HRP20141096A2 - Newcastle disease virus strain zg1999hds as an ocolytic agent - Google Patents

Abstract

The invention relates to the cytolytic and oncolytic effect of own strain of Newcastle disease virus ZG1999-HSD and its possible application in the treatment of human and animal tumor diseases. The results of preclinical studies are described. Concomitant parenchyral administration of the virus with tumor cells is only one way of delivering the virus, but it is expected to work in combination with conventional treatments for tumor disease.

Description

Field of invention

This patent application protects the invention in the following areas of technology, according to the International Patent Classification:

A 61 K 39/17,

A 61 K 48/00,

A 01 N 63/00

Technical problem

The idea that viruses, otherwise pathogenic microorganisms, are used in the treatment of patients with tumors arose at the beginning of the last century, and was based on observations of tumor regression in patients who had overcome a viral infectious disease or were vaccinated against diseases caused by viruses. During the fifties of the last century, numerous experiments were carried out that showed that many viruses can infect and lyse tumor cells more effectively than normal cells, and at the end of the last century there was a renewed interest in the use of viruses in antitumor therapy.

The Newcastle Disease Virus (NDV) is an avian Paramyxovirus from the Paramyxoviridae family, less pathogenic for mammals, including humans, in which even highly virulent strains cause only mild conjunctivitis and laryngitis. Unlike the group of genetically modified viruses that acquired the property of specific destruction of tumor cells only after genetic manipulation of viral particles, NDV is included in the group of viruses with the so-called innate ability to selectively destroy tumor cells. Thanks to the mentioned properties, different strains of NDV were investigated in experiments on experimental tumor models on animals, and the results of clinical studies were also published in which this virus was used as an antitumor agent in the treatment of various tumors in humans. With regard to pathogenicity for the natural host (birds), NDV strains are divided into highly (velogenic), moderately (mesogenic) and weakly (lentogenic) pathogenic strains. Among these strains, the antitumor properties and mechanisms of the antitumor effect of moderately pathogenic NDV strains have been most investigated, while research related to the use of lentogenic NDV strains (e.g. LaSota, Hitchner...) in tumor growth control is relatively rare, as are the mechanisms of action. the NDV of these strains have not yet been fully elucidated.

State of the art

So far, research has been carried out on the effects of selected NDV strains on the proliferation of different types of mouse tumor and normal cells, control of tumor growth and animal survival on experimental models of mouse tumors, as well as their effect on the immune system of mice with tumors (Phuangsab et al., 2001, Lorence et al., 1994, Sinkovics and Horvath, 2000).

The results of research on cell cultures indicated an extremely cytotoxic (cytolytic) effect of NDV on tumor cells, while changes in normal cells were absent. Although it did not lead to the complete disappearance of the growing tumor, the application of NDV significantly slows down its growth, and the stimulatory effect of the virus on the immune system of mice with tumors was also recorded. The results of experiments in which a combined therapeutic approach was applied showed that NDV can significantly enhance the antitumor activity of smaller doses of radiation, which significantly reduces damage to normal tissue due to the application of high doses of radiation. Furthermore, the application of the virus in combination with significantly lower doses of cytostatics results in a statistically significant delay in tumor growth (Čović et al., 2006).

These results confirm the possibility of using lentogenic NDV strains in the treatment of malignant diseases, whether they are used individually or in a combined approach, and as such represent the basis for the creation of new, potentially clinically applicable, therapeutic modalities in the treatment of tumor diseases.

The following foreign patents are considered relevant prior art for the patent application in question:

Document 1. Patent US7,056,689, 06.06.2006, R. M. Lorence, K. W. Reichard: Methods of treating and detecting cancer using viruses.

Document 2. Patent US7, 7,736,640, 15.06.2010, R. M. Lorence, K. W. Reichard: Methods of treating and detecting cancer using viruses.

Document 3. US Patent app. number: 20100113335, 06.05.2010, Z. Zakay-Rones, A. Panet, E. Greenbaum, E. Galun, A. I. Freeman, L. Rasooly, C. S. Irving: Compositions and methods for treatment of cancer.

Documents 1 and 2 of the same applicant disclose a method of treating tumors by administering Newcastle disease virus in combination with known chemostatics, immunoadjuvants, etc.

Document 3 discloses the treatment of tumors using a lentogenic strain, i.e. a strain suitable as a vaccine strain, of the Newcastle disease virus.

The essence of invention

A proprietary strain of the Newcastle disease virus designated ZG1999HDS was isolated and proved to belong to a group of lentogenic and immunogenic viruses different from the known ones. The virus according to the subject invention has been deposited in the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM), INSTITUT PASTEUR, 25 RUE DU DOCTEUR ROUX 75724 PARIS CEDEX 15, FRANCE under number CNCM I-4811. Due to the generally expected cytolytic effect of the Newcastle disease virus, the effect of this strain was investigated in vitro on four types of tumor cell lines, namely:

FsaR – mouse fibrosarcoma

SCCVII – murine squamous cell carcinoma

CT26WT – mouse colon adenocarcinoma

4T1 – murine breast adenocarcinoma,

and compared with the effect on the growth of normal cells L929 - (mouse fibroblasts).

The dynamics of tumor growth induced by parenteral administration of tumor cells CT26WT – mouse colon adenocarcinoma and 4T1 – mouse mammary adenocarcinoma were investigated on in vivo models. The cytolytic effect of the ZG1999HDS virus was demonstrated by experiments in vitro on cell cultures of tumor cells and equally in vivo on mice, significantly better than the comparatively investigated cytolytic effect of the Newcastle disease virus strain.

Application

The purpose of these experiments is to prove that the Newcastle disease virus strain ZG1999HDS can significantly inhibit the reproduction of tumor cells, namely FsaR - mouse fibrosarcoma, SCCVII cells - mouse squamous cell carcinoma, CT26WT mouse colon adenocarcinoma cells and 4T1 - mouse breast adenocarcinoma in vitro , and delay and inhibit the growth of mouse colon adenocarcinoma CT26WT and mouse mammary adenocarcinoma 4T1 in an experimental tumor model in vivo. The ZG1999HDS strain of the Newcastle disease virus could, after completed clinical research, be used in the treatment of some human and animal tumor diseases, either alone or in combination with modern methods of treating these diseases (radiation, chemotherapy, etc.).

EXPERIMENT 1. Investigation of in vitro cytotoxic activity of Newcastle disease virus strain ZG1999HDS and comparative Newcastle disease virus strain LaSota, on tumor and normal cell cultures

The aim of the experiment is to prove the cytotoxic effects of different doses of the ZG1999HDS strain of Newcastle disease virus on tumor and normal cells and to compare the cytotoxic potential of this strain with the activity of the commercially available lentogenic LaSota strain of Newcastle disease virus.

Cell lines

The research was conducted on four types of tumor cells: mouse fibrosarcoma FsaR, mouse squamous cell carcinoma SCCVII, mouse colon adenocarcinoma CT26WT and mouse breast adenocarcinoma 4T1, and as a negative control, normal mouse fibroblast cells L929.

Viruses

The preparation of the Newcastle disease virus strain marked ZG1999HDS and the commercial Newcastle disease virus vaccine preparation from the LaSota strain (PESTIKAL® Pliva, Zagreb, Croatia) were used.

The viruses were grown on the chorioallantoic membrane of ten-day-old chicken SPF (specific pathogen free) embryos (VALO-SPF, Lohmann, Cuxhaven, Germany). After infection, the eggs were incubated at 37oC for 72 hours, and then the virus-enriched allantoic fluid was isolated from the eggs, and the 50% infectious dose for the chicken embryo, EID50 (Eng. EID50 - Embryo-Infective Dose50), was determined. Virus preparations containing at least 109 EID50 virus per vial were used.

Cell cultivation and exposure of cell cultures to virus action

Two days (48 hours) before the start of the experiment, tumor and healthy cells were removed from liquid nitrogen and thawed. After centrifugation, the cells were resuspended in RPMI-1640 cell culture medium (Sigma, USA) to which 10% fetal calf serum FCS (FCS; Sigma, USA) had previously been added, and then they were seeded into plastic 250 ml cell culture flasks (Greiner, Germany) and left in an incubator (Heraeus 6000, Germany) under standard conditions (5% CO2 in a humidified atmosphere at 37oC) to adapt to in vitro growth. When the cells covered the bottom of the flask in which they were growing, the supernatant was poured off, and 1 ml of trypsin was added to the flask to detach the cells from the substrate. After adding the medium, the cells were centrifuged for 5 minutes at 150 x g, and then the number and viability of the cells was determined by staining with trypan blue.

Exposure of cells to viruses was performed in 96-well cell culture microplates (Greiner, Germany). Approximately 10 4 cells in 100 µl of RPMI-1640 medium supplemented with 10% FCS were added to each well, and then the cells were incubated under standard conditions to obtain a monolayer cell culture. When the cells covered the bottom of the well (in 24 hours), the old medium was discarded and replaced with a new one (225 µl), and then 25 µl of a virus sample with a concentration of 20 or 200 EID50/cell was added. 25 µl of virus-free RPMI-1640 medium was added to the control groups of cells. The cells were incubated in the presence of the virus under standard conditions for 48 hours, and then the cytotoxic effect was determined by determining the degree of cell survival using the cytotoxicity test with crystal violet.

Assessment of the degree of cell survival by cytotoxicity test with crystal violet

At the end of exposure of the cells to the virus, the cells were fixed for 15 minutes with a 3% formalin solution. Then the cells were washed in redistilled water and left in the air to dry. The cells fixed in this way were stained by adding a 0.1% solution of crystal violet for 20 minutes, washed several times with redistilled water and left overnight to dry. The dye bound in the cells was extracted using a 10% acetic acid solution, and then the absorbance at 540 nm was measured using a photometer (Anthos Microplate Reader HT3). The absorbance value is proportional to the number of surviving cells. The obtained results are shown in a drawing as mean values with associated standard deviations.

In addition, the percentage of cell growth inhibition compared to the control was calculated according to the following formula: cell growth inhibition (%) = (K-T/K) x100,

where T denotes the mean absorbance value in treated cells (cells exposed to the virus), and K denotes the mean absorbance value in control cells (cells not exposed to viruses).

the results

SCCVII mouse carcinoma cells, FsaR fibrosarcoma, CT26WT colon adenocarcinoma, 4T1 adenocarcinoma and L929 fibroblasts were exposed for 48 hours under in vitro conditions to 20 and 200 EID50/cell of Newcastle disease virus strain ZG1999HDS and LaSota strain, respectively. The degree of cell survival was monitored depending on the strain and concentration of the virus.

Figures 1-5 show the effects of Newcastle disease virus strains ZG1999HDS and LaSota on the degree of survival of the investigated cells after 48 hours of incubation of the cells in the presence of the virus, while in Tables 1-5 the survival is expressed relatively as a percentage of inhibition compared to the control.

Both investigated strains of the Newcastle disease virus significantly reduced the number of surviving tumor cells compared to the survival of cells in the control group, with the fact that at the same dose, the ZG1999HDS strain showed a statistically significantly higher inhibition % compared to the LaSota strain. The effect was dose-dependent (200 EID50/cells > 20 EID50/cells), and the sensitivity of cells to viruses decreased in the following order: FsaR>CT26WT>SCCVII>4T1. Application of the virus to L929 fibroblasts also reduced the survival of these cells to a certain extent, with the fact that the intensity of inhibition was significantly lower compared to fibrosarcoma cells.

EXPERIMENT 2. The effect of Newcastle disease virus strain ZG1999HDS and in comparison Newcastle disease virus strain LaSota on the growth of mouse colon adenocarcinoma CT26WT and mouse mammary adenocarcinoma 4T1

The aim of the experiment is to investigate the effect of the Newcastle disease virus strain ZG1999HDS on the growth of mouse colon adenocarcinoma CT26WT and mouse mammary adenocarcinoma 4T1 in an experimental tumor model in vivo and to compare the antitumor potential of this strain with the commercially available lentogenic strain of Newcastle disease virus, LaSoto.

Experimental animals

Mice of the BALB/c strain, aged between 3 and 4 months and body weight 20-23 g, were used. The mice were fed standard food for laboratory mice (4 RF 21 GLP Mucedola srl, Italy) and drank water from the city water supply. Access to food and water was free (ad libitum). The temperature in the experimental room was 22oC, and the air humidity was 55%. The lighting rhythm was 12 hours of light and 12 hours of darkness. The experiment was conducted in accordance with the Croatian Animal Welfare Act (Official Gazette 19/99).

Tumor transplantation and virus administration

Tumor cell viability was previously checked using the trypan blue dye exclusion test and was >95%. The dried virus was stored in a refrigerator at 4oC before use. Immediately before injection, the virus was suspended in RPMI-160 and mixed with the medium containing the cells so that the final virus dose was 200 EID50/cell. Then, 200 µl of a suspension containing 106 tumor cells and 200 EID50/cells of Newcastle disease virus strain ZG1999HDS or LaSote strain was injected subcutaneously into the shaved right thigh of the mouse, using a tuberculin needle and syringe. Control mice received 200 µl of RPMI-160 at the same time and in the same way.

Monitoring of the antitumor effect

Tumors were measured on the 3rd, 5th, 7th, 10th and 13th days of the experiment, using a caliper (Lange Skinfold Caliper, Cambridge Scientific Industry, USA). Three mutually perpendicular tumor diameters were measured: length (A), width (B) and thickness (C). The following equation was used to calculate tumor volume: Tumor volume (mm3) = π/6 x (A x B x C).

Statistical data processing

The achieved results are expressed as mean values with the associated standard deviation. Testing of differences between groups was performed using the ANOVA test in combination with the Holm-Sidak post hoc test of multiple comparisons, in the case when the comparison was made for more than two groups, while the Student's t-test for independent samples was used to compare two groups. The results were processed with the computer program SigmaStat 3.5. Values of p<0.05 were considered statistically significant.

the results

In an in vivo experiment, a suspension of tumor cells and Newcastle disease virus strain ZG1999HDS or LaSote was applied subcutaneously to BALB/c mice, and their effect on the delay in tumor onset as well as the inhibition of tumor growth after tumor onset was monitored.

The growth curve of CT26WT colon adenocarcinoma in BALB/c mice after the simultaneous application of cells and virus is shown in Figure 6. Tumor growth in the group that simultaneously received the Newcastle disease virus strain ZG1999HDS was completely delayed in all mice until the 10th. days from the injection of cells and virus. After that, the appearance of tumors was recorded, and the average tumor volume in this group on the 13th day was statistically significantly smaller compared to the control group and the group that received the LaSota strain (p<0.05; Holm-Sidak test). In the group that received Newcastle disease virus strain LaSota, tumor growth was delayed until day 5 in all mice. On the seventh day after the injection of tumor cells, growth was recorded in 2 mice, and on the 13th day, tumors grew in all mice of this group (Figure 6).

The growth of adenocarcinoma 4T1 in BALB/c mice after the simultaneous application of tumor cells and virus is shown in Figure 7. The ZG1999HDS strain completely delayed tumor growth until day 10 after the injection of tumor cells. In this group, tumor growth was recorded in all mice on the 13th day of the experiment, but the tumor volume was significantly smaller compared to the control and the group that received the LaSotu strain (p<0.05; Holm-Sidak test). Tumor growth was delayed for 5 days in mice that received the LaSotu strain concurrently with tumor cells. After that, a gradual tumor growth was observed in all individuals, with the fact that the tumor volumes in all measurement points were significantly smaller compared to the control (p<0.05; Student's t-test).

Findings

The Newcastle disease virus strain deposited in the Collection nationale de cultures de microorganismes (cncm), Institut Pasteur, 25 Rue du docteur roux 75724 Paris Cedex 15, France under number CNCM I-4811, internal designation ZG1999-HSD can be used in the treatment of tumor diseases and either alone or in combination with other recognized procedures for the treatment of tumor diseases in humans and animals. In particular, the strain of the virus according to the subject invention shows exceptional effectiveness in the treatment of fibrosarcoma, squamous cell carcinoma, colon adenocarcinoma, and breast adenocarcinoma. More precisely, the strain according to the subject invention lyses (decomposes) tumor cells of fibrosarcoma, squamous cell carcinoma, colon adenocarcinoma and breast adenocarcinoma cells. In addition to the above-mentioned effect, the Newcastle disease virus strain ZG1999-HSD has a pronounced oncolithic effect in a way that delays the growth of tumors such as fibrosarcoma, squamous cell carcinoma, colon adenocarcinoma and breast adenocarcinoma cells.

The ZG1999-HSD strain of the Newcastle disease virus, compared to the LaSota strain of the Newcastle disease virus, has a more pronounced cytolytic and oncolytic effect in such a way that it kills tumor cells significantly faster in vitro and also statistically significantly slows down the growth of tumors induced by four different types of tumor cells.

Brief description of figures and tables

Figure 1. Cytotoxic effect of NDV strain ZG1999HDS and NDV strain LaSote on murine squamous cell carcinoma SCCVII cells after 48 hours of cell incubation in the presence of 20 and 200 EID50/cell of virus.

Figure 2. Cytotoxic effect of NDV strain ZG1999HDS and NDV strain LaSote on FsaR mouse fibrosarcoma cells after 48 hours of cell incubation in the presence of 20 and 200 EID50/cell virus.

Figure 3. Cytotoxic effect of NDV strain ZG1999HDS and NDV strain LaSote on CT26WT mouse colon adenocarcinoma cells after 48 hours of cell incubation in the presence of 20 and 200 EID50/cell of virus.

Figure 4. Cytotoxic effect of NDV strain ZG1999HDS and NDV strain LaSote on murine mammary adenocarcinoma 4T1 cells after 48 hours of cell incubation in the presence of 20 and 200 EID50/cell of virus.

Figure 5. Cytotoxic effect of NDV strain ZG1999HDS and NDV strain LaSote on L929 mouse fibroblast cells after 48 hours of cell incubation in the presence of 20 and 200 EID50/cell of virus.

Figure 6. Growth of CT26WT colon adenocarcinoma in BALB/c mice after simultaneous administration of a suspension of 106 CT26WT tumor cells and 200 EID50/cells of Newcastle disease virus strain ZG1999HDS or LaSota.

Figure 7. Growth of 4T1 mammary adenocarcinoma in BALB/c mice after the simultaneous administration of a suspension of 106 4T1 tumor cells and 200 EID50/cells of the Newcastle disease virus strain ZG1999HDS or LaSota.

Table 1. Mean values of absorbance at 595 nm (A595) and cell growth inhibition expressed as % of control on SCCVII cells.

Table 2. Mean values of absorbance at 595 nm (A595) and cell growth inhibition expressed as % of control on FsaR cells.

Table 3. Mean values of absorbance at 595nm (A595) and cell growth inhibition expressed as % of control on CT26WT cells.

Table 4. Mean values of absorbance at 595 nm (A595) and cell growth inhibition expressed as % of control on 4T1 cells.

Table 5. Mean values of absorbance at 595 nm (A595) and cell growth inhibition expressed as % of control on L929 fibroblasts.

An asterisk (*) in the tables indicates a statistically significant difference in inhibition between ZG1999HDS-20 and LaSota-20, and a sign (#) between ZG1999HDS-200 and LaSota-200 (p<0.05; Student's t-test).

Claims (6)

1. Newcastle disease virus strain deposited in Collection nationale de cultures de microorganismes (cncm), Institut Pasteur, 25 Rue du docteur roux 75724 Paris Cedex 15, France under number CNCM I-4811, internal designation ZG1999-HSD, indicated to be used in the treatment of tumor diseases, either alone or in combination with other recognized procedures for the treatment of human and animal tumor diseases. 2. Newcastle disease virus strain according to claim 1, characterized in that it is used to treat fibrosarcoma, squamous cell carcinoma, colon adenocarcinoma, breast adenocarcinoma in humans and animals. 3. Newcastle disease virus strain according to claims 1 or 2, characterized in that it lyses tumor cells. 4. Newcastle disease virus strain according to claim 3, characterized in that it lyses fibrosarcoma cells, squamous cell carcinoma, colon adenocarcinoma and breast adenocarcinoma cells. 5. Newcastle disease virus strain according to claims 1 or 2, characterized in that it has a pronounced oncolithic effect and delays tumor growth. 6. The Newcastle disease virus strain according to claim 5, characterized in that it has an oncolytic effect and delays the growth of fibrosarcoma, squamous cell carcinoma, colon adenocarcinoma and breast adenocarcinoma cells.