Method to obtain granulocytes as bioactive agent able to inhibit the growth of tumor cells

BACKGROUND OF THE INVENTION

1) Field of the Invention

The present invention relates on a new approach in cancer treatment. More specifically, the present invention relates to a method of obtaining agent inhibiting the growth of tumor cells based on the role of granulocytes in tumor development / regression and its use in treatment of cancer bearing host including parenteral granulocyte application.

2) Description of the Related Art

While it is well known that granulocytes play an essential role in host defense against micro organisms (Babior B.M., Am J Med 109 [2000] 33-44), mechanisms by which granulocytes may also participate in immune reactions against cancer are not well understood. Several researchers have reported cytotoxicity of granulocytes against tumor cells in vitro (Dallegri F, Frumento G, Ballestrero A, Goretti R and Patrone F, Clin Exp Immunol 70 ]1987] 479-483.; Dallegri F, Patrone F, Frumento G and Sacchetti C, J Natl Cancer Inst 73 [1984] 331-339.; Reali E, Guiliani AL, Spisani S, Moretti S, Gavolini R, Masucci G, Gambari R and Traniello S, Clin Immunol Immunopathol 71 [1994] 105-112.; Valerius T, Repp R, Wit TPM, Berthold S, Platzer E ₃ Kalden JR, Gramatzki M and Winker JGJ, Blood 82 [1993] 931- 939) and in vivo (Katano M and Torisu M, Cancer 50 [1982] 62-68.). The activation process of phagocytosis at the site of inflammation is accompanied by the intense production of reactive oxygen species (ROS) (Sbarra AJ. and Karnovsky M. J., J Biol Chem 234 [1959] 1355-1362.) and an extended release of destructive hydrolytic enzymes (Stipancic I. and Zarkovic N., Lijec Vjesn 119 [1997] 279-290.; Niwa Y., Kasama T., Miyachi Y. and Kanoh T., Life Sci 44 [1989] 1655-1664.). Overproduction of ROS is cytotoxic and damages macromolecules (Esterbauer H., Schaur RJ. and Zollner H., Free Radic Biol Med 11 [1991] 81-128.), however, at low concentrations; ROS may function as physiological mediators of cellular responses to various stimuli (Schreck R. and Baeuerle P.A.., Trends Cell Biol 1 [1991] 39-42.). ROS generated by phagocytes act as regulators of other immune reactive cells (Hellstrand K., Asea A., Dahlgren C. and Hermodsson S, J Immunol 153 [1994] 4940-4947.)

and of apoptotic processes (Lundqvist-Gustafsson H. and Bengtsson T., J Leukoc Biol 65

[1999] 196-204.).

ROS have been identified as effector molecules in the mechanisms of oxygen dependent killing of cancer cells by granulocytes (Lichtenstein A., Seelig M, Berek J and Zighelboim J, Blood 74 [1989] 805-809.; Lichtenstein A. and Kahle J., Int J Cancer 35 [1985] 121-127.). In spite of many data showing that granulocytes can participate in the destruction of malignant cells, little is known about the function of granulocytes in tumor-bearing hosts.

The cancer therapy and immunology researches have made a remarkable progress in the last fifty years. The attentions of the researches have first been focused on cytokines and their role in stimulation of mononuclear cells (eg. lymphocytes, monocytes and macrophages). Anti-tumor activity was proven for various cytokines such as tumor necrosis factor (TNF), interferon (IFN), interleukin-1 (IL-I) and IL-2, while some cytokines were determined to promote the T cell mitosis (e.g. TNF, GM-CSF (granulocyte-macrophage colony stimulating factor), IL-I, IL-2, IL-4, IL-6 and IL-7). Majority of researches have otherwise focused on the anti-tumor activity of mononuclear cells, and according to the most of the publications and patents involving leukocytes are based on the biology of mononuclear cells - lymphocytes and macrophages. This patent is based on the anticancer effects of granulocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention features would be clearer from the following detailed description of the invention taking in to account accompanying drawings whereby:

Fig.l. Scheme describing essential aspects of the preparation of granulocytes as bioactive anticancer agent.

Fig.2. A. The survival of tumor-bearing rats, according to Kaplan-Meier method and the incidence of tumor progression or regression; B. The functional activity of circulating phagocytes measured by the whole blood chemiluminescence assay 30 minutes after PMA activation. Light emission is expressed as relative light units per second (RLU/s) on the day 4, 7 and on the day 10 after W256 injection. Average chemiluminescence values are given ± SE: (a) significance p<0.05 in comparison to the earlier day, (b) significance p<0.05 in

comparison to the values obtained from the rats in tumor progression, (c) significance p<0.05 in comparison to the healthy controls.

Fig.3. Histological changes at the site of tumor transplantation observed during spontaneous tumor regression (Magnification: 10Ox - Day 2 and 6; 20Ox - 5h and Day 1; 40Ox - Day 4 and 9).

Fig.4. Anti-tumor activity of granulocytes in vitro. Proliferation of tumor cells in vitro measured by 3H-TdR incorporation assay was referred to as 100% cpm (count per minutes). Mean values (± SE) for the respective triplicates of cultures of granulocytes (significance p<0.05 if compared with cytotoxic effect of granulocytes from normal healthy controls).

Fig.5. The comparative results based on survival curves of tumor-bearing rats, according to Kaplan-Meier method are shown.

Fig.6. A. Survival curves of tumor-bearing animals, according to Kaplan-Meier method and B. tumor volume growth dynamics (± SE per group are given).

DESCRIPTION OF PREFFERED EMBODIMENTS OF THE INVENTION

For the purpose of isolation of granulocytes present invention uses absorbing agent that produces inflammation and absorbs the humoral and cellular exudates. For the same purpose effective granulocytes can be also obtained from the blood by any method preserving their biological activity (such as those mentioned in Boyum A, Scand J Clin Lab Invest 97 [1968] 77-89, in Yui N, Sanui K ₅ Ogata N ₅ Kataoka K ₅ Okano T ₅ Sakurai Y. Biomaterials. [1985] 6:409-415 and in Rock G. Berger R. Romans RA. Russell NM. Owens WA. Transfusion [2000] 40: 1442-1445). The essential aspects of the present method are presented as a scheme on Figure 1.

Hereafter, a detailed description will be given for the use of the granulocyte absorbing agent. Any sterile absorbing agent which is non-toxic for the host may be used, preferably dextran polymer particles (DPP), e.g. Sephadex G. The size of the particles is not specifically limited, but the density should be high. Particles of a diameter of approximately 40 to 120μm may be employed.

The granulocyte absorption step will now be explained more specifically. A 4 to 5 % suspension of DPP in a 0,9% sterile NaCl solution, pH=7,2, is prepared. Since sterility of implanted DPP is a prerequisite for the standardization of the inflammation the DPP suspension is left to swell for 3 days at room temperature subsequent to sterilization in an autoclave for 20 rnin at 110 ⁰ C before injection. We have shown that 24 hours after injection majority of granulocytes are distracted from the circulation and adsorbed hi the subcutaneously formed papula. Afterwards the granulocyte number increases and nine days later reach the control values.

In DPP treated organism, granulocytes are absorbed in subcutaneously formed papula and removed from the blood. As previously described, tumor growth dynamic, W256 (rats) or B16F10 (mice), is faster in DPP treated Wistar rats or C57B1 mice, respectively. We have also shown that granulocytes distracted from tumor bearing-animals into subcutaneously formed papula have significant cytotoxic effect on the in vitro tumor cell proliferation.

Antitumorous activity of granulocytes was not only associated with oxidative stress at the site of tumor transplantation, but also on the systemic level, as verified by the whole blood oxidative burst of granulocytes (see Example 1) and inhibition of tumor cell proliferation in vitro (see Example 3). The obtained results suggest that granulocytes play extremely important role in defense against cancer in the early stages of the tumor development (see Example 2), while their oxidative burst is essential mechanism of the anticancer response which might even lead to the tumor regression. According to the present invention, it has been found that administration of granulocytes as bioactive anticancer agent can lead to tumor regression (see Example 4) or slower tumor growth volume and extend the overall survival (see Example 5).

EXAMPLES

The few embodiments of the present invention will now be explained more specifically in examples. However, it is assumed that scope of the invention as defined by patent claims covers all analogous modification that can be done by person skilled in the art, and therefore Examples do not represent any kind of limitation to the present invention.

Example 1

Effects of DPP on rats with transplanted tumor

Examination was carried out on the following two groups of 46 Sprague Dawley rats in total; W group consisting of 23 tumor bearing animals and WS group of 23 tumor bearing DPP treated animals. Both groups received 10 ⁷ W256 tumor cells in the hind right limb.

The comparative results based on survival curves, according to Kaplan-Meier method are shown in Fig.2A. The incidence of continuous tumor progression / regression in these animals, in percentage is also shown in Fig.2A.

Fig.2A shows that the survival rate of W group is significantly higher than that of WS group. Since Sprague Dawley rats survived (87%) the initial challenge with W256 cancer cells without any prior exposure implies that the anticancer response is innate immunity. Tumor bearing rats that were also DPP treated had survival as low as 30%.

The functional activity of circulating phagocytes, in W and WS group, was measured by the chemiluminescence (CL) assay as described previously (Zivkovic M, Poljak-Blazi M, Zarkovic K, Mihaljevic D, Schaur RJ and Zarkovic N, Cane Lett 246 [2007] 100-108). Briefly, oxidative burst of granulocytes was assessed by mixing 10 μL of heparinised blood samples (isolated from the jugular vein) with 10 μL of PMA (1 μg/mL) and diluted in 600 μL of CL mixture. The CL assay was carried out at 37°C using a Berthold FB12 Luminometer. The results obtained for each sample were evaluated as average values of relative light units per second (RLU/s) at the time point of 30 minutes incubation.

The comparative results based on functional activity of granulocytes in animals with tumor regression / progressions are shown in Fig.2B. The activity of circulating granulocytes differed corresponding to tumor regression or progression. The functional activity of circulating phagocytes during tumor regression was significantly increased on the 3rd day after tumor transplantation and afterwards it decreased with the time on the control values. In animals with tumor progression the granulocyte activity constantly increased. These results implied that granulocytes have a significant role in the early tumor development.

Example 2

Influence of granulocytes in the early tumor development

Twenty seven Sprague Dawley rats were intramuscularly injected with 10 ⁷ W256 tumor cells in the right hind limb. Every day few rats were sacrificed and upon death tumor mass was surgically removed, placed in 10% buffered formalin, embedded in paraffin and used for histopathologycal analysis. Sections made from paraffin blocks were stained with haematoxylin and eosin. The sections were examined by a pathologist well experienced in the histopathology of tumors but without prior knowledge of the experimental groups.

Five hours after tumor transplantation huge granulocyte influx can be seen at the site of tumor transplantation. On the day 1 and 2 after tumor injection a bunch of granulocytes are present inside the tumor mass. Two days later strong inflammation is still present, the beginning of heeling process can be clearly noticed as well. The inflammation and reparation process in tumor are still present on the day 6, while on the 9th day no tumor cells are observed, but inflammation retained. These results suggest that granulocytes have ability to detect unique signals from tumor cells and to migrate to the tumor site resulting in the destruction of the tumor cells. The results described above are shown in Fig.3.

Example 3

Effect of granulocytes on tumor cells proliferation

Six Sprague Dawley rats by means of the same experimental system described in the Example 1 together with two control healthy rats were used. On day 18 animals were anesthesiaed and used in experiment.

Granulocytes were isolated from peripheral blood after dextran sedimentation and centrifugation on Ficoll-Paque cushions as described previously (Boyum A, Scand J Clin Lab Invest 97 [1968] 77-89).

The seeding density of granulocytes cultured in 96-well microcytoplates was 2x10 ^s cells per culture in RPMI 1640 medium with 10% FCS. The seeding density of the tumour cells was 2x10 ⁴ cells per culture irrespective if cultured alone or if added to the granulocytes. The cell cultures were incubated for 24 h at 37°C in a humidified air atmosphere with 5%

CO ₂ . After 24 h, radioactive ³ H-thymidine ( ³ H-TdR) was added to each culture and left for an additional 48 hours. The incorporation of ³ H-TdR, determined with a β-liquid-scintillation counter (Beckman, LS 3800 Series), was used as a indicator of tumour cell proliferation (DNA synthesis) (Zarkovic N, Zarkovic K, Grainca S, Kissel D and Jurin M, Anticanc Drugs 8 (1997) 17-22).

Effect of granulocytes on the inhibition of tumor cell proliferation is shown on Fig.4. Whole blood granulocytes obtained from animals during tumor regression and after showed extreme cytotoxic activity when compared to granulocytes of healthy rats. Granulocytes from animals in regression have a capacity in inhibiting more than 50% tumor cell proliferation, while granulocytes from healthy rats inhibit less than 20%. We have therefore confirmed the anti-tumor effects of granulocytes on tumor cell proliferation in vitro, as well.

Example 4

Granulocyte transplantation in progressive tumor animals 1

Examination was carried out on 16 tumor-bearing DPP treated Sprague Dawley rats. On the 18th day we administrate the granulocytes as a biotherapeutical agent on these rats. As a source for granulocytes healthy rats were used. Those healthy rats were DPP treated and 24 hours later the papula content was surgically removed, placed into sterile 0,9% NaCl and filtered over filter paper that allows granulocytes to pass. Isolated granulocytes were injected into the site of tumor progression into 8 tumor-bearing DPP treated Sprague Dawley rats (group A) while other 8 were left as a control tumor progressive animals (group B).

In Fig.5, survival rate in % is shown on axis of ordinates and the time after tumor transplantation is shown in days on axis of abscissa. The survival rate increased with granulocyte therapy from 30% to 75%.

Example 5

Granulocyte transplantation in progressive tumor animals 2

In thirty three Balbc mice 10 ⁶ Erlich tumor cells were transplanted in the hind right limb. Afterwards, on twenty mice we administrate the granulocytes as a biotherapeutical

agent. As a source for granulocytes healthy Balbc mice were used. Those healthy mice were DPP treated and 24 hours later the papula content was surgically removed, placed into sterile 0,9% NaCl and filtered over filter paper that allows granulocytes to pass. Isolated granulocytes were injected into the site of tumor progression at different time points.

Fifteen mice received granulocyte transplantat on a one-time basis as follows; B group, consisting of 5 mice, was transplanted on the 1st day while other 10 mice, group C, were transplanted with granulocytes on the 6th day. Furthermore, 5 mice (group D) were treated with granulocytes twice (on the day 1, 4 and 8). The rest of mice (n=13) were left as a control tumor progressive animals (group A).

In Fig.6, survival rate in percent of the total number is shown on axis ordinates and the time after tumor transplantation is shown in days on abscissa (Fig.όA). The survival rate was better in mice that were transplanted with granulocytes. On Fig.6B the tumor growth dynamic in these mice is presented. As can be observed, tumors grew faster in control animals (group A) that were not treated with granulocytes as a bioactive anticancer agent.

Example 6

Pharmaceutical application of the peripheral blood granulocyte suspension

Granulocytes should be collected from the peripheral blood of healthy donors using the least aggressive procedures that would preserve functional activity of the cells. Thus obtained granulocyte suspension should be sterile and of physiological pH. To obtain optimal viability and efficiency of granulocytes, the suspension should contain 10-20% human plasma or albumin and must be kept on cold, preferably in refrigerator.

Thus prepared pharmaceutical solution of granulocytes may be used as bioactive agent against cancer. It should be delivered to the recipient in the shortest possible time to be used as potentially bioactive agent against cancer. Before the application the suspension should gain the room temperature.