Greek orf strain 155, New Zealand (NZ) isolates such as NZ2, NZ7 and NZ10, and also to the use of Baypamun  obtained from D1701 for humans.

In addition to the original strain, the invention also relates to progeny obtained by passaging and/or adapting to specific cells, such as WI 38. In addition to whole viruses, the present invention also relates to portions or fragments of these viruses. By parts is understood genomic or subgenomic fragments which are expressed in a suitable system, for example in fibroblast cell culture, using a suitable vector, for example vaccinia. By fragments are understood fractions obtained by biochemical purification, such as chromatography, or particles obtained after disruption using physical methods, such as with sonication.

Parapoxviruses are known to stimulate non-specific immune responses in vertebrates. Baypamun^Is a chemically inactivated parapoxvirus ovis strain D1701 preparation for the prophylaxis, post-disease regulation and treatment of infectious diseases and for the prevention of stress-induced diseases in animals。

Patent document DE 3504940 a (Mayr, Ant on) teaches the beneficial effects of symptoms like high energy radiation, chemotherapy, AIDS, immunosuppression, age-related injuries and detoxification induced immunodeficiency in Baypamun  subjects, but does not teach the direct reduction of liver fibrosis. DE 3504940 also teaches Baypamun  as an adjunct to the efficacy of tumor therapy and which prevents diseases in the newborn due to inadequate immune defence of the mother.

Taking the present knowledge as a starting point, it was surprisingly found that administration of an inactivated parapoxvirus reduces or prevents liver fibrosis. In animal models, this effect was found in the case of carbon tetrachloride-induced liver fibrosis based on toxic liver injury, and also in the case of liver fibrosis induced by heterologous serum and in which there is no liver infection. The extent of this therapeutic effect is likewise surprising: overproduction of collagen associated with liver fibrosis was inhibited by 60% in the carbon tetrachloride model and almost completely in the serum model. Consistent with the results of these long-term experiments, it was possible to demonstrate from acute administration of carbon tetrachloride that Baypamun  and the preparations obtained from the above parapoxvirus ovis strain inhibited the conversion of hepatic stellate cells to collagen-producing myofibroblast cell types.

Different etiologies can induce liver fibrosis and/or cirrhosis, such as viral infections and alcohol abuse, but different pathomechanisms enter a common final pathway, namely collagen production. Animal experimental results obtained on the basis of the non-infectious model described above demonstrate that the administration of inactivated parapoxvirus surprisingly prevents collagen deposition independently of the inducing etiology.

Parapoxviruses thus provide a novel therapeutic principle for exerting an effect on the final pathway common to all diseases leading to fibrosis.

This effect suggests that a particularly effective treatment is achieved when using parapoxvirus preparations, even in the case of virus-induced liver fibrosis, since such preparations are known to have an additional immunostimulatory effect.

The discovery of the potent anti-fibrotic effect of Baypamun  now opens the possibility of using Baypamun  or NZ2 formulations as reference criteria for evaluating anti-fibrotic effects in assays for identifying anti-fibrotic substances.

Thus, the inactivated parapoxviruses or their progeny, and the preparations obtained from these strains have a broad spectrum of anti-fibrotic effects and are therefore suitable not only for the prevention and treatment of fibrotic diseases of the liver, but also for fibrotic diseases of other organs, such as the lung, pancreas, heart and skin. In the prevention and treatment of liver fibrosis and cirrhosis, the use of isolates of parapoxviruses is particularly preferred.

Depending on the clinical question, parapoxvirus-based therapeutic drugs are administered systemically, i.e., for example intramuscularly, subcutaneously, intraperitoneally, intravenously, orally or by inhalation, but also topically. The parapoxvirus is purified and lyophilized and is suspended in a suitable solvent directly prior to administration, or is present in another suitable formulation, or is present in a gastric juice-resistant oral administration form or some other oral administration form.

In connection with this, multiple administrations or long-term treatment according to a schedule of time-administration corresponding to the requirements of clinical problems may be necessary.

The present invention relates to the use of isolates of parapoxviruses from the strain D1701, the strain orf-11, the strain greek orf 176, the strain greek orf 155, the strain New Zealand (NZ) for the preparation of a medicament having a preventive or therapeutic effect on organ fibrosis in humans. It is preferable to use the New Zealand (NZ) strains, i.e., NZ2, NZ7 and NZ10 strains, for the preparation of a medicament having a preventive or therapeutic effect on human organ fibrosis, with NZ2 strain being particularly preferred. In addition to this, the above-mentioned parapoxviruses can be modified by passaging or adaptation to suitable cells, and those parapoxviruses which have been obtained by passaging or adaptation can be used for the preparation of medicaments having a preventive or therapeutic effect on organ fibrosis in humans, in connection with which human cells, such as WI-38, MRC-5, bovine cells, such as BK-K13A47/Reg or MDBK, and ovine cells, such as MDOK, can be used for the passaging or adaptation. The use of parts or fragments of the above-mentioned parapoxviruses for the preparation of a medicament for the prophylaxis or therapy of fibrosis in human organs is also possible. By parts is understood genomic or subgenomic fragments expressed in a suitable system, for example in fibroblast cell culture, using a suitable vector, for example vaccinia virus, by fragments are understood expressed viral particles or fractions obtained by biochemical purification, for example chromatography, by physically disrupting the viral particles, for example by sonication. The invention further relates to the use of the above parapoxvirus ovis strain or the modification obtained therefrom as described above in combination with other drugs for the preparation of a medicament and pharmaceutical preparation having a prophylactic or therapeutic effect on human organ fibrosis, and to the use of Baypamun  per se or in combination with other drugs for the preparation of a medicament and pharmaceutical preparation having a prophylactic or therapeutic effect on human organ fibrosis. The invention preferably relates to the use of the abovementioned parapoxvirus ovis strain or of the modifications obtained therefrom as described above in combination with other drugs for the preparation of formulations for oral administration, for example for the preparation of gastric juice-resistant capsules.

The invention further relates to the use of a Baypamun  or NZ2 preparation as a reference standard for the identification of an anti-fibrotic substance test for the evaluation of anti-fibrotic effects.

The parapoxvirus ovis NZ-2 mentioned here by way of example was deposited at 10.7.2001 in the center for cell cultures Europe, applied microbiology and research center, Porton Down, Salisbury, Wiltshire, SP 40 JG, United Kingdom. The accession number is.

Example 1 Effect of parapoxvirus ovis, strain D1701, Bavpamun  Method

The preparation obtained from the chemically inactivated parapoxvirus ovis, strain D1701, Baypamun  was driedThe dry substance was dissolved in water as indicated for injection (titer according to TCID50 (50% tissue culture infectious dose) was about 10⁷Per ml).

The control animals were administered a polygeline solution containing the same amount of protein as Baypamun  solution as a placebo solution.

Each animal was administered intraperitoneally 0.5ml of solution each time. Three times per week, but never on consecutive days.

Baypamun  was tested in two animal models, which differ in the origin of fibrosis, namely the carbon tetrachloride model and the porcine serum model.

Chronic treatment with carbon tetrachloride is the standard method for experimental induction of liver fibrosis with subsequent cirrhosis (McLean EK, McLean AEM, Sutton PM. instant cirrhosis. Br. J exp. Pathol. 1969; 50: 502-. It is generally considered to be a model of human liver fibrosis and cirrhosis. Female Sprague-Dawley rats were used. To ensure maximum induction of microsomal metabolism of carbon tetrachloride, animals were given 1 gram of isoniazid/liter and drinking water one week prior to starting treatment. Every five days, carbon tetrachloride (carbon tetrachloride: mineral oil 1: 1) was administered orally at a dose of 0.1 ml/100 g body weight. After seven weeks of treatment, animals were sacrificed and examined. Baypamun  treatment was performed in parallel with the carbon tetrachloride treatment.

Treatment with heterologous serum, for example porcine serum in the case of rats, is also a commonly used method in the literature to induce liver fibrosis with subsequent cirrhosis, using which, in contrast to other models, only minor damage and inflammation are caused to the parenchymal cells of the liver (Bhunche, E. and Wake, K. (1992): the Role of the stromal cell population in porcine serum-induced liver fibrosis in rats (Hepatology) 16: 1452-. Female Sprague Dawley rats were treated twice weekly intraperitoneally with 0.5ml of sterile pig serum (Sigma) per animal and control animals were administered a sterile physiological solution of sodium chloride (twice weekly, 0.5 ml/animal, intraperitoneally). Baypamun  treatment was performed in parallel with porcine serum treatment, but never on the same day. After seven weeks of treatment, animals were sacrificed and livers were removed for quantitative determination of collagen amount.

For histological examination of liver tissue, standard transverse tissue cylinders (approximately 10 x 2mm) were punched out of the right front page of the liver. Frozen sections were stained with 0.1% piscirius red solution for detection of collagen plaques produced by liver fibrosis.

Fast green was used as a counterstain for contrast magnification. The degree of liver fibrosis was measured for each section as a percentage of the total area measured as the area stained by Picrosirius red. Visual microscope color inspection parameters were standardized and kept constant throughout the experiment. Measured at 31mm at 50 times final magnification²The normalized 64 squares in the grid.

To quantify the extent of hepatic stellate cell (HSC; also known as Ito cells or vitamin a storage cells) transformation following acute treatment of rats with carbon tetrachloride, the α -smooth muscle actin-positive area was examined immunohistologically. The α -smooth muscle actin-positive zone was determined in the 248 × 180 μm area in the center of the 16 leaflets at 200 x final magnification for each section in each case. It is known that the transformation of HSCs into myofibroblast-like cells that produce collagen and produce growth factors is an important step in the induction of liver fibrosis. Thus transformed HSCs are an early indicator of hepatic fibrinogen activity.

Semi-automated morphometric assays were performed using Leica Quantimed 500MC (Leica Germany).

To determine OH-proline, 50-100mg of liver tissue were in each case dried and boiled with 6N HCl for approximately 17 hours. After the acid was evaporated in the vacuum oven, the residue was dissolved in 5ml of distilled water and the solution was filtered. 200 microliters of this filtered solution was incubated with 200 microliters of ethanol and 200 microliters of an oxidizing solution (7% chloramine T hydrate in water, diluted 1: 4 with acetate-citrate buffer pH 6.0) at room temperature for 25 minutes. Thereafter, 400. mu.l of Ehrlich's reagent (12 g of 4-dimethylaminobenzaldehyde in 20 ml of ethanol + 2.74 ml of concentrated sulfuric acid in 20 ml of ethanol) was added. After 3 hours incubation at 35 ℃ the absorbance was measured at 573 nm. Aqueous OH-proline (Sigma) was used in the standard series. The OH-proline content in the liver samples was calculated in milligrams per gram of dry weight of the liver.

To monitor the formation of reactive oxygen radicals, the concentration of reduced alpha-tocopherol (alpha-TOC), a free radical-trapping agent, in the liver was measured, along with the activity of the free radical-sensitive enzyme 7-ethoxyresorufin deethylase (EROD) in the serum. These two parameters are characteristically down-regulated in the case of carbon tetrachloride poisoning, and the severity of oxidative damage to the tissue can be estimated.

The liver status of animals was determined by measuring several standard serum parameters:

alanine Aminotransferase (ALT), Alkaline Phosphatase (AP), aspartate Aminotransferase (AST), gamma-glutamyl transferase (GGT), glutamate dehydrogenase (GLDH), and Total Bilirubin (TBIL). And (4) conclusion:

treatment with Baypamun  significantly reduced the degree of fibrotic degeneration of the liver in carbon tetrachloride-treated rats (figure 1). In addition, an almost complete inhibition of HSC transformation could be observed (fig. 2). The number of proliferating parenchymal tissue cells in the liver of Baypamun  treated animals was greatly reduced (fig. 3). Non-parenchymal tissue cells include HSC and Kupfer cells also involved in fibrogenesis.

Serum indicators of hepatocyte injury, such as ALT, AP, AST, GGT, GLDH and TBIL (table 1) indicate a normalization trend.

The same reduction in EROD and alpha-tocopherol concentrations in the control and Baypamun  treated groups provided evidence of the presence of toxic reactive oxygen radicals generated by carbon tetrachloride poisoning in both groups (tables 1 and 2). Thus, the possibility that the "anti-fibrotic" effect of Baypamun  is due to detoxification was excluded.

In the serum model, Baypamun  showed almost complete inhibition of fibrosis (fig. 4): in Baypamun  treated rats, hydroxyproline content and sirius red-stainable area in the liver were almost at the level of healthy control animals, whereas they were increased several-fold in serum-treated control rats. In the Baypamun group, the increase in collagen content induced by the pig serum treatment was only 10% of the corresponding value in the control group.Example 2 Parapoxvirus ovis, strain NZ2 The method comprises the following steps:

replication of NZ2 virus in stacked tanks. For this, BK clone 3A cells were cultured in EMEM2gr + 10% FCS in cell culture dishes (37 ℃) for 3-5 days until the cell layer reached 90-100% confluence. Four cell culture dishes were used as inoculum for each stacked pot, the latter being filled with medium (EMEM 2gr + 10% FCS) to a volume of 2.5 liters. Incubate at 37 ℃ for 3-5 days (90-100% confluent cell layers), replace the medium with EMEM2g without serum added, and infect the medium with NZ2 virus (MOI, 0.001-0.01).

After 100% CPE was reached (incubation at 37 ℃ for approximately 7-8 days), the virus was collected. For this purpose, the virus suspension was filled into sterile medium sachets and frozen at-80 ℃. The suspension was then thawed in the culture chamber at 37 ℃ and the cells were freed by means of depth bed filtration (pore size 5 μm). Thereafter, the viral suspension was concentrated 20-40 fold using ultrafiltration (100kDa cut-off). Alternatively, the virus can be concentrated by ultracentrifugation (Ti45, 30,000rpm, 4 ℃, 60 minutes).

The titer of the virus contained in the suspension achieved by concentration was determined by titration of BK clone 3A cells. After adjusting the virus titer to 6.0 using EMEM medium without FCS, the virus was heat inactivated at 58 ℃ for 2 hours. Inactivation was checked using an inactivation control on BK clone 3A cells.

Parapoxvirus ovis was tested in a model in which porcine serum that had been used in example 1 induced liver fibrosis in rats, strain NZ 2:

0 twice weekly for each animal5ml of sterilized pig serum (Sigma) female Sprague Dawley rats were treated intraperitoneally and control animals were administered a sterilized sodium chloride physiological solution (twice weekly, 0.5 ml/animal, intraperitoneally). At 1.5X 10⁵Or 5.0X 10⁵TCID₅₀Dose per animal strain NZ2 was administered intraperitoneally three times a week (dosing volume: 0.5 ml/animal). For lower doses, the starting material was diluted with cell culture medium (Eagle's minimal medium, Sigma). Control animals were treated with cell culture medium. Treatment with strain NZ2 was performed in parallel with treatment with serum, but never on the same day. After seven weeks of treatment, animals were sacrificed and livers removed; liver fibrosis was then determined morphologically and quantitatively by OH-proline content. This method has already been described in example 1.And (4) conclusion:

the results of the collagen assay are given in figure 5. Surprisingly, treatment with strain NZ2 inhibited the development of liver fibrosis: serum treated control animals showed a significant increase in OH-proline content and Sirius red-stained collagen compared to healthy animals. NZ2 reduced this increase in a dose-dependent manner. The extent of this effect is also surprising: 5X 10⁵TCID₅₀The dose reduced the increase in collagen content in the liver to 10% less than the control. Qualitative analysis of histological preparations showed that the proportion of animals with collagen septa was 1.5X 10⁵TCID₅₀The group was reduced from 93% (14/15) to 33% (5/15) at 5.0X 10⁵TCID₅₀The group was reduced to 0%.Example 3 Anti-fibrotic effects following oral administration of PPVO

PPVO was formulated as a dry lyophilizate in gastric juice-resistant capsules (Elanco, Indianapolis, USA).

Four groups of six experimental animals were treated as follows: one control group (group 1) was orally administered with gastric juice-resistant capsules (without PPVO) alone and additionally injected intraperitoneally with sterile sodium chloride solution (0.5 ml/animal). Group 2, gastric juice-resistant capsules (without PPVO) were orally administered with 0.5ml of carbon tetrachloride/animal, and further intraperitoneally injected with 0.5ml of physiological sodium chloride solution. Animals of group 3 were orally administered gastric juice-resistant capsules (without PPVO) with 0.5ml of carbon tetrachloride per animal. In addition, the animals of group 3 were given PPVO D1701 (dose: 5X 10) as an intraperitoneal injection in 0.5ml of water for injection⁶TCID₅₀Animal). PPVO D1701 was orally administered to the animals of group 4 (dose: 5X 10)⁶TCID₅₀Animal, formulated as gastric juice-resistant capsules) and 0.5ml of carbon tetrachloride. Animals in group 4 were also administered 0.5ml of sterile sodium chloride solution by intraperitoneal injection.

After 48 hours, livers were removed and the central area of α -smooth muscle actin (α -SMA) positive leaflets was determined immunohistochemically for each animal using representative tissue sections, expressed as a percentage of the total area measured (Johnson S J, Hines J E, Burt A D., parasinus pericyte Phenotypic modulation following acute liver injury: quantitative analysis (photoplastic modulated epithelial cells following sinus input: acquired analysis), int.J exp.Path.1992; 73: 765-. This value is a measure of hepatic stellate cell transformation.

Two experimental series were performed as described above. Table 3 gives the results of the first series of experiments and table 4 gives the results of the second series of experiments.

In the first series of experiments, the proportion of the area of the central region of the α -SMA-positive lobules in liver tissue of animals intraperitoneally administered PPVO D1701 (group 3) was decidedly higher than in animals not receiving PPVO (in contrast to the other experimental results). For this reason, a second series of experiments was performed as a repeat experiment.

In both experimental series, it was found that, after oral administration of PPVO D1701 (group 4 in each case), the conversion was consistently and unexpectedly inhibited by about 50%, compared to the control group (group 2 in each case). In the second experimental series, the inhibition of transformation after oral administration of PPVO D1701 (group 4) was similar to that observed with intraperitoneal administration of PPVOD1701 (group 3).

From these results, it can be concluded that PPVO also plays an anti-fibrotic role after oral administration.

TABLE 1

BaypamunEffects on serum parameters of liver status and toxic indications
			ALT AST AP GGT GLDH TBIL ERODU/l U/l U/l U/l U/l μmol/l nmolgx min
Control SEM carbon tetrachloride SEMCt + BaypamunSEM	49.1 45.7 162.8 0.8 12.5 1.6 0.303.7 8.7 14.76 0.2 7.9 0.2 0.0295.6 92.4 392.7 6.1 37.1 2.9 0.1514.7 14.1 43.0 1.3 14.8 0.3 0.0172.0 65.9 329.5 4.5 18.2 1.9 0.179.9 9.1 26.9 0.8 4.0 0.2 0.03

ALT: alanine aminotransferase; GLDH: a glutamate dehydrogenase;

AST: an aspartate aminotransferase; TBIL: total bilirubin;

AP: alkaline phosphatase; EROD: 7-ethoxy resorufin deethylation enzyme;

GGT: gamma-glutamyl transferase

TABLE 2

BaypamunInfluence of (2)
		Para-hepatic alpha-tocopherol
	Alpha-tocopherol nmol/g tissue
		Control SEM	73.12.2
Carbon tetrachloride SEM	35.72.3
		T.+BaypamunSEM	38.56.1

TABLE 3

Effect of PPVO on hepatic stellate cell transformation following administration of fibrogenic doses of carbon tetrachloride either intraperitoneally or orally (first experimental series)

Fibrosis of fiber	Application of drugs	Dosage form	α-SMA(％)
				Group 1 complete	Oral empty capsules + intraperitoneal administration of water for injection	---	0.28±0.04
Group 2 carbon tetrachloride	Oral empty capsules + intraperitoneal administration of water for injection	---	2.76±0.79
				Group 3 carbon tetrachloride	Intraperitoneal administration of PPVO + oral empty capsules in water for injection	5×106TCID50Animal	5.05±2.00
Group 4 carbon tetrachloride	Oral administration of PPVO + in capsules intraperitoneal administration of Water for injection	5×106TCID50Animal	1.46±0.34

TABLE 4

Effect of PPVO on hepatic stellate cell transformation following administration of fibrogenic doses of carbon tetrachloride either intraperitoneally or orally (second series)

Fibrosis of fiber	Application of drugs	Dosage form	α-SMA(％)
				Complete group 1	Oral empty capsules + intraperitoneal administration of water for injection	---	0.20±0.04
Group 2 of carbon tetrachloride	Oral empty capsules + intraperitoneal administration of water for injection	---	3.18±0.56
				Group 3 of carbon tetrachloride	Intraperitoneal administration of PPVO + oral empty capsules in water for injection	5×106TCID50Animal	1.22±0.35
Group 4 of carbon tetrachloride	Oral administration of PPVO + in capsules intraperitoneal administration of Water for injection	5×106TCID50Animal	1.55±0.34

Claims (10)

1. Use of an isolate of parapoxvirus for the preparation of a medicament for the prophylaxis or treatment of fibrosis in a human organ.

2. Use of isolates of parapoxviruses, such as the D1701 strain, the orf-11 strain, the Greek orf strain 176, the Greek orf strain 155 and the New Zealand (NZ) strain, for the preparation of a medicament having a prophylactic or therapeutic effect on organ fibrosis in humans.

3. Use of an isolate of parapoxvirus according to claims 1-2, characterized in that the New Zealand (NZ) strains used for the preparation of a medicament having a prophylactic or therapeutic effect on human organ fibrosis are NZ2, NZ7 and NZ10 strains.

4. Use of the parapoxvirus according to claims 1 to 3 modified by passaging or adaptation to suitable cells for the preparation of a medicament having a preventive or therapeutic effect on human organ fibrosis.

5. Use of the parapoxvirus according to claims 1 to 3 modified by passaging or adaptation to suitable cells for the preparation of a medicament having a preventive or therapeutic effect on human organ fibrosis, characterized in that human cells, such as WI-38 or MRC-5, bovine cells, such as BK-K13a47/Reg or MDBK, and ovine cells, such as MDOK, are used for the passaging or adaptation.

6. Use of parts or fragments of the virus according to claims 1-5 for the preparation of a medicament having a preventive or therapeutic effect on human organ fibrosis, characterized in that said parts are understood as genomic or subgenomic fragments expressed in a suitable system, such as fibroblast cell culture, using a suitable vector, such as vaccinia virus, the so-called fragments being understood as fractions obtained by biochemical purification, such as chromatography, of the expressed or physically disrupted viral particles.

7. Use of an isolate according to claims 1-6 together with other drugs for the manufacture of a medicament and pharmaceutical preparation for the prevention or treatment of fibrosis in the human organ.

8. The use of parapoxvirus ovis D1701 as such or together with other medicaments for producing medicaments and pharmaceutical preparations which have a preventive or therapeutic effect on fibrosis in the human organs.

9. Use of a preparation of parapoxvirus ovis D1701 or NZ2 as a reference standard for evaluating anti-fibrotic effects in an assay for identifying anti-fibrotic substances.

10. The use of the parapoxvirus ovis D1701 according to claim 8, characterized in that the pharmaceutical preparations and medicaments are suitable for oral administration.