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Efficacy, safety, quality

EFFICACY: Case Reports

CASE REPORTS

Besides clinical studies, NSC 631570 has been also used by many physicians in the treatment of various tumors, for example cervical cancer (96), ovarian cancer (97), testicular cancer (98), esophageal carcinoma (99), urethral carcinoma (100), and neuroblastoma (116). Such reports complete the pattern of the clinical using NSC 631570.

Breast cancer

Physicians report on the successful treatment of a stage IV breast cancer patient. After the NSC 631570 therapy the tumor size decreased and the surgical removal was alleviated (162).

There are also more reports on the successful treatment of breast cancer (94, 95), including a relapsing breast carcinoma with lung metastases (94).

Sarcomas

Beside common solid cancers, infrequent sarcomas present a real problem in oncology because of therapy resistance (93). All the more interesting is the case of retroperitoneal sarcoma successfully treated with NSC 631570. At the time of the publication, e.g. four years after the start of the NSC 631570 therapy, the patient was in full remission (163).

The Ewing’s sarcoma is a tumor from connective tissue cells of bone marrow. It is a rare disease with the incidence of 3 new cases per million. Ewing sarcoma can occur on every site of skeleton. In most cases, legs, pelvic bone, scapula and ribs are concerned. Usually, children between 10-15 years suffer from it, but also children under 10 years can be affected.  

A 9 year-old girl from Poland was diagnosed with Ewing's sarcoma. The girl received chemotherapy and radiotherapy but the tumour growth could not be stopped. The girl was declared to have exhausted all possible forms of treatment (i.e. had been given up by mainstream medicine) and was sent home with infaust prospects. The parents took their daughter to Vienna to St. Anna Children Hospital because they hoped to receive the state-of-the-art treatment. The doctors carried out new examinations and had to declare that they too could not help her. The complete repertoire of mainstream medicine was used up but the tumour growth continued. At this point her parents heard about Ukrain by chance and contacted Dr. Wassil Nowicky. On 21 January 1984 treatment with Ukrain was begun.
After six months of treatment with Ukrain the girl was examined again at St. Anna Children's Hospital and to the great astonishment of the doctors not only had the tumour growth been stopped but even better, the tumour had become smaller. However, there was still no interest in Ukrain.
Treatment with Ukrain was continued and every six months the progression of the disease was checked at St. Anna Children's Hospital at the instigation of Dr. Nowicky. In this way Dr. Nowicky wanted to arouse the interest of doctors in Ukrain (unfortunately to no avail).
Gradually the tumour disappeared completely and at the X-ray examination on October 31, 1990 it was seen that even the bone which had been eaten away had regenerated (28).

Ewing’s sarcoma, first diagnosed 22.11.1983, histologically verified, tumour resistant both to chemotherapy and radiotherapy. UKRAIN therapy started on 21 January 1984.

Ewing-Sarkom

A 9 year-old girl had felt marked pain below the right knee joint in November 1983 following a slight injury. X-ray revealed Ewing’s sarcoma in the proximal portion of the right fibula. Hospital treatment included chemotherapy and cobalt therapy. X-rays confirmed that the patient’s tumour had not responded to radiation or chemotherapy and the tumour mass increased rapidly. One month after the end of chemotherapy, UKRAIN treatment was started at a dose of 5 mg i.m. for a total of 10 injections, combined with regional deep hyperthermia. The first series of UKRAIN therapy included three identical courses with a two-week pause between them. Six series of UKRAIN treatment were administered over the course of one year. Repeated x-rays showed reduction of the tumour mass.

In a 10 year girl the Ewing’s was verified histologically. She was treated in the high risk group of the EICESS 92 study. The MRI of the pelvis revealed the disease progression; the tumor was resistant to chemotherapy and to radiotherapy as well. The treatment with NSC 631570 was started: 15 mg mixed with 250 ml dextrose 5% and 5 g vitamin C followed by local hyperthermia on alternate days, 10 sessions totally. The control MRI revealed the stop of the progression. The followed therapy cycles brought about the regression of the tumor. At the MRI after four years no signs of a relapse or metastases could be revealed (115). The patient is in remission till now.

Ewing’s sarcoma, first diagnosed 18.3.1996; the tumour was resistant to both chemotherapy and radiotherapy. UKRAIN therapy started on 13 October 1997 (115).

Ewing-Sarkom

The patient, a 10 year-old girl, was treated in the high-risk arm of the EICESS 92 study. MRI examination of the pelvic region on 1.9.1997 showed progression in the cystic-edematous process. She was then treated with combined Ukrain and local hyperthermia therapy. The therapy series consisted of 15 mg Ukrain in an infusion with 250 ml glucose and 5 g vitamin C, followed by local hyperthermia treatment. Treatment was administered every second day up to a total of 10 therapy sessions. MRI examination on 8.1.1998 showed no progression of the tumour. Subsequent therapy cycles caused regression of the tumour (see MRI on 15.6.1999 and 1.2.2000). MRI on 1.2.2001: Cystic residual defect in right femur, as observed in previous examinations. No sign of a relapse or of metastases.

In vitro studies (studies on cell cultures) by scientists at the University of Tübingen have demonstrated the effectiveness of Ukrain with Ewing's sarcoma. These studies and clinical successes, such as with Dr. Aschhoff, were repeatedly submitted to the Ministry of Health. At Dr. Aschhoff's clinic ‘Cancer patients were treated whose disease had already been treated with all mainstream medicine therapy protocols and as a result of relapse and/or progress of the disease no further mode of therapy was available for them and who had thus exhausted all modes of therapy’ (161). In these patients, Dr. Aschhoff achieved high remission rate using NSC 631570, for example, 50% for Ewing's sarcoma.

Renal cell carcinoma

NSC 631570 has been used for the treatment of renal cell cancer after the therapy with vinblastin had failed and the tumor had spread metastases. Full remission was achieved with Ukrain therapy and lasted 32 months at the time of publication (164).

 Testicular cancer

NSC 631570 has been used in the combined treatment of a nonseminoma patient and improved his immune parameters (98).

Esophagus carcinoma

The therapy of an esophagus carcinoma patient with NSC 631570 induced a prolonged remission (99).

Bladder cancer (urothelial cell carcinoma)

A case of a successful treatment of urothelial cell carcinoma was described. The authors reported on a sustained remission and improved quality of life of the patient (100).

Neuroblastoma

After chemotherapie had failed, the patient was treated with NSC 631570. The tumor markers decreased, the metastases responded well on the therapy and the quality of life of the patient improved (116).

 

Hereditary diseases

Tuberous sclerosis

As an example of the using NSC 631570 in hereditary diseases, a case of a girl with tuberous sclerosis can be considered. After successful treatment the patient is now an adult woman and has given birth to a healthy son (210).

Generalised lymphangiomatosis

Another publication reports on the successful treatment of a severe hereditary disease generalised lymphangiomatosis (211). The three year-old Stefan Dan with the diagnosis of generalised lymphangiomatosis was sent home by doctors in 1995 as having exhausted all forms of therapy. To “comfort” his parents they were told that Stefan would never be able to speak and walk in his life. After two years of UKRAIN therapy with a general practitioner the child was most certainly able to speak and also to walk. However, treatment with UKRAIN was stopped. The tumour then began to grow and brought about a compression of the spinal cord. Stefan – in the meantime eight years-old – underwent surgery. Despite the surgical intervention the condition of the young patient deteriorated continually, resulting in him having to be connected to a lung ventilator. Paraplegia appeared. In this condition the child was discharged to be cared for at home. He had to be given morphine four times a day to relieve his pain and his breathing had to be supported with a home respirator. The doctors then after all recommended his parents to turn to UKRAIN therapy again. Although the child’s condition could be improved with UKRAIN therapy, Stefan can speak, read, is very intelligent, but will no longer be able to walk.

Xeroderma pigmentosum

Xeroderma pigmentosum (XP) is a genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient. Multiple basal cell carcinomas (basaliomas) and other skin malignancies frequently occur at a young age in those with XP. In fact, metastatic malignant melanoma and squamous cell carcinoma are the two most common causes of death in XP victims.

This is a very rare disease. The incidence differs regionally and is between 1:40000 (Japan) and 1:250000 (USA). About 250 XP patients live in the USA, about 50 in Germany, mostly children. The life expectance is low; usually they die in the first decade. If left unchecked, damage caused by UV light can cause mutations in individual cells DNA. XP patients are at a high risk (more than 2000 times over the general population) for developing skin cancers, such as basal cell carcinoma, for this reason.
A report on the successful using NSC 631570 in a XP patient suggests this drug can be very useful also in this hereditary disease (212).

Ukrain (NSC-631570) bei Xeroderma pigmentosum

Patient S.S., an eight year old boy, was presented with an ulcering lesion of the nose. As he was 10 month old, xeroderma pigmentosum was diagnosed. Until the age of three years the number of skin lesions increased considerably. In May 2002 skin cancer (squamous cell carcinoma) at the nose was diagnosed, T4NXM0, histologically verified. From May till June 2002 three cycles of chemotherapy were administered (cyclophosphamide, vincristine, and vinblastine). The therapy failed and the tumors grew up. Clinical investigation in April 2004 revealed deforming malignant melanoma of the nose with invasion into the cartilage of nasal septum, measuring 3x3 cm. On 20 May 2004 the therapy with Ukrain was started, 5 mg intravenously twice a week, up to a total dose of 85 mg. One month after the last administration of Ukrain a complete regression of the tumor was revealed. The skin defect was partially replaced with connective tissue. Xeroderma skin lesions improved throughout the body.

Xeroderma pigmentosum

WIRKSAMKEIT: Bibliography

BIBLIOGRAPHY

  1. Nowicky JW. New Immuno-stimulating anti-cancer preparation ‘Ukrain’. 13th International Congress of Chemotherapy, Vienna, 28th August to 2nd September, 57, 1983, PS 12 5 33/A-6, part 288.
  2. Nowicky JW. Cancer Treatment Using Anticancer Preparation Alkaloid Derivative Ukrain. IV Mediterranean Congress of Chemotherapy, 19-25 October 1984, Rhodos, Greece, Chemioterapia, Supplement to n. 2, Volume 4, 1169, April 1985.
  3. Nowicky J, Greif M, Hamler F, Hiesmayr W, Staub W. Biological Activity of Ukrain in Vitro and in Vivo. V Mediterranean Congress of Chemotherapy. 26 October-1 November 1986 Cairo, Egypt, Chemioterapia, Supplement to n. 2, Volume 6, 683, June 1987.
  4. Nowicky JW, Greif M, Hamler F, Hiesmayr W, Staub W. Macroscopic UV-Marking through Affinity.Journal of Tumor Marker Oncology, Volume 3, Number 4, 463, 1988.
  5. Nowicky J, Hiesmayr W, Nowicky W. Sensitisation for Specific Lysis in Target-Effektor-System with Derivatives of Chelidonium majus Alkaloids - Ukrain.Extracted from the Proceedings of the 16th International Congress of Chemotherapy; 852. 1, June 1989, Israel.
  6. Nowicky JW. Biological and physiological effects of Ukrain. Journal of Cancer Research and Clinical Oncology, Supplement Vol. 116, A3. 112. 46, 1990.
  7. Slesak B, Nowicky JW, Harlozinska A. In vitro effect of thiophosphoric acid derivatives from Chelidonium majus L. on normal lymphocyte subpopulations.Journal of Cancer Research and Clinical Oncology, Supplement Vol. 116, A3. 112. 50,1990.
  8. Liepins A. Enhancement of cell mediated lysis of tumor cells by Chelidonium majus L. Alkaloids (Ukrain). Journal of Cancer Research and Clinical Oncology, Supplement Vol. 116, A3. 118. 10, 1990.
  9. Nowicky JW, Liepins A, Zbroja-Sontag W, Staniszewski A, Danilos J. Evaluation of clinical studies of Ukrain in cancer patients. Art:Bar.206, Zona:Barcel-fos, 29-Nov-90,09:37:310.
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  11. Nowicky JW, Liepins A, Slesak B, Staniszewski A, Harlozinska-Szmyrka A, Zbroja-Sontag W, Danilos J. Evaluation of clinical studies of Ukrain in cancer patients. Journal of Chemotherapy, Supplement n. 4, 522, 1991.
  12. Liepins A, Nowicky JW. Ukrain is Selectively Cytostatic and/or Cytotoxic to Human Tumor and HIV-Infected Cells but not to Human Normal Cells. Recent Advances in Chemotherapy, Anticancer Section, Proceedings of the 17th International Congress of Chemotherapy, Berlin, 2660, 1991.
  13. Vatanasapt V, Wongpratoom W, Mairiang P, Mairiang E, Chaiyakam C, Buddhisawasd V, Pairojkul C, Nowicky JW. Preliminary report on clinical experience in the use of Ukrain.Thai Cancer Journal, Volume 17 No. 1-2, 20, 1991.
  14. Nowicky JW, Liepins A, Staniszewski A, Slezak B, Nowicky W, Hiesmayr W. The malignotoxic and immune modulating property of the alkaloid derivative Ukrain. Journal of Cancer Research and Clinical Oncology, Suppl. Vol. 118, V1. 09. 05, 1992.
  15. Kleinrok Z, Jagiello-Wojtowicz E, Matuszek B, Chodkowska A. Basic Central Pharmacological Properties of Thiophosphoric acid alkaloid derivatives from Chelidonium Majus L. Pol. J. Pharmacol. Pharm., Vol. 44, 227, 1992.
  16. Remiszewska M, Wutkiewicz M, Jastrzebski Z, Czyzewska-Szafran H, Danysz A. Pharmacological Effects of Ukrain in Rats and Rabbits. Acta Poloniae Pharmaceutica-Drug Research, Vol. 49 no. 4, 43, 1992.
  17. Liepins A, Nowicky JW. Activation of Spleen Cell Lytic Activity by the Alkaloid Thiophophoric Acid Derivative: Ukrain.International Journal of Immunopharmacology, 14, 8, 1437-1442, 1992.
  18. Slesak B, Nowicky JW, Harlozinska A. In Vitro Effects of Chelidonium Majus L. Alkaloid Thiophosphoric Acid Conjugates (Ukrain) on the Phenotype of Normal Human Lymphocytes. Drugs Exptl. Clin. Res., XVIII, 17, 1992.
  19. Chlopkiewicz B, Marczewska J, Ejchart A, Anuszewska E, Koziorowska J. Evaluation of Mutagenic; Genotoxic and Transforming Properties of Ukrain. Drugs Exptl. Clin. Res., XVIII, 31, 1992.
  20. Kleinrok Z, Jagiello-Wojtowicz E, Nowicky JW, Chodkowska A, Feldo M. Interaction between Ukrain and Aminophenazone in Analgesic Test in Rodents. Drugs Exptl. Clin. Res., XVIII, 97, 1992.
  21. Musianowycz J, Judmajer F, Manfreda D, Spängler P, Albrecht H, Hoffmann J, Meijer D. Clinical Studies of Ukrain in Terminal Cancer Patients (Phase II). Drugs Exptl. Clin. Res., XVIII, 45, 1992.
  22. Danilos J, Zbroja-Sontag W, Baran E, Kurylcio L, Kondratowicz L, Jusiak L. Preliminary studies on the effect of Ukrain (Tris 2-5BS-(5BA,6B, 12BA)-5B,6,7,12B,13,14-hexahydro-13-methyl1,3 benzodioxolo 5,6-C-1-3-dioxolo4,5,-i phenanthri-dinium-6-ol]-ethaneaminyl phosphinesulfide.6HCl) on the immunological response in patients with malignant tumours. Drugs Exptl. Clin. Res., XVIII, 55, 1992.
  23. Wyczolkowska J, Czuwaj M, Maslinski C. The immunomodulating preparation Ukrain does not induce anaphylactic sensitization in mice and guinea pigs. Drugs Exptl. Clin. Res., XVIII, 35, 1992.
  24. Nowicky JW, Manolakis G, Meijer D, Vatanasapt V, Brzosko WJ. Ukrain both as an anticancer and immunoregulatory agent. Drugs Exptl. Clin. Res., XVIII, 51, 1992.
  25. Staniszewski A, Slesak B, Kolodziej J, Harlozinska-Szmyrka A, Nowicky JW. Lymphocyte subsets in patients with lung cancer treated with thiophosphoric acid alkaloid derivatives from Chelidonium majus L. (Ukrain). Drugs Exptl. Clin. Res., XVIII, 63, 1992.
  26. Sotomayor E, Rao K, Lopez DM, Liepins A. Enhancement of macrophage tumouricidal activity by the alkaloid derivative Ukrain. In vitro and in vivo studies. Drugs Exptl. Clin. Res., XVIII, 5, 1992.
  27. Pengsaa P, Wongpratoom W, Vatanasapt V, Udomthavornsuk B, Mairieng E, Tangvorapongchai V, Pesi M, Krusan S, Boonvisoot V, Nowicky JW. The effects of thiophosphoric acid (Ukrain) on cervical cancer, stage IB bulky. Drugs Exptl. Clin. Res., XVIII, 69, 1992.
  28. Lohninger A, Hamler F. Chelidonium majus L. (Ukrain) in the treatment of cancer patients. Drugs Exptl. Clin. Res., XVIII, 73, 1992.
  29. Kleinrok Z, Jagiello-Wojtowicz E, Nowicky JW, Chodkowska A, Feldo M, Matuszek B. Some pharmacological properties of prolonged administration of Ukrain in rodents. Drugs Exptl. Clin. Res., XVIII, 93, 1992.
  30. Juszkiewicz T, Minta M, Wlodarczyk B, Biernacki B. Teratological evaluation of Ukrain in hamsters and rats.Drugs Exptl. Clin. Res., XVIII, 23, 1992.
  31. Jagiello-Wojtowicz E, Kleinrok Z, Surmaczynska B, Baran E, Feldo M, Nowicky JW. Effect of single and three months treatment with Ukrain on amino-transferases (ALT and AST) and on the serum protein level in rodents. Drugs Exptl. Clin. Res., XVIII, 85, 1992.
  32. Jagiello-Wojtowicz E, Kleinrok Z, Nowicky JW, Matuszek B, Baran E, Surmaczynska B. Effect of single and prolonged administration of Ukrain on prolactin concentration in rats. Drugs Exptl. Clin. Res., XVIII, 89, 1992.
  33. Jagiello-Wojtowicz E, Kleinrok Z, Matuszek B, Surmaczynska B, Baran E, Nowicky W, Nowicky JW. Effect of three months treatment with Ukrain on peripheral blood morphology in rodents. Drugs Exptl. Clin. Res., XVIII, 79, 1992.
  34. Jagiello-Wojtowicz E, Kleinrok Z, Feldo M, Chodkowska A, Nowicky JW. Effect of Ukrain on the efficacy of anti-epileptic drugs against maximal electroshock-induced seizures in mice. Drugs Exptl. Clin. Res., XVIII, 107, 1992.
  35. Jagiello-Wojtowicz E, Kleinrok Z, Chodkowska A, Feldo M, Nowicky JW. Modification of antinociceptive action of morphine by Ukrain in rodents. Drugs Exptl. Clin. Res., XVIII, 101, 1992.
  36. Hohenwarter O, Strutzenberger K, Katinger H, Liepins A, Nowicky JW.Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain. Drugs Exptl. Clin. Res., XVIII, 1, 1992.
  37. Danysz A, Kokoschinegg M, Hamler F. Clinical studies of Ukrain in healthy volunteers (phase I). Drugs Exptl. Clin. Res., XVIII, 39, 1992.
  38. Brüller W. Studies concerning the effect of Ukrain in vivo and in vitro. Drugs Exptl. Clin. Res., XVIII, 13, 1992.
  39. Nowicky JW. Biological Properties of Ukrain in Experimental and Clinical Investigations.International Medical Reviews, Avalanche Ltd. St. Petersburg, Russia, 1, 1, 5, 1993.
  40. Nowicky JW, Nowicky W, Liepins A. Cytostatic and cytotoxic effects of Ukrain on malignant cells. VIII Mediterranean Congress of Chemotherapy, 24-29 May 1992, Athens, Greece, Journal of Chemotherapy, Supplement n. 1, Volume 5, 797, 1993.
  41. Nowicky JW. Ukrain Antineoplastic Immunomostimulant. E09/029, NSC-631570, NSC-B238865, UKSR-222, W122, Drugs of the Future, Prous Science Publishers, Copyright Prous Science, 18 (11), 1015, November 1993.
  42. Kamyshentsev MV, Voltchek IV, Btailovskaya IV, Leschev AL, Lavinsky YC and Nowicky JW. Testing Ukrain as an Anti-Influenza Remedy. Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 645, 1993.
  43. Liepins A, Sotomayor EM, Lopez DM, JW Nowicky. Biological Response-Modifying Properties of the Alkaloid Derivative Ukrain (NSC 631570). Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 783, 1993.
  44. Nowicky JW, Manolakis G, Meijer D, Vatanasapt V, Brzosko WJ, Lohninger A. Immunological and Tumoricidal Properties of Ukrain. Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 793, 1993.
  45. Lohninger A, Musianowyc J, Judmaier F, Manfreda D, Spängler P, Albrecht H, Hoffmann J, Mejer D. Results of Phase II Clinical Studies with Ukrain. Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 794, 1993.
  46. Voltchek IV, Nowicky JW, Zolotukhin NN, Kamyshentsev MV, Miroshnichenko AG, Leschev AL, Belskikh AN. Some Immunohematological Effects of Ukrain. Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 798, 1993.
  47. Nowicky JW, Markowska J, Brzosko WJ. Ukrain and Natural Killer Cells. Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 863, 1993.
  48. Zemskov VS, Yaremchuk OYa, Susak YaM, Deneka ER, Kravchenco OV, Kamenets LYa. Experience of the Application of Ukrain in Oncological Practice in Ukraine. Recent Advances in Chemotherapy, American Society for Microbiology, Proceedings of the 18th International Congress of Chemotherapy, Stockholm, Sweden, June 27-July 2, 870, 1993.
  49. Nowicky JW, Cisak E, Liepins A, Susak JaM, Zemskow W. Stimulation of phagocytic activity in vitro, in vivo and in the clinic by Ukrain. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 68, April 1994.
  50. Kurik MV, Susak YM, Kravchenko OV. Some biophysical properties of Ukrain. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 79, April 1994.
  51. Lisnyak OI, Lozjuk RM. Biological activity of some thiophosphamide derivatives of alkaloids with respect to influenza virus. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 96, April 1994.
  52. Lozjuk RM, Lisnyak OI, Lozjuk LV. Theoretical grounds and experimental confirmation of antiviral effect of the preparation Ukrain. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 95, April 1994.
  53. Brzosko WJ, Graczyk A, Konarski J, Nowicky JW. Synergic influence of Ukrain and protoporphyrine amino conjugates on human malignant cell lines. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 110, April 1994.
  54. Brzosko WJ, Uglianica K, Fomin K, Nowicky JW. Influence of Ukrain on breast cancers. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abs. 109, April 1994.
  55. Zemskov VS, Susak YaM, Zemskov SV. Ukrain monotheraphy for treatment of colorectal cancer. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abs. 78, April 1994.
  56. Liepins A., Nowicky J.W. Selective induction of programmed cell death (apoptosis) in malignant cells by the alkaloid derivative Ukrain (NSC-613570). 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 93, April 1994.
  57. Jagiello-Wojtowicz E., Kleinrok Z., Nowicky J., Hodkowska A., Feldo M., Matuszek B., Jablonska M., Gorzelak M. Effect of six month treatment with Ukrain on early osteoporosis induced by ovariectomy in rats. 11th Future Trends in Chemotherapy, Interdisciplinary World Congress on Antimicrobial and Anticancer Drugs, 24-27 April 1994, Palexpo Geneva (Switzerland), Abstracts: abs. 49, April 1994.
  58. Nowicky J.W., Wladyslawa Nowicky, Hiesmayr W., Potopalsky A.. Alterations of DNA; RNA and protein synthesis in malignant cells under the influence of Ukrain. XVI International Cancer Congress 1994, 30 October - 5 November 1994, New Delhi, India, abs. PSB 15 -17, 319.
  59. Voltchek I. Ukrain - Drug of the Future in the Cancer Treatment? Terra Medica, Nr. 1, 1995, 24 - 25 (in Russian).
  60. Ciebiada I., Korczak E., Denys A., Nowicky J. W. Effect of Ukrain preparation on immune response in mice affected by influenza virus. Journal of Chemotherapy, Vol. 7 (Suppl.), n. 4, 1995 101-104.
  61. Voltchek I., Kamyshentsev M., Lavinsky Y., Nowicky J., Medvedev Y., Litvinchuk L. Comparative Study of the Cytostatic Effects of Oliphen and Ukrain. Journal of Chemotherapy, Vol. 8 - n. 2, 1996, 144-146.
  62. Liepins A., Nowicky J.W., Bustamante J.O., Lam E. Induction of Bimodal Programmed Cell Death in Malignant Cells by the Derivative Ukrain (NSC-631570).Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 1 - 7.
  63. Nowicky JW, Hiesmayr W, Nowicky W, Liepins A. Influence of Ukrain on DNA, RNA and Protein Synthesis in Malignant Cells. Drugs Exptl. Clin. Res., XXII (Suppl.), 1996, 9-19.
  64. Nowicky J.W., Hiesmayr W., Influence of Ukrain on Human Xenografts in vitro.Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 21 - 25.
  65. Jin Y.M., Nowicky J.W., Liepins A. Mitogenic Properties of Ukrain (NSC-631570) on Human Peripheral Blood Monocytes: Clinical Implications. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 27 - 30.
  66. Liepins A., Nowicky J.W. Modulation of Immune Effector Cell Cytolytic Activity and Tumour Growth Inhibition in vivo by Ukrain (NSC-631570). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 31 - 42.
  67. Susak Y.M., Zemskov V.S., Yaremchuk O.Y., Kravchenko O.B., Yatsyk I.M., Korsh O.B. Comparison of Chemotherapy and X-ray Therapy with Ukrain Monotherapy for Colorectal Cancer. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 43 - 50.
  68. Uglanica K.N., Fomin K.A., Nefyodov L.I., Nowicky J.W., Brzosko W.J., Jankowski A. Influence of Ukrain on Patients with Surgically Treated Breast Cancer (Introductory Remarks). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 51 - 54.
  69. Brzosko W.J, Uglanica K.N., Fomin K.A., Nowicky J.W. Influence of Ukrain on Breast Cancer. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 55 - 62.
  70. Uglanica K.N., Fomin K.A., Nefyodov L.I., Nowicky J.W., Brzosko W.J., Jankowski A. Influence of Ukrain on Patients with Surgically Treated Breast Cancer. Part I. Clinical and Laboratory Parameters. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 63 - 66.
  71. Uglanica K.N., Fomin K.A., Nefyodov L.I., Vilkiewich T.W., Nowicky J.W., Brzosko W.J., Jankowski A. Influence of Ukrain on Patients with Surgically Treated Breast Cancer. Part II. Hormonal Profile. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 67 - 70.
  72. Uglanica K.N., Fomin K.A., Nefyodov L.I., Djurd T.I., Nowicky J.W., Brzosko W.J., Jankowski A. Influence of Ukrain on Patients with Surgically Treated Breast Cancer. Part III. The Immune System. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 71 -74.
  73. Uglanica K.N., Maciuk J.R., Fomin K.A., Nefyodov L.I., Kravchuk R.I., Vinogradova L. M., Nowicky J.W., Brzosko W.J. Influence of Ukrain on Patients with Surgically Treated Breast Cancer. Part IV. Electromicroscopic and Cytochemical Evaluation. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 75 - 82.
  74. Nefyodov L.I., Uglanica K.N., Smirnov V.Y., Doroshenko Y.M., Fomin K.A., Nowicky J.W., Brzosko W.J. Amino Acids and Their Derivatives in Blood Plasma of Patients with Breast Cancer Treated with Ukrain. Part V. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 83 - 86.
  75. Nefyodov L.I., Uglanica K.N., Smirnov V.Y., Doroshenko Y.M., Fomin K.A., Nowicky J.W., Brzosko W.J. Amino Acids and Their Derivatives in Tumour Tissue from Patients with Breast Cancer Treated with Ukrain. Part VI. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 87 - 90.
  76. Nowicky J.W., Hiesmayr W., Liepins A. Influence of Ukrain on Immunological Blood Parameters in vitro and in vivo. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 91 - 94.
  77. Boyko V.N., Voltchek I.V., Petrov A.S., Bubnov V.P. Action of Ukrain, a Cytostatic and Immunomodulating Drug, on Effects of Irradiation. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 95 - 100.
  78. Jagiello-Wojtowicz E., Kleinrok Z., Nowicky J.W., Jablonski M., Gorzelak M., Chodkowska A., Feldo M., Matuszek B. Effect of Six-Month Treatment with Ukrain on Early Osteoporosis Induced by Ovariectomy in Rats. Part I: Preliminary Studies of Bone Parameters. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 101- 104.
  79. Jagiello-Wojtowicz E., Kleinrok Z., Nowicky J.W., Chodkowska A., Feldo M., Surmaczynska B., Gorzelak M., Jablonski M. Effect of Six-Month Treatment with Ukrain on Early Osteoporosis Induced by Ovariectomy in Rats. Part II: Preliminary Studies of Peripheral Blood Parameters. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 105-108.
  80. Jagiello-Wojtowicz E., Kleinrok Z., Nowicky J.W., Baran E. Effect of Six-Month Treatment with Ukrain on Early Osteoporosis Induced by Ovariectomy in Rats. Part III: Preliminary Studies of Some Hormone Levels. Drugs Exptl. Clin. Res., XXII (Suppl.), 1996, 109-113.
  81. Susak Y.M., Kurik M.V., Kravchenko O.V., Zemskov S.V. Certain Biophysical Properties of Ukrain. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 113 - 116.
  82. Zhalilo LI, Susak YM, Zemskov SV, Susak IA. Influence of Ukrain on the Redox Processes of Hepatocytes. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 117-120.
  83. Brzosko W.J., Graczyk A., Konarski J., Nowicky J.W. Synergic Influence of Ukrain and protoporphyrin Amino Acids Conjugates on Human Malignant Cell Lines. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 121 - 122.
  84. Wyczólkowska J., Michon T., Nowicky J.W. Inhibitory Effect of Thiophosphoric Acid Alkaloid Derivatives from Chelidonium majus L. (Ukrain) on Ovalbumin Antigenicity and Antiovalbumin IgE Antibody Response in Mice. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 123 - 128.
  85. Jagiello-Wojtowicz E., Kleinrok Z., Chodkowska A., Nowicky J.W., Piper H., Kubiatowski T. Antinociceptive Effect of Ten Day Administration of Ukrain in Mice and Interaction of the Treatment with Morphine. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 129 - 132.
  86. Jagiello-Wojtowicz E., Kleinrok Z., Nowicky J.W., Chodkowska A., Kubiatowski T, Piper H. Interaction Between Ukrain and Morphine in Their Ten-Day Treatment in Mice in the Writhing Syndrome Test. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 133 - 134.
  87. Ciebiada I, Korczak E., Nowicky JW, Denys A. Does the Ukrain Preparation Protect Mice Against Lethal Doses of Bacteria? Drugs Exptl Clin Res, XXII (Suppl), 1996, 135-140.
  88. Lozjuk R.M., Lisnyak O.I., Lozjuk L.V. Theoretical Grounds and Experimental Confirmation of the Antiviral Effect of the Preparation Ukrain. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 141 - 146.
  89. Ciebiada I., Korczak E., Nowicky J.W., Denys A. Estimation of Direct Influence of Ukrain Preparation on Influenza Viruses and the Bacteria E. coli and S. aureus. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 147 - 152.
  90. Lisnyak OI, Lozjuk RM. Biological Activity of Some Thiophosphamide Derivatives of Alkaloids with Respect to Influenza Virus. Drugs Exptl. Clin. Res., XXII (Suppl.), 1996, 153-156.
  91. Stabuc B., Benedicic D. Ukrain with Chemotherapy in Malignant Melanoma (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 159-162.
  92. Hamler F., Hiesmayr W., Korsh O.B., Melnyk A. Ukrain Monotherapy in Malignant Melanoma(Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 163 - 166.
  93. Kotsay B., Lisnyak O., Myndiuk O., Romanyshyn J., Fabri O. Ukrain Treatment of Rhabdomyosarcoma (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 167 - 170.
  94. Kadan P., Korsh OB, Melnyk A. Ukrain Therapy of Recurrent Breast Cancer with Lung Metastases (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 171-174.
  95. Nowicky J.W., Schramm E., Godysh Y. Biophysical Effects of Ukrain Therapy in a Patient with Breast Cancer (Case Report). Drugs Exptl Clin Res., Vol XXII (Suppl), 1996, 175-182.
  96. Kroiss T., Melnyk A., Korsh O.B. Ukrain Treatment in Carcinoma of the Cervix (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 183 - 186.
  97. Lohninger A, Korsh OB, Melnyk A. Combined Therapy with Ukrain and Chemotherapy in Ovarian Cancer (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 187 - 190.
  98. Sakalo V.S., Korsh O.B., Melnyk A. Ukrain Treatment in a Patient with Non-Seminomatous Germ-Cell Tumour of Testis (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 191 - 194.
  99. Vyas J.J., Jain V.K. Ukrain Treatment in Carcinoma of the Oesophagus (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 195 - 198.
  100. Kadan P., Korsh O.B., Hiesmayr W. Ukrain in the Treatment of Urethral Recurrent Carcinoma (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 199 - 202.
  101. Steinacker J., Kroiss T., Korsh O.B., Melnyk A. Ukrain Treatment in a Frontal Anaplastic Grade III Astrocytoma (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 203-206.
  102. Steinacker J., Korsh O.B., Melnyk A. Ukrain Therapy of a Recurrent Astrocytoma of the Optic Nerve (Case Report). Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 207 - 210.
  103. Voltchek I.V., Liepins A., Nowicky J.W., Brzosko W.J. Potential Therapeutic Efficacy of Ukrain (NSC 631570) in AIDS Patients with Caposi’s Sarcoma. Drugs Exptl. Clin. Res., Vol. XXII (Suppl.), 1996, 211 - 214.
  104. Thakur M.L., De Fulvio J., Tong. J., John E., McDevitt M.R. and Damjanov I. Evaluation of biological response modifiers in the enhancement of tumor uptake of technetium-99m labeled macromolecules. Journal of Immunological Methods, 152 (1992), 209 – 216.
  105. Jagiello-Wojtowicz E., Kleinrok Z., Chodkowska A., Misztal G., Jagiello G. Preliminary pharmacokinetic studies of Ukrain in rats.
  106. Zemskov V.S., Yaremchuk O.Y., Susak Y.M., Deneka E.R., Kravchenko O.V., Yatsyk I.M. The first experience in the using of the Ukrain preparation in the treatment of oncological patients in Ukraine. Likarska Sprava, 1-2, (1024), 1996, 158 – 161.
  107. Zemskov S.V., Susak Y.M., Todor I.N., Khasanova L.T., Mosienko V.S. Antimetastatic effect of Ukrain and its influence on the oxygen and energy metabolism of mice with melanoma B-16. Experimental Oncology, Vol. 18 (1996), 4, 405 – 408.
  108. Zemskov V.S., Yaremchuk O.Y., Susak Y.M., Kravchenko O.V., Yatsyk I.M., Voltchek I.V. Ukrain – noviy effectivniy preparat dla lecheniya raka tolstoy i pryamoy kishki. In: Actualniye Voprosy Oncologii, St. Petersburg, 1996, 175 – 177 (in Russian).
  109. Nefyodov L.I., Uglanica K.N., Smirnov V.Y., Doroshenko Y.M., Fomin K.A., Nowicky J.W., Brzosko W.J. Swobodnie aminokisloty i ich proisvodnie v plasme krovi i opuholevoy tkani bolnih rakom molochnoy zhelezi na fone lecheniya novym protivoopuholevim preparatom Ukrain. In: Actualniye Voprosy Oncologii, St. Petersburg, 1996, 212 – 214 (in Russian).
  110. Uglanica K.N., Fomin K.A., Nefyodov L.I., Nowicky J.W., Brzosko W.J. Electronnomicroscopicheskiy i morphohistohimicheskiy analis vosdeystvia Ukrain na opuholevuyu tkan molochnoy zhelezy. In: Actualniye Voprosy Oncologii, St. Petersburg, 1996, 175 – 177 (in Russian).
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  112. Bondar G.V., Borota A.V., Yakovets Y.I., Zolotukhin S.E. Comparative evaluation of the complex treatment of rectal cancer patients (chemotherapy and X-ray therapy, Ukrain monotherapy). Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 221-226.
  113. Uglianitsa K.N., Nechiporenko N.A., Nefyodov L.I., Brzosko W.J. Ukrain therapy of stage T1N0M0 bladder cancer patients.Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 227-230.
  114. Uglianitsa K.N., Nefyodov L.I., Brzosko W. Evaluation of the efficacy of Ukrain in the treatment of breast cancer: Clinical and laboratory studies. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 231-239.
  115. Aschhoff B. Ukrain and hyperthermia treatment in a patient with Ewing's sarcoma (case report). Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 241-242.
  116. Aschhoff B. Ukrain treatment in a patient with stage IVneuroblastoma. A case report. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 243-245.
  117. Todor I.N., Kazmin S.D., Susak Ya.M., Ztmskov S.V. The influence of glucose, succinate, pH of the medium and higher temperature on the cytotoxic activity of the preparation Ukrain. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 247-252.
  118. Korolenko T.A, Svechnikova I.G, Filjushina E.E, Kaledin V.I., Vakulin G.M., Usynin I.F., Tsyrendordjiev D.D. Macrophage stimulation and antitumor effect of Ukrain. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 253-260.
  119. Svechnikova I.G., Korolenko T.A., Stashko J.F., Kaledin V.I., Nikolin V.P., Nowicky J.W. The influence of Ukrain on the growth of HA-1 tumor in mice: The role of cysteine proteinases as markers of tumor malignancy. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 261-269.
  120. Korolenko T.A., Kaledin V.I., Svechnikova I.G., Li X.V., Stashko J.F., Ilnitskaya S.I., Nikolin V.P. Study of the antitumor effect of ukrain: The role of macrophage secretion of α-1-proteinase inhibitor. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 271-276.
  121. Kulik G.I. Comparative in vitro study of the effects of the new antitumor drug Ukrain and several cytostatic agents on the thiol groups in the tissue of Guerin carcinoma and its resistance to cisplatin variant. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 277-280.
  122. Deneka E.R. Morphometric and kinetic analysis of the growth of experimental sarcoma-45 in the presence of Ukrain. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 281-285.
  123. Kulik GI, Deneka ER, Todor IN, Karmozina LG. Study of acute toxicity of Ukrain in rats after intravenous injection. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 287-293.
  124. Jagiello-Wojtowicz E., Kleinrok Z., Feldo M., Chodkowska A., Szponar J., Urbanska E.M. Six-week treatment with Ukrain in rabbits. Part 1: Morphological parameters. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 295-299.
  125. Jagiello-Wojtowicz E., Kleinrok Z., Feldo M., Chodkowska A., Szklarczyk V., Urbanska E.M. Six-week treatment with Ukrain in rabbits. Part II: Serum levels of gonadal hormones. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 301-304.
  126. Jagiello-Wojtowicz E., Kleinrok Z. Feldo M., Chodkowska A., Szklarczyk V., Urbanska E.M. Six-week treatment with Ukrain in rabbits. Part III: Serum levels of thyroid hormones. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 305-308.
  127. Jagiello-Wojtowicz E, Kleinrok Z, Chodkowska A, Misztal G., Jagiello G. Preliminary pharmacokinetic studies of Ukrain in rats. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 309-311.
  128. Gorzelak M., Jablonski M., Patyra M., Jagiello-Wojtowicz E. Effect of intermittent three month treatment with different doses of Ukrain on subregional femoral bone mineral density of sexually mature female rats. Drugs Exptl Clin Res, XXIV (5/6), 1998, 313-316.
  129. Jablonski M., Gorzelak M., Patyra M., Jagiello-Wojtowicz E. Effect of intermittent three-month treatment with different doses of Ukrain on subregional bone mineral density of the femur of ovariectomized rats. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 317-320.
  130. Jagiello-Wojtowicz E, Kleinrok Z, Chodkowska A, Szkodziak A, Siembida E, Gustaw K, Urbanska E. Modification of antinociceptive action of Ukrain by endogenous nitric oxide in the writhing syndrome test in mice. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 321-325.
  131. Jagiello-Wojtowicz E., Kleinrok Z., Gustaw K. Interaction between Ukrain and Naltrexone in the writhing syndrome test in mice. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 327-330.
  132. Boyko V.N., Zholus R.B. A comparative evaluation of the influence of the complex drug Ukrain and its components on the effects of radiation. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 331-333.
  133. Boyko V.N., Belskiy S.N. The influence of the novel drug Ukrain on hemo-and immunopoiesis at the time of its maximum radioprotective effect. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 335-337.
  134. Boyko V.N., Levshina Ye.V. A study of the influence of a novel drug Ukrain on in vivo effects of low-dose ionizing radiation. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 339-341.
  135. Boyko V.N., Zholus R.B., Legeza V.I. A study of the influence of different types of radioprotectors on the survival of mice treated with ionizing radiation over a wide dose range. Drugs Exptl. Clin. Res., XXIV (5/6), 1998, 343-347.
  136. Koshelnick Y., Moskvina E., Binder B.R., Nowicky J.W. Ukrain (NSC-631570) inhibits angiogenic differentiation of human endothelial cells in vitro. 17th International Cancer Congress, Rio de Janeiro, August 24-28, 1998, Monduzzi Editore, 91-95.
  137. Uglyanitsa K.N., Nefyodov L.I., Nowicky J.W., Brzosko W.J. The effect of Ukrain on cancer of the urinary bladder.17th International Cancer Congress, Rio de Janeiro, August 24-28, 1998, Monduzzi Editore, 1065-1068.
  138. Voltchek I., Sologub T., Gontcharova L., Pokrovskaya L., Belozyorova L., Lamanova E. Choice of Ukrain and interferon-alpha doses for the cancer patients therapy.
  139. Panzer A, Seegers JC. Ukrain, a semisynthetic alkaloid of Chelidonium majus, is selectively toxic to malignant cells by causing a methapase block which results in apoptosis. Proceedings of the American Association for Cancer Research, vol. 39, March 1998, New Orleans, LA, USA.
  140. Panzer A., Joubert AM, Bianchi PC, Seegers JC. The antimitotic effects of Ukrain™, a Chelidonium majus alkaloid derivative, are reversible in vitro. Cancer Letters 150 (2000), 85-92.
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  145. Panzer A, Joubert AM, Eloff JN, Albrecht CF, Erasmus E, Seegers JC. Chemical analyses of ukrain, a semi-synthetic Chelidonium majus alkaloid derivative, fail to confirm its trimeric structure. Cancer Lett. 2000 Nov 28;160(2):237-41.
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  147. Roublevskaia IN, Polevoda BV, Ludlow JW, Haake AR. Induced G2/M arrest and apoptosis in human epidermoid carcinoma cell lines by semisynthetic drug Ukrain. Anticancer Res. 2000 Sep-Oct;20(5A):3163-7.
  148. Kazmin SD, Todor IN. The effect of antitumor drug Ukrain on malignant proliferating cells being at different stages of cell cycle. Intl J Med Biol Environ 28(1), 57-64 (2000).
  149. Roublevskaia I.N., Haake A.R., Ludlow J.W., Polevoda B.V. Induced apoptosis in human prostate cancer cell line LNCaP by Ukrain. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 141-147.
  150. Roublevskaia I.N., Haake A.R., Polevoda B.V. BCL-2 overexpression protects human keratinocyte cells from Ukrain induced apoptosis but not from G2/M arrest Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 149-155.
  151. Hruby R. Ukrain: Acute toxicity after intravenous, intramuscular and oral administration in rats.Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 157-161.
  152. Doroshenko Y.M., Hodysh Y.Y., Uglyanitsa K.N., Nefyodov L.I. A method for determination of Ukrain in blood plasma for monitoring and pharmacokinetic study.Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 163-170.
  153. Doroshenko Y.M., Karavay A.V., Hodysh Y.Y., Uglyanitsa K.N., Nowicky W.M., Nefyodov L.I. The dynamics of concentration of the main fluorescent component of Ukrain in the tissues and blood plasma of rats with W-256 tumor after a single intravenous injection. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 171-177.
  154. Zemskov V.S., Procopchuk O.L., Susak Y.M., 1 Zemskov S.V., Hodysh Y.Y., Zemskova M.V. Ukrain (NSC 631570) in the treatment of pancreas cancer. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 179-190.
  155. Uglyanitsa K.N., Nechiporenko N.A., Nefyodov L.I., Doroshenko Y.M., Brzosko W., Nowicky W. Results of Ukrain monotherapy of prostate cancer. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 191-193.
  156. Nefyodov L.I., Uglyanitsa K.N., Nechiporenko N.A., Smirnov V.Y., Brzosko W., Karavay N.L. New biochemical mechanisms of the anticancer effect of Ukrain in the treatment of cancer of the urinary bladder. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 195-199.
  157. Uglyanitsa K.N., Nefyodov L.I., Doroshenko Y.M., Brzosko W.J. Comparison of the efficacy of different doses of Ukrain in the combined treatment of breast cancer. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 201-221.
  158. Uglyanitsa K.N., Nefyodov L.I., Brzosko V. Comparative evaluation of the efficiency of various Ukrain doses in the combined treatment of breast cancer Report 1 Clinical aspects of Ukrain application. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 223-230.
  159. Nefyodov L.I., Uglyanitsa K.N., Smirnov V.Y., Karavay A.V., Brzosko W. Comparative evaluation of blood plasma and tumor tissue amino acid pool in radiation or neoadjuvant preoperative therapies of breast cancer with the antitumor drug Ukrain. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 231-237.
  160. Uglyanitsa K.N., Nefyodov L.I., Karayedova L.M., Nowicky J.W., Brzosko W. Clinical aspects of cancer treatment and new biochemical mechanisms of the drug Ukrain. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 239-247.
  161. Aschhoff B. Retrospective study of Ukrain treatment in 203 patients with advanced-stage tumors. Drugs Exptl. Clin. Res., XXVI(2000), 249-252.
  162. Zemskov S.V., Prokopchuk O.L., Susak Y.M. Ukrain treatment in a patient with breast carcinoma. Case report. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 253-254.
  163. Prokopchuk O.L., Zemskov S.V., Susak Y.M. Ukrain treatment of a patient with retroperitoneal synovial sarcoma. Case report. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 255-256.
  164. Prokopchuk O.L., Zemskov S.V., Susak Y.M. Ukrain treatment in a patient with metastatic renal cell carcinoma extending to the vena cava inferior. Case report. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 257-259.
  165. Voltchek I., Sologub T., Nowicky J.W., Grigoryeva T., Belozyorova L., Belopolskaya M., Semenyako N., Lamanova E. Preliminary results of individual therapy of chronic hepatitis C by Ukrain and interferon-a. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 261-266.
  166. Votrin I.I., Voltchek I.V., Kurochkin S.N. Kolobkov S.L. Effects of Ukrain on the activities of DNA-nicking enzymes. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 267-273.
  167. Kurochkin S.N., Kolobkov S.L., Votrin I.I., Voltchek I.V. Induction of apoptosis in cultured Chinese hamster ovary cells by Ukrain and its synergistic action with etoposide. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 275-278.
  168. Korolenko T.A., Djanayeva S.J., Falameyeva O.V., Wevers R.A., Filjushina E.E., Buzueva I.I., Kaledin V.I., Sandula J., Nowicky J. Chitotriosidase as a new marker of macrophage stimulation in a tumor model treated with cyclophosphamide and Ukrain. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 279-283.
  169. Korolenko T.A., Poteryaeva O.N., Djanayeva S.J., Svechnikova I.G., Kaledin V.I., Timofeyeva O.A., Filipenko M.L., Nowicky J.  Cystatin C in LS lymphosarcoma and HA-1 hepatoma treated with Ukrain and cyclophosphamide and involvement of apoptosis. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 285-292.
  170. Djanayeva S.J., Korolenko T.A., Svechnikova I.G., Falameyeva O.V., Korolenko E., Kaledin V.I., Nowicky J. Influence of Ukrain and cyclophosphamide administration on HA-1 murine hepatoma and LS lymphoma on aspartic proteinase cathepsin D. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 293-299.
  171. Poteryaeva O.N., Falameyeva O.V., Korolenko T.A., Kaledin V.I., Djanayeva S.J., Nowicky J.W., Sandula J. Cysteine proteinase inhibitor level in tumor and normal tissues in control and cured mice. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 301-306.
  172. Luksa-Lichtenthaler G.L., Ladutko E.I., Nowicky J.W. Influence of Ukrain on the nuclear thyroid hormone receptors after short-term g-irradiation. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 307-310.
  173. Luksa-Lichtenthaler G.L., Ladutko E.I., Nowicky J.W. Radiomodification effects of Ukrain, a cytostatic and immunomodulatmg drug, on intracellular glucocorticoid reception during short-term g-irradiation. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 311-315.
  174. Jablonski M. Ukrain (NSC 631570) influences on bone status: A review. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 317-320.
  175. Jablonski M., Gorzelak M., Patyra M., Jagiello-Wojtowicz E. Intermittent three-month treatment with Ukrain in intact and ovariectomized rats. Part I: Effect on selected biomechanical parameters of the femur. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 321-325.
  176. Jablonski M., Gorzelak M., Patyra M., Jagiello Wojtowicz E. Intermittent three-month treatment with Ukrain in intact and ovariectomized rats. Part II: Effect on bone mineral density of the femur. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 327-331.
  177. Jablonski M., Korczak W., Gorzelak M., Jagiello-Wojtowicz E. Intermittent three-month treatment with Ukrain in intact and ovariectomized rats. Part III: Effect on the native electron paramagnetic resonance signal intensity of the femur. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 333-336.
  178. Jagiello-Wojtowicz E., Dudka J., Dawidek-Pietryka K. Effect of Ukrain on human liver alcohol dehydrogenase activity in vitro. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 337-339.
  179. Uglyanitsa K.N., Nefyodov L.I., Doroshenko Y.M., Nowicky J.W., Volchek I.V., Brzosko W.J., Hodysh Y.J. Ukrain: A novel antitumor drug. Drugs Exptl. Clin. Res., XXVI (5/6), 2000, 341-356.
  180. Grinevich Yu.A., Bendyug G.D., Khranovska N.M., Martinenko S.V., Kadkalenko G.G., Novicky J.W. Investigation of immunotropic activity of Ukrain in experiments.Imunolohiya ta Alerholohiya, 1, 2001, 22-26 (in Ukrainian).
  181. Gansauge F, Ramadani M, Gansauge S, Pressmar J, Muehling B, Strecker K, Cammerer G, Leder G, Beger HG. Pancreatic Cancer and chemotherapy. New developments from bench to bedside. Progress in Pancreatology and GI-Tract Surgery. Gastrointestinal Surgery Today – the Ulm Surgical School, September 2001.
  182. Gansauge F, Ramadani M, Pressmar J, Gansauge S, Muehling B, Stecker B, Cammerer G, Leder G, Beger HG. NSC-631570 (Ukrain) in the palliative treatment  of pancreatic cancer. Results of a phase II trial. Langenbeck’s Archives of Surgery (2002), 386:570-574.
  183. Andrievski OM, Smalukh NV, Nowicky WM, Krivitski AK. Vyvchennia vplyvu preparatu “Ukrain” na proyav actyvnosti trypsynopodibnymy proteasami in vitro.Odeskyi Medychnyi Journal, 1 (69), 2002:6-9.
  184. Cordes N, Plasswilm L, Bamber M, Rodemann HP. Ukrain®, an alkaloid thiophosphoric acid derivative of Chelidonium majus L. protects human fibroblasts but not human tumour cells in vitro against ionizing radiation. Int J Radiat Biol 2002, vol. 78, No. 1, 17-27.
  185. Zemskov V, Prokopchuk O, Susak Y, Zemskov S, Tkachenko O, Hodysh Y, Nowicky W. Efficacy of Ukrain in the treatment of pancreatic cancer. Langenbeck’s Archives of Surgery (2002) 387:84-89.
  186. Gansauge F., Ramadani M., Beger H.G. Ukrain beim fortgeschrittenen Pankreaskarzinom. Ars Medici 2002, 22, 1056-1062.
  187. Gansauge F. Ukrain in Pancreatic cancer: Study Final Report. University Ulm, 2002.
  188. Aschhoff B. Using UKRAIN in cancer patients: a clinical experience. www.villamedica.de.
  189. European Organization for Research and Treatment of Cancer. New Drug Development Office. Ukrain (E90/029, W122, UKSR-222, NSC B238865): Screening results – human tumor xenografts in vitro.
  190. National Cancer Institute, National Institute of Health: Developmental Therapeutics Program. NSC 631570: results of the Human Cell Line Screen. Available at www.dtp.nci.nih.gov.
  191. Aschhoff B. Tumor-Arten. www.villamedica.de.
  192. Izdebska M, Jagiello Wojtowicz E. Protective effects of thiophosphoric acid alkaloid from Chelidonium majus L. in rats intoxicated by methanol. Pol J Eur, 2003, submitted.
  193. Jagiello-Wojtowicz E, Macijewska-Kozak H, Chodkowska A, Radej R. The influence of chelidonine and Ukrain on the β2-microglobulin concentration in the serum of lead-poisoned rats. Herba Polonica, 2003.
  194. Wielosz-Tokarzewska E, Jagiello-Wojtowicz E. The effect of Ukrain on the serum VIP level in diabetic mice. International Journal of Immunotherapy, XIX (2-4), 2003, 189-191.
  195. Jagiello-Wojtowicz E, Kleinrok A, Jurek D. Effects of Ukrain in acute intoxication with streptozotocin in rats.International Journal of Immunotherapy, XIX (2-4), 2003, 193-196.
  196. Jagiello-Wojtowicz E, Izdebska M, Piatkowska-Chmiel I. The effect of Ukrain on selected biochemical parameters in the serum of methanol intoxicated rats.International Journal of Immunotherapy, XIX (2-4), 2003, 197-200.
  197. Yagodina OV, Nikolskaya EB, Faddejeva MD. Inhibition of liver mitochondrial monoamine oxidase activity by alkaloids isolated from Chelidonium Majus and Macleaya and by derivative drugs “Ukrain” and “Sanguirythrine”. Citologiia. 2003;45(10):1032-7 (in Russian).
  198. Cordes N, Blaese MA, Plasswilm L, Rodemann HP, Van Beuningen D. Fibronectin and laminin increase resistance to ionizing radiation and the cytotoxic drug Ukrain in human tumour and normal cells in vitro. Int J Radiat Biol. 2003 Sep;79(9):709-720.
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  200. Zhanaeva SY, Korolenko TA, Shilov AG, Halikova TA, Guthova IV, Margulis BA, Yarygina ES. Effect of Ukrain on human lymphoma cell growth with different expressions of heat shock protein 70 (hsp70). Int J Immunother 2003, XIX(2-4): 33-39.
  201. Aschhoff B. Ukrain in the treatment of prostate cancer patients.Int J Immunother 2003, XIX(2-4): 41-45.
  202. Zahriychuk O. Ukrain, a thiophosphoric acid derivative of alkaloids of Chelidonium majus L., is effective in the treatment of recurrent bronchopulmonary pathology in children from areas contaminated after the Chernobyl accident.Int J Immunother 2003, XIX(2-4): 47-53.
  203. Sologub TV, Voltchek IV, Kivisepp NA, Grigoryeva T. Efficacy and safety of the drug Ukrain in chronic hepatitis C patients.Int J Immunother 2003, XIX(2-4): 55-59.
  204. Usova TA, Poteryaeva ON, Pospelova TI, Korolenko TA. Serum cystatin C in patients with hemoblastosis. Int J Immunother 2003, XIX(2-4): 61-65.
  205. Gansauge F. Treatment of pancreatic cancer patients with Ukrain: four case reports.Int J Immunother 2003, XIX(2-4): 67-71.
  206. Susak Y. Ukrain stimulates fibrotic and sclerotic transformations of pancreatic cancer tissue: comparative description of six cases.Int J Immunother 2003, XIX(2-4): 73-80.
  207. Aschhoff B. Retrospective study of Ukrain treatment in 28 patients with unresectable pancreatic cancer.Int J Immunother 2003, XIX(2-4): 81-85.
  208. Kleef R. Ukrain (NSC 631570) in combination with locoregional hyperthermia in the treatment of pancreatic cancer with liver metastases: a case report.Int J Immunother 2003, XIX(2-4): 87-90.
  209. Kroiss T. Ukrain in the therapy of advanced metastatic pancreatic cancer: a case report.Int J Immunother 2003, XIX(2-4): 91-94.
  210. Nowicki G, Zahriychuk O. Ukrain treatment of astrocytomas in girl with tuberous sclerosis: a case report.Int J Immunother 2003, XIX(2-4): 95-97.
  211. Langer A, Zahriychuk O, Hodysh Y. Treatment of generalized lymphangiomatosis with Ukrain: a case report.Int J Immunother 2003, XIX(2-4): 99-103.
  212. Aschhoff B. Clinical improvement of a patient with xeroderma pigmentosum after treatment with Ukrain. Int J Immunother 2003, XIX(2-4): a case report. Int J Immunother 2003, XIX(2-4): 105-108.
  213. Nefyodov LI, Doroshenko YM, Smirnov VY, Uglyanitsa KN, Nowicky W. Metabolic control and treatment of malignant growth: tumor development, patterns of amino acid imbalance and Ukrain.Int J Immunother 2003, XIX(2-4): 109-114.
  214. Melnichenko N, Sundikovich E, Makarchikov A, Zverinsky I. Antioxidative status and lipid peroxidation in rats after administration of Ukrain.Int J Immunother 2003, XIX(2-4): 115-119.
  215. Zverinsky I, Maximchuk Y, Melnichenko N, Zaloga I. Effect of Ukrain on hepatic drug-metabolizing enzymes in rats: pilot studies. Int J Immunother 2003, XIX(2-4): 121-124.
  216. Egorov A., Belanovskaya E, Rudyak T, Melnichenko N, Zverinsky I. Assessment of the functional state and morphological structure of the liver of rats after administration of the drug Ukrain.Int J Immunother 2003, XIX(2-4): 125-128.
  217. Levina OA, Goncharova IA, Filatova TG, Nadeev AP, Nowicky W, Sukhenko TG, Kolesnikova OP, Korolenko TA. Protective effect of Ukrain against acute acetaminophen-induced hepatitis in rats. Int J Immunother 2003, XIX(2-4): 129-134.
  218. Usova TA, Poteryaeva ON, Falameyeva OV, Zhanaeva SY, Levina OA, Yarygina ES, Korolenko TA, Nowicky W. Serum cystatin C in murine tumors treated by combinations of cyclophosphamide with Ukrain or glucans. Int J Immunother 2003, XIX(2-4): 135-140.
  219. Zhanaeva SJ, Falameyeva OV, Filatova TG, Goncharova IA, Sukhenko TG, Kolesnikova OP, Kaledin VI, Korolenko TA, Nowicky WM. Effect of different biological response modifiers on growth and metastazing of murine Lewis adenocarcinoma. Int J Immunother 2003, XIX(2-4): 141-150.
  220. Korolenko TA, Falameyeva OV, Poteryaeva OM, Zhanaeva SY, Levina OA, Nowicky W. Cysteine protease inhibitor stefin A in murine tumors during treatment by Ukrain and/or cyclophosphamide. Int J Immunother 2003, XIX(2-4): 151-157.
  221. Todor I. The effect of the antineoplastic drug Ukrain on the electrokinetic potential of malignant and normal cells.Int J Immunother 2003, XIX(2-4): 159-167.
  222. Andrievsky A, Smalyukh N, Krivitsky A, Zahriychuk O. In vitro effects of Ukrain on the activity of trypsin-like proteases. Int J Immunother 2003, XIX(2-4): 169-175.
  223. Jablonski M. Selected calciotropic hormones in serum of sexually mature female rats treated with Ukrain.Int J Immunother 2003, XIX(2-4): 177-182.
  224. Jablonski M. Serum levels of corticosterone, progesterone, parathyroid hormone and calcitonin in ovariectomized rats after 3-month treatment with Ukrain.Int J Immunother 2003, XIX(2-4): 183-188.
  225. Wielosz-Tokarewska E, Jagiello-Wojtowicz E. The effect of Ukrain on the serum vasoactive intestinal polypeptide level in diabetic mice. Int J Immunother 2003, XIX(2-4): 189-191.
  226. Jagiello-Wojtowicz E, Kleinrok A, Jurek D. Effects of Ukrain in acute intoxication with streptozotocin in rats.Int J Immunother 2003, XIX(2-4): 193-196.
  227. Jagiello-Wojtowicz E, Izdebska M, Piatkowska-Chmiel I. Effect of Ukrain on selected biochemical parameters in the serum of methanol-intoxicated rats.Int J Immunother 2003, XIX(2-4): 197-200.
  228. Jagiello-Wojtowicz E., Izdebska M.: Effect of CNS-631570 (Ukrain) on selected biochemical parameters indicating liver dysfunction in acute methanol intoxication of rats. Pol. J. Environ., 2004.
  229. Jagiello-Wojtowicz E., Piatkowska-Chmiel I.: Effects of chelidonine on some biochemical parameters in the serum of rats subacutely intoxicated by copper. Herba Polonica, 2004, Vol. 50, No 3/4.
  230. Zemskov S.V. Efficacy of adjuvant treatment with combination of Ukrain (NSC-631570) and Gemcitabin in cases of pancreas cancer. Ukrainian Chemotherapeutic Journal 2005, #1-2.
  231. Mozhenok T.P., Belyaeva T.N., Leontieva E.A., Faddejeva M.D. Modulation of vesicular membrane fusion and of actin cytoskeleton in mouse macrophages inducing by alkaloid sanguinarine and a derivative drug Ukrain. Cytologia 2005, 10:860-865.
  232. Tomaszewski M., Olchowik M. Influence of Ukrain on some parameters in pelvis bone tissue of rats exposed to subacute microwave radiation. Krajowe Sympozjum Studenckich Kół Naukowych Akademii Medycznych, Lublin, 15 stycznia 2005.
  233. Olchowik M., Tomaszewski M. Effect of Ukrain on biochemical parameters of rats subjected to subacute microwave radiation (mW). Krajowe Sympozjum Studenckich Kół Naukowych Akademii Medycznych, Lublin, 15 stycznia 2005.
  234. Piątkowska-Chmiel I. Porównanie działania chelidoniny i leku Ukrain w ostrych zatruciach miedzią i cyną u szczurów. Krajowe Sympozjum Studenckich Kół Naukowych Akademii Medycznych, Lublin, 15 stycznia 2005.
  235. Izdebska M., Jagiełło-Wójtowicz E. Protective effects of NSC-631570 in rats acutely intoxicated by ethylene glycol. VII Sympozjum „Postępy Toksykologii Klinicznej i Sądowej", Kraków, 2-4 czerwca 2005, Przegl. Lek., 2005, t. 62, 6, s. 638.
  236. Piątkowska-Chmiel I., Jagiełło-Wójtowicz E. Comparison of actions of Chelidonine and Ukrain in acute copper chloride intoxication in rats. VII Sympozjum „Postępy Toksykologii Klinicznej i Sądowej", Kraków, 2-4 czerwca 2005, Przegl. Lek., 2005, t. 62, 6, s. 638.
  237. Izdebska M., Jagiełło-Wójtowicz E. Effect of single or 10-day treatment with Ukrain on some biochemical parameters in the serum of rats acutely intoxicated with alcohols. 42nd Congress of Toxicology EUROTOX, Kraków, 11-14.9.2005, Vol. 158, Suppl. 1, s. 56-57.
  238. Ernst E, Schmidt K: Ukrain – a new cancer cure? A systematic review of randomised clinical trials. BMC Cancer. 2005; 5: 69. doi: 10.1186/1471-2407-5-69.
  239. Kleinrok A., Jagiełło-Wójtowicz E. Effects of Ukrain on some biochemical parameters In mice pretreated with streptozotocin. IV Lwowsko-Lubelska Konferencja Biochemii Eksperymentalnej i Klinicznej, Lublin, 11-13 maja 2006.
  240. Izdebska M., Jagiełło-Wójtowicz E. Effect of thiophosphoric acid alkaloid derivatives from Chelidonium majus L. on some biochemical parameters in the serum of rats acutely intoxicated by ethylene glycol. 5th International Symposium on Chromatography of Natural Products (ISCNP). The Application of Chromatographic Methods in Phytochemical & Biomedical analysis, Lublin -Kazimierz Dolny, June 19-22, 2006, P-58, s. 110.
  241. Jagiełło-Wójtowicz E., Piątkowska-Chmiel I. Protective effects of chelidonine and Ukrain in rats acutely intoxicated with tin chloride. 5th International Symposium on Chromatography of Natural Products (ISCNP). The Application of Chromatographic Methods in Phyto-chemical & Biomedical analysis, Lublin - Kazimierz Dolny, June 19-22, 2006, P-60, s. 112.
  242. Jagiełło-Wójtowicz E., Chodkowska A. Estimation of biochemical parameters in blood serum of rats subjected to 10-day treatment with alkaloid thiophosphoric acid derivative of Chelidonium majus L. (Ukrain) with simultaneous exposition to ionizing radiation. 5th International Symposium on Chromatography of Natural Products (ISCNP). The Application of Chromatographic Methods in Phytochemical & Biomedical analysis, Lublin -Kazimierz Dolny, June 19-22, 2006, P-61, s. 113.
  243. Lanvers-Kaminsky C, Nolting DM, Köster J, Schröder A, Sandkötter J, Boos J (2006): In-vitro toxicity of Ukrain against human Ewing tumor cell lines. Anticancer Drugs 17(9): 1025-1030.
  244. Ernst E. Why there will never be an alternative cancer cure. Anti-Cancer Drugs 2006, 17: 1023-1024.
  245. Gagliano N, Moscheni C, Torri C, Magnani I, Bertelli AAE, Nowicky W and Gioia M (2006): Effect of Ukrain on matrix metalloproteinase-2 and Secreted Protein Acidic and Rich in Cysteine (SPARC) expression in human glioblastoma cells. Anti-Cancer Drugs 17:189-194.
  246. Habermehl D, Kammerer B, Handrick R, Eldh T, Gruber C, Cordes N, Daniel PT, Plasswilm L, Bamberg M, Belka C, Jendrossek V: Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway. BMC Cancer 2006; 6:14, http://www.biomedcentral.com/1471-2407/6/14.
  247. Gansauge F, Ramadani M, Schwarz M, Beger HG, Lotspeich E, Poch B: The Clinical Efficacy of Adjuvant Systemic Chemotherapy with Gemcitabine and NSC-631570 in Advanced Pancreatic Cancer. Hepato-Gastroenterology 2007; 54:917-920.
  248. Jagiełło-Wójtowicz E., Izdebska M.: Effect of thiophosphoric acid alkaloids derivative from  Chelidonium majus (L.) on antioxidant status in alcohol -intoxicated rats. J. Ethnopharmacol., 2007.
  249. Jagiełło-Wójtowicz E, Chodkowska A, Jabłoński M, Olchowik G: Serum RANKL in rats treated with Ukrain and simultaneous exposition to ionizing radiation. J. Ethnopharmacol., 2007.
  250. Gagliano N, Moscheni C, Torri C, Donetti E, Magnani I, Costa F, Nowicky W, Gioia M: Ukrain modulates glial fibrillary acidic protein, but not connexion 43 expression, and induces apoptosis in human cultured glioblastoma cells. Anti-Cancer Drugs 2007, 18: 669-676.
  251. Rebscher H: Off-label use in oncology. Quackery or progress? MMW Fortschr Med. 2002 Oct 10; 144(41): 25-6. German.
  252. Kuznetsova LP, Sochilina EE, Faddeeva MD, Iagodina OV: Effect of some isoquinoline alkaloids on enzymatic activity of acetylcholinesterase and monoamine oxidase. Ukr Biokhim Zh. 2005 Mar-Apr; 77(2): 147-53. In Russian.
  253. Dudka J, Burdan F, Szumilo J, Tokarska E, Korobowicz A, Klepacz R, Gieroba R, Madej B, Korobowicz E: Effect of selected alcohol dehydrogenase inhibitors on human hepatic lactate dehydrogenase activity – an in vitro study. J Appl Toxicol. 2005 Nov-Dec; 25 (6): 549-53.
  254. Grinevich Y, Shalimov S, Bendyuh G, Zahriychuk O, Hodysh Y. Effect of Ukrain on the growth and metastasizing of Lewis carcinoma in C57BL/6 mice. Drugs Exp Clin Res. 2005; 31(2): 59-70.
  255. Mendoza J., Zamora R., Gallardo J., Ceballos G., Aldana A., Espinosa M., Maldonado V., Melendez-Zajgla: NF-kappaB Does Not Influence the Induction of Apoptosis by Ukrain. Cancer Biology & Therapy 5:7, July 2006: 788-793.
  256. Izdebska M, Jagiello-Wojtowicz E. Evaluation of antioxidative activity of Ukrain in alcohol-intoxicated rats. Ann Universitatis Mariae Curie-Sklodowska, Vol. XX No.2, 22.
  257. Izdebska M, Piatkowska-Chmiel I, Jagiello-Wojtowicz E. Effect of Ukrain on selected biochemical parameters indicating kidney functioning of rats pretreated with ethanol. Farm Przegl Nauk, 2008, 11-12: 42-44.
  258. Shvets Yu, Zemskov S, Skachkova O, Rivis O, Khranovska N. Anticancer drug „ukrain“ stimulates dendritic cells maturation in vitro. Immunology & Allergology 4:2008, 24-27.
  259. Wolanska E, Bachanek T, Jarmolinska K, Jagiello-Wojtowicz E, Radzki S. Influence of Ukrain and strontium on the rat tooth intertubular dentine. I. XRF microprobe study. Bull Vets Inst Pulawy 52, 655-657, 2008.
  260. Wolanska E, Bachanek T, Jarmolinska K, Jagiello-Wojtowicz E, Radzki S. Influence of Ukrain and strontium on the rat tooth intertubular dentine. II. Atomic force microscopy study. Bull Vets Inst Pulawy 52, 659-665, 2008.
  261. Skivka LM, Trompak OO, Kudryavets YI, Bezdenezhnykh NA, Susak YM. The effect of NSC-631570 (Ukrain) alone in combination with pathogen-associated molecules on cell cycle distribution and apoptosis induction of mouse melanoma cells with different biological properties. Experimental Oncology, 2010, 32, 2:92-96.
  262. Susak YM, Skivka LM, Rudik MP, Pozur VV, Liubunya AV. Comparative investigation of the effect of Ukrain on growth of ascite and solid forms of Ehrlich’s carcinoma. Experimental Oncology, 2010, 32, 2:107-110.
  263. Skivka L, Susak Y, Trompak O, Kudryavets Y, Bezdenezhnykh N, Semesiuk N, Lykhova O. The effect of monotherapy and combined therapy with NSC-631570 (Ukrain) on growth of low- and high-metastasizing B16 melanoma in mice. J Oncol Pharm Pract. 2010 Sep 3.
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EFFICACY: Quality

III. QUALITY

The proof of quality has been described in the Pharmacopoea Austriaca VIII, European Pharmacopoeia and in the Deutsches Arzneibuch (German Pharmacopoeia), 6th edition.

Greater celandine - Extract from the European Pharmacopoeia 6.0
6.0/1861
Greater celandine
Chelidonii herba
DEFINITION
Dried, whole or cut aerial parts of Chelidonium majus L. collected during flowering.
Content: minimum 0.6 per cent of total alkaloids, expressed as chelidonine (C20H19NO5; Mr 353.4) (dried drug).
CHARACTERS
Macroscopic and microscopic characters described under identification tests A and B.
IDENTIFICATION
A. The stems are rounded, ribbed, yellowish to greenish-brown, somewhat pubescent, about 3 mm to 7 mm in diameter, hollow and mostly collapsed. The leaves are thin, irregularly pinnate, the leaflets ovate to oblong with coarsely dentate margins, the terminal leaflet often three-lobed; the adaxial surface is bluish-green and glabrous, the abaxial surface paler and pubescent, especially on the veins. The flowers have 2 deeply concavo-convex sepals, readily removed, and 4 yellow, broadly ovate, spreading petals about 8 mm to 10 mm long; the stamens are numerous, yellow, and a short style arises from a superior ovary; long, capsular, immature fruits are rarely present.
B. Reduce to a powder (355). The powder is dark greyish-green to brownish-green. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters: numerous fragments of leaves in surface view, the epidermal cells with sinuous walls; anomocytic stomata (2.8.3) occur on the abaxial surface only; covering trichomes long, uniseriate, with thin walls and usually fragmented; vascular tissue from the leaves and stems with groups of fibres, pitted and spirally thickened vessels and associated latex tubes with yellowish-brown contents; occasional fragments of the corolla with thin-walled, partly papillose cells containing numerous pale yellow droplets of oil; spherical pollen grains about 30 µm to 40 µm in diameter with 3 pores and a finely pitted exine.
C. Thin-layer chromatography (2.2.27).
Test solution. To 0.4 g of the powdered drug (710) add 50 ml of dilute acetic acid R. Boil the mixture under a reflux condenser in a water-bath for 30 min. Cool and filter. To the filtrate add concentrated ammonia R until a strong alkaline reaction is produced. Shake with 30 ml of methylene chloride R. Dry the organic layer over anhydrous sodium sulphate R, filter and evaporate in vacuo to dryness. Dissolve the residue in 1.0 ml of methanol R.
Reference solution. Dissolve 2 mg of papaverine hydrochloride R and 2 mg of methyl red R in 10 ml of alcohol R.
Plate: TLC silica gel plate R.
Mobile phase: anhydrous formic acid R, water R, propanol R (1:9:90 V/V/V).
Application: 10 µl as bands.
Development: over a path of 10 cm.
Drying: in air.
Detection: spray with potassium iodobismuthate solution R and dry the plate in air; spray with sodium nitrite solution R and allow the plate to dry in air; examine in daylight.
Results: see below the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other weaker zones may be present in the chromatogram obtained with the test solution.

Top of the plate

_______

_______

Methyl red: a red zone

A brown zone

 

A brown zone

Papaverine: a greyish-brown zone

A greyish-brown zone

_______

_______

 

 

 

A brown zone

 

A brown zone

Reference solution

Test solution

TESTS
Foreign matter (2.8.2): maximum 10 per cent.
Loss on drying (2.2.32): maximum 10.0 per cent, determined on 1.000 g of the powdered drug (355) by drying in an oven at 100-105 °C for 2 h.
Total ash (2.4.16): maximum 13.0 per cent.
ASSAY
Test solution. To 0.750 g of the powdered drug (710), add 200 ml of dilute acetic acid R and heat on a water-bath for 30 min, shaking frequently. Cool and dilute to 250.0 ml with dilute acetic acid R. Filter. Discard the first 20 ml of the filtrate. To 30.0 ml of the filtrate add 6.0 ml of concentrated ammonia R and 100.0 ml of methylene chloride R. Shake for 30 min. Separate the organic layer, place 50.0 ml in a 100 ml round-bottomed flask and evaporate to dryness in vacuo at a temperature not exceeding 40 °C. Dissolve the residue in about 2-3 ml of alcohol R, warming slightly. Transfer the solution to a 25 ml volumetric flask by rinsing the round-bottomed flask with dilute sulphuric acid R and dilute to 25.0 ml with the same solvent. To 5.0 ml of the solution, add 5.0 ml of a 10 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R in a 25 ml volumetric flask, stopper the flask and mix carefully. Dilute to 25.0 ml with sulphuric acid R and stopper the flask.
Compensation solution. At the same time and in the same manner, place in a 25 ml volumetric flask 5.0 ml of dilute sulphuric acid R and 5.0 ml of a 10 g/l solution of chromotropic acid, sodium salt R in sulphuric acid R, stopper the flask and mix carefully. Dilute to 25.0 ml with sulphuric acid R and stopper the flask.
Place both solutions on a water-bath for 10 min. Cool to about 20 °C and dilute if necessary to 25.0 ml with sulphuric acid R. Measure the absorbance (2.2.25) of the test solution at 570 nm.
Calculate the percentage content of total alkaloids, expressed as chelidonine, from the expression:

A x 2.23
m

i.e. taking the specific absorbance of chelidonine to be 933.

A

absorbance at 570 nm,

m

mass of the substance to be examined, in grams.

 

Thiotepa – Extract from the US Pharmacopoeia, 24th edition

„Thiotepa

C6H12N3PS        189.22
Aziridine, 1,1',1"-phosphinothioylidynetris-,
Tris(1-aziridinyl)phosphine sulfide     [52-24-4]

» Thiotepa contains not less than 97.0 percent and not more than 102.0 percent of C6H12N3PS, calculated on the anhydrous basis.
Caution — Great care should be taken to prevent inhaling particles of Thiotepa or exposing the skin to it.

Packaging and storage — Preserve in tight, light-resistant containers, and store in a refrigerator.
USP Reference Standards (11) — USP Thiotepa RS.
Identification, Infrared Absorption (197S) — Solution:  3 in 400. Medium: carbon disulfide. Melting range (741): between 52° and 37°
Water, Method I (92I): not more than 2.0%.
Assay
Mobile phase — Prepare a suitable filtered and degassed mixture of water and acetonitrile (9:1). Make adjustments if necessary (see System Suitability under Chromatography (621)).
Standard preparation — Dissolve an accurately weighed quantity of USP Thiotepa RS in Mobile phase to obtain a solution having a known concentration of about 1.5 mg per mL.
Assay preparation — Transfer about 75 mg of Thiotepa, accurately weighed, to a 50-mL volumetric flask, dissolve in Mobile phase, dilute with Mobile phase to volume, and mix.
Resolution solution — Transfer about 10 mg of USP Thiotepa RS to a 4-mL vial, add 2 mL of methanol, and mix. Add 50 µL of 0.1% phosphoric acid solution. Place a cap on the vial, and heat at 65° for 50 seconds. Cool the solution, add 1 mL of methanol, and mix.
Chromatographic system (see Chromatography (621) — The liquid chromatograph is equipped with a 215-nm detector and a 4-mm x 15-cm column that contains packing L1. The flow rate is about 0.8 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed under Procedure: the relative retention times are about 1.25 for methoxythiotepa and 1.0 for thiotepa, and the resolution, R, between the methoxythiotepa peak and the thiotepa peak is not less than 3.0. Chromatograph the Standard preparation, and record the responses as directed under Procedure: the tailing factor for the thiotepa peak is not more than 1.8, the column efficiency is not less than 2600 theoretical plates, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure — Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C6H12N3PS in the portion of Thiotepa taken by the formula:

50C(ru/rs),

in which C is the concentration, in mg per mL, of USP Thiotepa RS in the Standard preparation, and ru and rs are the thiotepa peak responses obtained from the Assay preparation and the Standard preparation, respectively.”

During the production of Ukrain thiotepa is washed out so that there is no thiotepa in the end product. This has been proved using the most sensitive method of gas chromatography.

 

Stability

The stability of the Ukrain solution for injection was investigated in a real time stability study with 3 batches stored for 60 months at 25°C and 60% relative humidity as well as for 6 months at 40°C and 75% relative humidity (accelerated condition stability study). The study was performed according to the OECD GLP Guidelines. At the end of the study all parameters were within reference range having proved the stability of the end product (Stability Study of Ukrain Ampoules Stored under Controlled Conditions. Final Study Report. March 2003. SGS Lab Simon S.A. Healthcare and Bioscience Services 796.529).

EFFICACY: Safety

II. SAFETY

 

At therapeutic dose NSC 631570 has no appreciable adverse effects and does not damage healthy cells but only attacks cancer cells. Due to its very high therapeutic index of 1250 – in contrast to common cytostatics with a low TI of 1.4-1.8 – there is no danger of an overdose with Ukrain therapy (therapeutic index is the ratio between the toxic dose and the therapeutic dose of a drug, used as a measure of the relative safety of the drug for a particular treatment. From ‘The American Heritage® Dictionary of the English Language, 4th Edition’). Ukrain also does not cause necroses when administered intramuscular, which is a proof for its safety (37).

The Austrian Research Center Seibersdorf is the leading Austrian research institution in life sciences. Now it is a part of the Austrian Institute of Technology (AIT), the largest non-university research institution in Austria. At the Seibersdorf branch of AIT the state-of-art toxicological studies are performed according to the GLP guidelines (Good Laboratory Practice). Following GLP conformed studies with NSC 631570 were performed at the ARCS.

 

Acute Intravenous Toxicity with Rats

The study was performed in conformance with the EC-Guideline 92/69 and the OECD-Guideline 401. The test substance was administered undiluted as a slow intravenous injection to male and female Him:OFA rats at doses of 33 mg/kg, 57 mg/kg, or 100 mg/kg (only females) body weight (b.w.). The administration induced immediate effects, which, if not lethal, soon lead to an almost complete recovery. Most probable cause of death was a shift in the blood pH or in the blood ion homeostasis. Males were more susceptible than females. Based on the results obtained, the LD50 (intravenous) of the active substance was calculated as 43 mg/kg b.w. for males and 76 mg/kg b.w. for females (151, OEFZS-A-4483, October 1998).

 

Acute Intravenous Toxicity with Mice

The study was performed according to the Directive 92/69/EEC and the OECD Guideline 401, 1987. NSC 631570 was administered as a slow intravenous injection to three groups of five male and of five female Him:OF1, SPF mice at doses of 33 mg/kg b.w., 74 mg/kg b.w., and 165 mg/kg b.w. All animals in the high dose group and two males and three females in the mid dose group died. All other animals survived until 14 days after administration. All survived animals were normal at the end of the study. All animals were normal at necropsy. There was no marked sex difference in the response to the test substance. Based on the results obtained, the LD50 (intravenous) was calculated as 80 mg/kg b.w. for males and 68 mg/kg b.w. for females (OEFZS-L-0400, May 2000).



Acute Intramuscular Toxicity with Rats

The aim of the study was to reveal acute toxic effects of NSC 631570 after a single intramuscular administration to rats. The study was performed according to the EC-Directive 87/176/EWG and the OECD-Guideline 401, as far as these were useful for intramuscular administration. The test substance was administered undiluted as an intramuscular injection to five male and five female Him:OFA rats at the highest technically feasible dose for the intended route, i.e. 150 mg active ingredient per kg b.w. All animals survived until the scheduled termination of the study. Body weight gain was not impaired. Sedation and less pronounced disturbed locomotion was noted on the day of the administration. Local changes at the injection site (crusts) were present in about half of the animals. There was no sex difference in the response to the test substance. The authors concluded intramuscular injection of NSC 631570 in the highest feasible dose induced some transient signs of minor clinical importance but was otherwise well tolerated and did not become life threatening. Based on the results obtained in this study the LD50 (intramuscular) of NSC 631570 was calculated to be higher than 150 mg/kg b.w. (151, OEFZS-L-0194, October 1999).

 

Acute Oral Toxicity Study with Rats

This study aimed to investigate acute toxic effects of NSC 631570 after a single oral administration to rats and was performed according to the EC-Directive 92/69 and the OECD Guideline 401. NSC 631570 concentrated was administered undiluted once by stomach intubation to three groups of five female Him:OFA Sprague Dawley rats and to one group of five male Sprague Dawley rats at the doses of 450 mg/kg b.w., 810 mg/kg b.w., and 1500 mg/kg b.w. (females) or 810 mg/kg b.w. (males). All females in the highest dose group died spontaneously on the day of administration. All females in both other dosage groups as well as all males survived until the scheduled termination. All animals of the mid and high dosed groups were affected with most of the signs observed on the day of administration of the test substance. The effects lasted until maximal three days after administration. All surviving animals were normal at the end of the study. There was no relevant sex difference in the response to the test substance.
Hence the test substance induced immediately effects which, if not lethal, led to an almost complete recovery soon. The LD50 (oral, females) was calculated as 1110 mg/kg b.w. (151, OEFZS-L-0195, October 1999).

 

Local Tolerability Study

NSC 631570 was administered as a slow intravenous, intraarterial, paravenous or intramuscular injection to two male and two female rabbits per route to study its local tolerability. Normal saline was administered correspondingly to the other ear or muscle of each animal as a control. NSC 631570 was well tolerated at intravenous and intraarterial administration. The paravenous injection caused mild local irritation, and the intramuscular administration caused a mild to moderate local inflammation. There was no sex difference in the response to the test substance (OEFZS-A-4204, October 1997).

Micronucleus Test with Mice

The study was performed to detect the possible production of micronuclei induced by NSC 631570 as a result of chromosomal damage or of damage to the mitotic apparatus in an in vivo test system. NSC 631570 was diluted with normal saline and administered once at doses of 1.25, 2.50, or 5.00 ml/kg b.w. intravenously to three groups of five male and five female Crl:NMRI BR mice each. Additionally, one high dose group of one male and one female spare animal was included to replace possible unscheduled deaths in the high dose group. Two negative control groups (normal saline) and one positive control group (thiotepa) were also included into the study. Preparation of bone marrow cells and investigations were performed in conformance with the OECD Guideline 474.
All animals survived until scheduled sacrifices. Thiotepa (positive control group) caused cytotoxicity and produced micronuclei in polychromatic erythrocytes. NSC 631570 did not cause cytotoxicity. All data were in the range of historical negative control data. There were no significant differences in micronucleated normochromatic erythrocytes (MNE) between the test substance group animals of both sexes and the corresponding negative controls, neither 24 nor 38 hours after administration. Also no statistically significant differences in the amounts of micronucleated polychromatic erythrocytes (MPE) were noted in any sex at any dose used. The rates of MPE were at both sampling times within the limits of historical negative control groups. No sex difference in the response to the test substance was noted. The authors concluded NSC 631570 not to cause cytotoxicity to the bone marrow at doses up to 5 mg/kg b.w. and not to produce micronuclei in the polychromatic erythrocytes in mice of both sexes at the doses used (OEFZS-L-0225, November 1999).
Similar results were obtained in the second micronucleus study with NSC 631570 concentrated (OEFZS-L-0224, November 1999).

 

Salmonella typhimurium Reverse Mutation Test

NSC 631570 was tested for mutagenic activity in the S. typhimurium reverse mutation test (Ames test) according to the OECD Guideline 471 and the EEC Guideline 92/69, part B14. The test substance was tested at the range of concentrations according to the direct plate incorporation method without and with external metabolisation system S9-mix. The bacterial strains S. typhimurium TA97a, TA98, TA100, and TA1535 were used as test system. Negative and positive controls were included and an independent repetition of the experiment was performed. The test substance was not toxic to the S. typhimurium strains used. According to the results obtained in this study, NSC 631570 is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102, and TA1535 (OEFZS-L-0003, January 1999).

 

Liver Toxicity

As the source materials for the production of NSC 631570 - celandine alkaloids and thiotepa are toxic for liver cells, a study was initiated to evaluate the possible toxic potential of NSC 631570 to the liver. The study was performed in rats and proved NSC 631570 is not toxic to the rat liver (Müller 2004, original report).

 

Other Toxicity Studies

Many other toxicity studies were performed in Eastern Europe (15, 16, 105, 123, 127).
The first toxicity studies were performed on Albino Swiss mice and Wistar rats. NSC 631570 was administered intraperitoneally at the single daily dose of 0.025, 0.05, and 0.1 of LD50 (e.g. 4.75, 9.5, 19 mg/kg in mice and 7, 14 and 28 mg/kg in rats) during three months. The treatment had no effect on the organ weight with except of spleen in rats where the organ weight was up to 3fold higher than in the control group (29). The estimation of catecholamines in brain revealed the three month administration of NSC 631570 at middle and higher dose diminished the dopamine concentration in mice and rats. The concentrations of noradrenalin, 5-hydroxytryptamine and 5-hydroxyindolacetat were not changed (29). The single administration of NSC 631570 had no effect on the serum prolactin concentration in rats. On the contrary, after the three month administration of NSC 631570 the serum prolactin concentration increased in all dose groups compared to the control (32).

Regarding blood count, the administration of NSC 631570 had similar effects in the middle and higher dose groups: increase of white blood cells (WBC) and decrease of platelets. The changes in the differential count were also observed, e.g. lymphocytes increase and neutrophil bands decrease. No significant changes in red blood cells (RBC) were observed compared to the control (33).

Also there were no changes in the transaminase activity in rats. In mice, increase of ALT and AST activity occurred in the middle and higher dose groups. A slight decrease of the total protein was observed in male mice in the groups 9.5 and 19.5 mg/kg, in female mice only in the group 19 mg/kg. The single administration of NSC 631570 did not affect the evaluated parameters (31).

In the experiments with hamster cells it was revealed NSC 631570 is not mutagen as well as not genotoxic and does not cause morphologic cell transformations (19).

Tests in mice and guinea pigs have revealed NSC 631570 not to cause anaphylactic sensitization (23).

In the tests in rats, NSC 631570 was confirmed not to exert embryotoxic or teratogenic effect. In hamsters, the embryotoxic effect was observed at the very high dosage of 28 mg/kg body weight (30).

In a series of experiments the effect of the six week administration of NSC 631570 in rabbits was studied (subacute toxicity).
The daily treatment at three various dosage caused no effect on the organ morphology and body mass of the animals. In blood count, the numbers of red blood cells and white blood cells did not change. In the high dosage group, the percentages of lymphocytes, monocytes, and eosinophils increased. The blood chemistry parameters were not affected apart from increase of urea and uric acid (124).

Among sex hormones, estradiol increased in some groups as did testosterone in the lowest dose male group. In females, progesterone increased only in the highest dose group (125).

NSC 631570 caused the increase of thyroid hormones in males. In females, triiodothyronine increased and thyroxin concentration did not change (126).

In a study in male Wistar rats the researchers revealed that the six day administration of NSC 631570 in both doses of 1 mg/kg and 2 mg/kg caused no significant changes in the following parameters: albumin, mucopolysaccharides, aminotransferase activity, and thymol test. Similarly unaffected compared to the control group was the liver tissue morphology. Significantly lower was the blood concentration of the middle molecules (216).

In the Wistar rats with implanted Guerin carcinoma, NSC 631570 was revealed to reduce the number of thiol groups in the tumor homogenates (121).

 

PHARMACOKINETICS

Pilot studies of pharmacokinetics gave first suggestions on the methods which should be used in the later studies (105, 127).

To estimate the plasma concentration and pharmacokinetic parameters of NSC 631570 at intravenous administration, first a method for its determination in plasma was developed (152). This enabled consequently the study of pharmacokinetics of NSC 631570 in rats. It was revealed the tumor tissue and the liver had the strongest affinity to NSC 631570. In brain and muscles the NSC 631570 accumulation was the least (153).

 

BIOPHYSICAL PROPERTIES

The examinations with physical methods (spectrometry, polarized light microscopy) revealed NSC 631570 to be resistant against the UV light of middle intensity. The absorption peaks 210 and 230 nm can be used for the detection of NSC 631570 in plasma (50, 81).

In the tests with the rat liver homogenates the oxygen consumption was detected by means of polarography. The addition of NSC 631570 caused complex redox reactions in the substrate. This was dependant on the NADP induced oxidation (82).

EFFICACY: microwaves, toxicologic studies, the effect

MICROWAVES

Microwaves are electromagnetic waves with wavelengths ranging from as long as one meter to as short as one millimetre, equivalent to frequencies between 300 MHz and 300 GHz. As the devices operating in this wavelength range have been more and more used, including therapeutic applications, the importance of the studies of their health effects increases constantly.

In the experiments on male Wistar rats irradiated with microwaves 53.57 MHz during 14 days, NSC 631570 was revealed to normalise the activity of aminotransferases ALT and AST as well as the serum concentration of the alpha-fetoprotein compared to the irradiated control group (233).

The effect of NSC 631570 (7 mg/kg intraperitoneal for 10 days) on the serum chemistry parameters of male Wistar rats during concomitant microwave irradiation (53.57 GHz, 10 mW/cm2, 20 min daily for 10 days) was studied. At the end of the study no significant changes were observed in the combined treated group compared to the control group. The authors concluded NSC 631570 can be used combined with microwave therapies (242).

 

TOXICOLOGIC STUDIES

On an induced hepatitis model the researchers studied whether NSC 631570 can protect liver cells from the toxic effect of acetaminophen overdosage. Indeed, NSC 631570 exerted a protective effect with the stimulation of liver macrophages (217).

In the tests on rats, the effects of chelidonine (50 or 100 mg/kg intraperitoneal) and NSC 631570 (7 or 14 mg/kg intraperitoneal) on some blood chemistry parameters after intoxication with copper chloride or lead chloride were studied. Both agents normalised the serum values of beta-2-microglobulin, creatinine, and urea. The effect of chelidonine was more pronounced (229, 234, 236). The treatment with NSC 631570 normalised also increased values of ALT and alpha-fetoprotein (193, 241).

In male Wistar rats with experimental acute ethylene glycol intoxication (3 or 3.5 g/kg intraperitoneal), the effect of NSC 631570 (7, 14 or 28 mg/kg intraperitoneal) on blood chemistry parameters was studied. Compared to the control group, the 10 day administration of NSC 631570 caused a significant decrease of serum urea concentration and increase of serum beta-2-microglobulin concentration (235, 257). In the study with lower ethylene glycol dose (2.5 g/kg), an increased serum concentration of beta-2-microglobulin was observed (240).

In male Wistar rats with experimental acute alcohol intoxication (methanol 4.5 g/kg intraperitoneal, ethanol 3 g/kg intraperitoneal, or ethylene glycol 3.5 g/kg intraperitoneal) the effect of NSC 631570 (28 mg/kg intraperitoneal as single dose or 10 day administration) on the aminotransferase activity (ALT and AST) and the serum alpha-fetoprotein concentration was studied. Compared to the control group, the single administration of NSC 631570 caused the drop of the increased AFP concentration (except the ethylene glycol group). The serum AFP concentration increased after the 10 day administration of NSC 631570. There were no changes in other parameters compared to the control (237, 248, 256, 257).

To study the effect of NSC 631570 on some chemistry parameters in the acute methanol intoxication, Wistar rats were treated with NSC 631570 for 10 days. The therapy alleviated the unfavourable consequences of the methanol intoxication, expressed by the normalization of the increased serum concentrations of beta-2-microglobulin and urea (192, 196, 227, 228).

 

NORMALISATION OF THE METABOLISM

Bone Metabolism and Osteoporosis

A series of animal tests was aimed to study the effect of NSC 631570 on bone density and mineral metabolism.

In a 6 month study on female ovariectomized rats, NSC 631570 has been revealed to inhibit the development of some indications of early osteoporosis (78). The blood parameters and transaminase activity in the NSC 631570 group did not differ from ones in the control group (79). Prolactin and progesterone concentrations were elevated, those of corticosterone and aldosterone diminished compared to the control ovariectomized group (57, 80).

The treatment of mature female rats with NSC 631570 at high dosage had no negative effect on the bone mineral density. A slight decrease of bone mineral content was observed (128).

After intermittent 3 month administration of NSC 631570 at high dosage to ovariectomized female rats the bone mineral density decreased (129).

The effect of NSC 631570 on the bone metabolism in rats was reviewed in an article. The author concluded this drug to affect especially the estrogen dependant mechanisms and has protective effect against osteoporosis (174).

In a set of animal tests, rat femur was used as a model for the study of the long-term effect of NSC 631570 on various bone parameters. NSC 631570 was revealed not to diminish the bone strength and density also at higher dosage (175, 176).

It was also studied how various doses of NSC 631570 affect the intensity of electron spin resonance (ESR) signal in intact and ovariectomized rats. The intensity of the signal correlates directly with the amount of free radicals in the tissue. The signal intensity was the lowest in the ovariectomized group with highest NSC 631570 dose and the highest in the intact group at the lowest dose of NSC 631570 (177).

In intact female rats the effect of NSC 631570 on the hormones involved into calcium metabolism was studied. In the highest dosage group the serum concentrations of corticosterone and progesterone decreased significantly and non-significantly, respectively. In the lowest dosage group the serum concentration of parathormone increased significantly. The serum values of calcitonin were not affected in either group (223).

The effect of NSC 631570 on the same hormones was also studied in female ovariectomized rats. In the middle dose group the corticosterone serum concentration dropped. There were no other changes in hormone values in all dose groups (224).

In the tests in male Wistar rats irradiated with 53.57 MHz microwaves for 14 days, NSC 631570 exerted practically no effect on the water content as well as organic and inorganic phases of pelvis bone compared to the irradiated control group (232).

The effects of NSC 631570 at different doses and/or strontium on the rat tooth intertubular dentine were analysed in cuts perpendicular to the dentinal tubes. The tooth surfaces and cross-section morphology and roughness were investigated with atomic force microscopy. The cross-section surface of intertubular dentine was analysed by roughness and fractal parameters. Histograms were prepared for the typical samples from all groups teeth analysed. Dentine cross section surfaces showed significant differences between the nano structures of normal rat teeth and those from animal treated with NSC 631570 and strontium (260).

 

THE EFFECT ON VARIOUS ENZYMES

In the tests on liver cells NSC 631570 was revealed to inhibit the alcohol dehydrogenase activity (178, 253).

The effect of NSC 631570 on the activity of trypsin-like enzymes was also studied (183, 222).

The effect of NSC 631570 on the concentration of the vasoactive intestinal peptide (VIP) was studied on the diabetes model in mice as well as on intact animals. NSC 631570 exerted no effect on the VIP concentration in intact mice. In diabetic mice the 10 day administration of NSC 631570 caused a significant increase of the VIP concentration in the lowest dose group (194, 225).

In the experiments on the rat liver the researchers revealed that NSC 631570, beside chelidonine, caused the strongest inhibition of the enzyme monoamine oxidase (MAO), indicating its antidepressant properties (197, 252).

In a review the researchers summarized the articles on the effect of NSC 631570 on the amino acid metabolism. They noted NSC 631570 exerts different effects on the amino acid pools in the body and in the malignant tissue (213).

In a set of experiments in rats the effects of NSC 631570 on various liver enzymes were studied. In a test, the effects of the NSC 631570 seven day administration on the lipid peroxidation and antioxidative function of the rat liver were explored. At daily dose of 2 mg/kg NSC 631570 the serum concentration of reduced glutathione decreased at 25% and the antioxidative function of the rat liver decreased at 49%. The glutathione reductase activity in the postmitochondrial fraction of the rat liver increased at 43% compared to the control group. There was no activation of the lipid peroxidation after the administration of 2 mg/kg NSC 631570, and the catalase as well as peroxidise activity did not differ from those in the control group. The authors suggested an assumption NSC 631570 to cause an oxidative stress in the tumor resulting in apoptosis (214).

The effects of NSC 631570 on the liver enzymes involved into the drug metabolism were studied. After the six day administration of NSC 631570 at a daily dose of 2 mg/kg, the activity of aminopyrine-N-demethylase increased by 35% and the one of glutathione-S-transferase by 55%. The concentrations of microsomal cytochromes P450 and b5 as well as the rate of ethylmorphine-N-demethylation were not affected (215).

 

INTERACTION WITH OTHER DRUGS

Non-Opioid Analgesics

In mice and rats the interaction of NSC 631570 and aminophenazone was studied. It was found that NSC 631570 affects the analgesic effect of aminophenazone in various ways. In the writhing syndrome test in mice as well as in tail-flick test in rats the antinoceptive effect of the analgesic was potentiated, whereas in the hot-plate test the analgesic effect of aminophenazone was reduced (20).

The analgesic effect of NSC 631570 was augmented by the nitric oxide synthase inhibitors in mice. These results suggest the endogenous nitric oxide can modify the analgesic effect of NSC 631570 (130).

 

Opioid Analgesics

The tests on mice and rats revealed NSC 631570 to modify the antinoceptive effect of opiates. In mice, for example, NSC 631570 potentiated the analgesic action of morphine in the hot plate test and in the tail flick test, however, reduced this effect in the writhing syndrome test (35).

In the studies in mice was revealed the 10 day intraperitoneal administration of NSC 631570 at high dose has an analgesic effect in mice. Combined administration of morphine and NSC 631570 reduced their antinoceptive effects reciprocally. The authors propose to avoid combined clinical use of these drugs (85, 86).

The analgesic action of NSC 631570 in mice was completely abolished by naltrexon, a pure opioid antagonist acting on all opioid receptors as a competitive antagonist (131).

 

Streptozotocin

The streptozotocin diabetes model was used in rats to explore how changes the liver and kidney function in these animals under the impact of NSC 631570. The 10 day administration of NSC 631570 to streptozotocin rats did not change the serum creatinine concentration, but increased the urea value. The authors concluded NSC 631570 should not be combined with streptozotocin (195, 226, 239).

 

Porphyrin Derivates

In the tests on murine sarcoma, mammary carcinoma, human colon carcinoma and melanoma cell lines NSC 631570 and porphyrin amino acid derivates to have synergetic action against cancer cell lines (53, 83).

 

Anticonvulsants

In Albino mice the interaction of NSC 631570 and various anticonvulsants such as diazepam, carbamazepine, diphenylhydantoin, phenobarbital, and valproate was studied. NSC 631570 potentiated the protective effect of valproate, whereas the effects of other anticonvulsants were not affected (34).

 

 

INTERACTION WITH OTHER THERAPY MODALITIES

Local hyperthermia

Hyperthermia is a type of treatment in which body tissue is exposed to high temperatures (up to 45°C), to damage and kill cancer cells, or to make cancer cells more sensitive to the effects of radiation and certain anticancer drugs. Local hyperthermia treatment (heat applied to a very small area, such as a tumor) is a well-established cancer treatment method with a simple basic principle: If a rise in temperature to 45°C can be obtained for one hour within a cancer tumor, the cancer cells will be destroyed. Primary malignant tumors have a bad blood circulation, which make them more sensitive to changes in temperature. The therapy with NSC 631570 has been successfully combined with local hyperthermia since many years (115, 144, 208).

In his speech at the 5th Vienna Dialog on the Holistic Medicine, Dr. B. Aschhoff reported on his experience in the NSC 631570 therapy and local hyperthermia. Summing up, following theses were noted: significant improvement of the quality of life; few adverse effects; NSC 631570 is safe and can be used in children also; wide indication range of the therapy (142).

 

Endovascular Laser Therapy / Photodynamic Therapy

The endovascular laser therapy as a part of photodynamic therapy (PDT) is a new method for systemic laser treatment and energy transfer to the human body. For therapeutic application red, infrared, green, and blue lasers are used. The photon stream applied intravenously leads to an improved microcirculation, activation of the immune system and mitochondria. Recently, NSC 631570 has been used as a sensitizer (Weber M, ‘The intravenous laser blood irradiation, a new therapeutic approach in immunology and cancer therapy”, presentation at the 2nd International Conference on Drug Discovery and Therapy, SL-341, Dubai, UAE, 2010).

NSC 631570 was administered to the patients with rheumatic diseases or high susceptibility to infections. In the lymphocytes from both patients group and from a control group, expression of IgG, proliferation marker Ki-67 and other marker molecules were estimated. There were no significant changes in the control group whereas in the NSC 631570 group the expression of IgG and Ki-67 increased significantly. Further increase of marker expression was achieved using additional endovascular low-level laser therapy. Circulating tumor cells from cancer patients were incubated with NSC 631570. Patients were treated with NSC 631570 alone or with NSC 631570 and endovascular laser. The tumor mass reduction was achieved in both groups but in the combined group this effect was more pronounced (Andrae F, ‘Ukrain based laser therapy in oncology’, presentation at the 2nd International Conference on Drug Discovery and Therapy, SL-274, Dubai, UAE, 2010).

 

Ozone

Empiric experience revealed NSC 631570 should not be combined with the ozone therapy. Generally, NSC 631570 should preferably be used as monotherapy. Other therapy modalities can be used in the breaks between the treatment cycles.

 

Others

In a pilot study it was elaborated how a method for determination of an optimal dosage for the therapy with NSC 631570 could be elaborated (the same for interferon-alpha, too). The method is based on the estimation of SS/SH groups ratio in serum (138).

EFFICACY: radioprotective effect

RADIOPROTECTIVE EFFECT

When NSC 631570 has been used in clinic, it was observed that the patients treated with this drug tolerate the concomitant radiotherapy much better. The adverse effects of this aggressive treatment modality were significantly reduced to minimal. This gave reason to study radioprotective properties of NSC 631570 in the in vitro and in vivo tests.

To evaluate whether the radio protective effect of NSC 631570 originates from its raw materials, the tests in mice with various radiation doses were performed at the Research Institute of Military Medicine, St. Petersburg, Russian Federation. It was proven the radioprotective effect of NSC 631570 was far superior compared to such of its raw materials taken separately, both measured by survival of mice irradiated by different doses and by the protection coefficient. For example, at a dose of 5.25 Gy protection coefficient of NSC 631570 was 95.0 ± 4.6 vs 50.8 ± 4.6 in the control. These observations suggested that the radio protective effect of Ukrain differs significantly from such of its raw materials (132).

Further studies showed NSC 631570 to modulate the cell components of the hemopoietic system (stem cells, proliferating, maturing, and competent cells) so that their total radioresistance increases. In the study aiming to describe qualitative and quantitative changes in hemopoietic precursor cells and in myelokaryocytes and leukocytes in the blood, Ukrain was administered intraperitoneally to mice at a dose of 0.2 mg/kg. Control animals were injected with normal saline. Colony-forming units (CFU) were counted in the spleen and bone marrow of the mice, and myelokaryocyte and leukocyte (Iymphocyte and granulocyte) counts were determined. The results of this study suggest that Ukrain causes qualitative and quantitative alterations in different pools of hemopoietic cells (stem cells, proliferating cells, maturing cells, and competent cells). These alterations affect the size of the stem cell pool, the kinetics of stem cell proliferation, the direction of their differentiation pathways, the rate of circulation of stem cells and precursor cells, the efficiency of recolonization of cell-depleted sites, and other parameters, which in turn modify standard responses of hemopoiesis and immunogenesis to irradiation so that the radioresistance of the whole organism increases (77, 133, 249).

The radioprotective effect of NSC 631570 has been confirmed by the infection models in mice where its effect was superior to the effect of the known radioprotector cysteamine (134).

Compared to other agents, NSC 631570 exerted a strong radioprotective effect similar to such of lymphokinin. In this study, the effects of the preventive administration of cysteamine, naphthysin, Ukrain, lymphokinin, prodigiosan, and polyribonate on the 30-day survival of mice subject to doses of ionizing radiation were compared. Conventional radioprotectors were shown to prevent the death of experimental animals irradiated with doses ranging from LD50/30 to LD100/30 but were inefficient as a means of preventing mortality at doses with a medium lethal effect. In contrast, the bioregulators Ukrain and lymphokinin were more potent at doses ranging from LD50 to LD70/30 (1.5 times more potent than conventional radioprotectors). Polyribonate and prodigiosan were the least potent. The authors assumed the radioprotective effect of Ukrain as ‘strong’ (135).

These radioprotective properties of NSC 631570 were confirmed in further studies in rats at the Institute of Applied Cell Culture (Munich, Germany). After peritoneal administration, Ukrain had no effect on the concentration of thyroid hormones in rat blood and increased the concentration of thyroid hormone receptors in the liver of intact rats during the first two months after administration. Ukrain normalized the level of nuclear thyroid hormone receptors influenced by short-term whole body gamma-irradiation of rats with 1 Gy, beginning from the first day after administration of the drug. Thus, authors concluded Ukrain can minimize the consequences of irradiation on the endocrine system of experimental animals (172).

Administered intraperitoneally, NSC 631570 exerted a protective effect on the hormonal system of irradiated female rats. Ukrain caused the normalization of the intracellular glucocorticoid receptor system, affected by short-term whole-body gamma-irradiation of rats with 1 Gy beginning on the 10th day after administration of the drug. It was found that Ukrain minimized the consequences of irradiation in the endocrine system of the experimental animals (173).

The radio protective effect of NSC 631570 was also studied and confirmed on in vitro models on the human skin firbroblasts HSF1 and HSF2 as well as lung fibroblasts CCD32-LU. As evaluation parameters were chosen cytotoxicity, apoptosis induction, cell cycle course, and the expression of TP53 and p21. Additionally, following malignant cell lines were used: MDA-MB-231 (human breast tumor), PA-TU-8902 (pancreas cancer), CCL-221 (colorectal cancer), and U-138MG (glioblastoma). The cytotoxicity of NSC 631570 was time- and dose dependent. The combination of NSC 631570 plus ionizing radiation (IR) enhanced toxicity in CCL-221 and U-138MG cells, but not in MDA-MB-231 and PA-TU-8902 cells. Most strikingly, a radioprotective effect was found in normal human skin and lung fibroblasts. Flow cytometry analyses supported differential and cell line-specific cytotoxicity of NSC 631570. CCL-221 and U-138MG cells accumulated in G2 after 24h treatment with NSC 631570, whereas no alterations were detected in the other tumor cells and normal fibroblasts tested. Differential effects of NSC 631570 in modulating radiation toxicity of human cancer cell lines and its protective effect in normal human fibroblasts suggest that this agent may be beneficial for clinical radiochemotherapy (184).

In their next study on the role of the proteins fibronectin and laminin in the radiation protection mechanisms of the cells, the researchers from the University of Tübingen used NSC 631570 as a reference substance (198).

NSC 631570 was effective in the therapy of recurring respiratory diseases in children from the Chernobyl area. A total of 38 children from areas radiation contaminated after the Chernobyl accident and suffering from recurrent respiratory diseases were included into the study. Ukrain was administered intravenously at a dose of 5 mg twice a week, up to a total dose of 35 mg. The control group received standard therapy. In the Ukrain treated group marked anti-inflammatory effects was revealed comparing to the control: normalisation of WBC count and blood sedimentation rate. The immune modulating effect of Ukrain was indicated through the improvement in specific humoral and cellular immunity: increased the IgG concentration, the phagocytic activity of neutrophils, the number of total lymphocytes, T-lymphocytes and T-helpers, and the helpers/suppressors ratio (202).

EFFICACY: Modulation of the immune system

MODULATION OF THE IMMUNE SYSTEM

Unusual for an anticancer agent NSC 631570 possesses some distinct immune properties (24, 44). In several immune target-effector systems NSC 631570 significantly amplified the malignotoxic activity of macrophages (231), lymphocytes and NK cells (47), and stimulates dendritic cells maturation in vitro (258). While the parameters like B-lymphocytes count, immune globulin concentrations, complement and acute phase proteins did not changed significantly, it can be postulated NSC 631570 modulates the cellular part of the immune system whereas the humoral part remains unaffected.

 

Clinical Immunology

The incubation of peripheral lymphocytes of healthy blood donors with NSC 631570 resulted in the increase of lymphocytes with the T-helper phenotype, decrease of the lymphocytes with T-suppressor phenotype as well as increase of T-helper/T-suppressor ratio (7, 18).

NSC 631570 was administered to nine advanced stage cancer patients (4 with liver cancer, 4 with head and neck carcinomas, and one breast cancer). In three cases the tumors responded partially on the therapy, in one case a minimal response was noted, in 3 cases the disease was stabil, and in 2 cases the tumors did not respond on the treatment. After the therapy, the number of T-helper cells (CD4) as well as the CD4/CD8 ratio increased (13).

In eight oncological patients immune parameters were compared before and after the treatment with NSC 631570. It was revealed NSC 631570 affected basically the thymus dependant cells (T-cells). The number of rosette-forming T-lymphocytes was significantly higher after the treatment. No significant changes were observed in the humoral immune parameters (22).

In nine male lung cancer patients lymphocytes subpopulations were determined before and after the therapy with NSC 631570. The therapy resulted in increased total T-cells and reduced T-suppressor fraction. The helper-suppressor ratio normalized. There was no sign of the activation of NK cells, T-helpers as well as B-cells. The restoration of the cellular immunity correlated with the better clinical course of the disease (25).

The effect of NSC 631570 on the functional activity of monocytes from 20 patients with lung cancer or peritonitis was studied using nitro blue tetrazolium chloride test (NBT-test). The authors reported on the positive effect of NSC 631570 on the functional activity of the macrophages as well as antioxidant systems of monocytes and erythrocytes (46).

23 patients with various tumors were treated with NSC 631570 and the immune e parameters were evaluated before and after the therapy. The authors observed the increase of lymphocytes and the decrease of the blood sedimentation rate. Following immune changes were also noted: increase of T-lymphocytes, T-helpers, NK-cytotoxicity, phagocytic activity, normalisation of the T-helper/T-suppressor ratio, and occurance of large granular lymphocytes (48, 106).

NSC 631570 was effective in the therapy of recurring lung diseases in children from the Chernobyl area (202).

 

The Effect on the Dendritic Cells

Dendritic cells are immune cells that act as messengers between the innate and adaptive immunity. Their main function is to process antigen material and present it on the surface to other cells of the immune system, thus functioning as antigen-presenting cells and are seen as the most potent population executing this function.
In the experiments with the mononuclears from the peripheric blood of healthy persons the effect of NSC 631570 on the phenotypic and functional properties of dendritic cells was studied. The most prominent induction of the expression of the cell surface molecules CD86 and HLA-DR was achieved with NSC 631570 at the lowest and highest concentration, 0.6 µg/mL and 10 µg/mL, respectively. Lipopolysaccharide as standard comparative agent induced similar increase of the cell surface receptors.

The proliferation index of the incubated lymphocytes was used as the indicator of the dendritic cells activity. After addition of NSC 631570 to the incubated dendritic cells, the lymphocyte proliferation index increased from 22.6% up to 32.30% at 0.6 µg/mL or 29.34% at 10 µg/mL, respectively. These values are similar to the one of 31.82%, i.e. proliferation index achieved at the incubation of lymphocytes with the phytohemagglutinin. The authors concluded dendritic cells incubated with NSC 631570 are strong stimulators of the lymphocyte proliferation. They postulate also NSC 631570 can take an important part in the immune therapy of cancer (268).

 

Immunological properties of Ukrain (NSC 631570)
NSC 631570 modulates the natural defense system against developing cancer

Immunologische Eigenschaften von Ukrain

The Studies in vitro and in vivo

In the tests on alloimmunised mice NSC 631570 augmented the lytic activity of splenic lymphocytes by up to 48fold. The lytic activity of the IL-2 treated spleen cells and peritoneal exudate lymphocytes were also increased significantly by the addition of NSC 631570 to the assay medium (17, 66).

Human lymphocytes as well as guinea pig lymphocytes were activated more pronounced when incubated with Ukrain than with PHA. In rats NSC 631570 caused a clear increase of macrophages with NK-activity. A similar modulating effect on the macrophages was observed also clinically (49).

It was revealed in the tests on CBA mice and Wistar rats that NSC 631570 stimulates macrophages. As marker of this activity the enzyme chitotriosidase - a part of the native immunity was used (168).

In a study on intact and thymus-ectomised mice NSC 631570 augmented the endocrine function of thymus. After NSC 631570 administration, increased production of substances with thymosin-like activity was detected. Repeated administration of NSC 631570 caused a 2fold rise of T-cells in blood, a 4.5fold rise of large granulocytes and increase of the NK-activity of splenocytes. The production of interferon and antibodies after the antigen administration was increased as well (180).

The effect of well-known immune modulators interferon-gamma, NSC 631570 and pokeweed mitogen on the selective uptake of technetium-99m(99mTc)-labelled tumor necrosis factor (TNF) was studied in the intramuscular implanted murine embryonic carcinoma. The highest absolute tumor uptake of 99mTc- TNF was achieved when NSC 631570 was used, followed by IFN-γ and pokeweed mitogen (104).

In the experiments on BALB/c and F1 (BALB/c x C57BL/6J) mice it was revealed that NSC 631570 inhibits the allergic sensitization of animals against ovalbumin, expressed in the weakened IgE-reaction and decreased histamine release. The incubation of ovalbumin with NSC 631570 induced decreased antigenicity of this protein (84).

The immune modulating effect of NSC 631570 was studied in several studies in mice. Repeated subcutaneous injections of NSC 631570 to mice infected with the twofold LD50 of E. coli, S. aureus, or influenza virus increased the survival rate of the animals significantly (60, 87, 89).

When human lymphocytes were incubated with phytohemagglutinin (PHA) and NSC 631570, increased absorption of 3H-thymidin in the cells was observed. The authors point out the strong synergetic effect of NSC 631570 and phytohemagglutinin (76).

By means of the cell proliferation assay the mitogenic effects of PHA and NSC 631570 on human peripheral blood mononuclear cells (PBMC) were studied. It was revealed even a short pretreatment of the PBMC with NSC 631570 has a strong synergetic effect on the PHA-mitogenesis. Consequently, the cell stimulation parameters were much higher after combined stimulation than after using PHA alone (65).

In experiments with murine (CC57 Black/6) macrophages and rabbit G-actin, the effects of NSC 631570 and sanguinarine on phagosome-lysosome membrane fusion and actin cytoskeleton were studied. The most stimulating effect on the phagosome-lysosome fusion exerted sanguinarine at 10 µmol and NSC 631570 at 5 µmol. At the same dose NSC 631570 doubled the content of fibrillary actin in murine peritoneal macrophages. Moreover, NSC 631570 and sanguinarine induced the polymerisation of rabbit globular actin. These effects were dose-dependent. The authors suggest sanguinarine and NSC 631570 can alter intracellular membrane transport (231).

NSC 631570 (UKRAIN): efficacy, safety, quality

NSC 631570 (UKRAIN): EFFICACY, SAFETY, QUALITY

INTRODUCTION: THE FIRST MEDICAMENT THAT KILLS CANCER CELLS BUT NOT HEALTHY CELLS

General on cancer treatment

Surgery, chemotherapy and radiotherapy are the three most important types of cancer treatment. However, each of them have their limitations and as well as considerable adverse effects. Surgery is the oldest treatment option and, if possible, is still frequently the method of first choice. Unfortunately curative surgical intervention is only possible in few cases; the residual tumour usually remains unrecognised, leading to tumour recurrence and metastazing. Additional treatment then comes into consideration.
Both radiation and chemotherapy do not have a selective effect against tumour cells, they are themselves carcinogenic (they cause cancer) and mutagenic (they damage chromosomes and change the genetic make-up).
Radiation therapy works by damaging the DNA of cancerous cells. This DNA damage is then passed on through cell division, accumulating damage to the cancer cell's DNA, causing them to die or reproduce more slowly. Radiotherapy causes varying side effects during treatment (acute side effects), in the months or years following treatment (long-term side effects), or after re-treatment (cumulative side effects).
The first use of drugs to treat cancer was in the early 20th century. Mustard gas was used as a chemical warfare agent during World War I and was studied further during World War II. During a military operation, a group of people were accidentally exposed to mustard gas and were later found to have very low white blood cell counts. It was reasoned that this agent might have a similar effect on cancer. In 1942 mechlorethamine was first used as a cytostatic agent for cancer therapy. Most commonly, chemotherapy acts by killing cells that divide rapidly, one of the main properties of most cancer cells. This means that it also harms cells that divide rapidly under normal circumstances: cells in the bone marrow, digestive tract and hair follicles; this results in the most common side effects of chemotherapy: myelosuppression (decreased production of blood cells, hence also immunosuppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss). Moreover, various groups of cytostatic agents cause other specific adverse effects.
Complex concomitant therapies have being used to alleviate adverse effects of the chemotherapy, i.e. corticosteroids, antiemetic agents etc. Still, a part of chemotherapy cycles must be performed with reduced dose, postponed or stopped.
It is clear that the problem of the immense adverse effects of chemotherapy cannot be solved in a customary way. This could only be achieved by preparations which only kill cancer cells while leaving healthy cells undamaged, in other words which act selectively against cancer cells.
With the new generation of ‚targeted’ anticancer agents one tries to block specific molecules needed for tumor development and growth. Still, current results of chemotherapy are unsatisfactory. Cancer is responsible for 20,000 deaths per day across the world, or 7.6 million people a year, according to a report of the American Cancer Society (http://www.cancer.org/Research/CancerFactsFigures/index ).
It is evident that it has always been the greatest wish of all scientists and cancer researchers to find a preparation which kills only cancer cells and leaves healthy cells undamaged. In other words, a preparation with a selective effect only against cancer cells but not against healthy cells. All the efforts of scientists all over the world have been unsuccessful – there was the strong conviction that this problem was insoluble because the difference between a healthy cell and a cancer cell is too small.

 

Cancer cells can be killed without damaging healthy cells

A new preparation was presented at the 13th International Congress of Chemotherapy in Vienna in August-September 1983 – thiophosphoric acid derivative of alkaloids from greater celandine (NSC631570, trade name Ukrain). The development of this preparation was the first significant step on the way to solving the problem. In vitro tests showed that after incubation with NSC631570 normal liver cells and Ehrilch tumour ascitic cells demonstrate different oxygen consumption: after initial increase, oxygen consumption in cancer cells falls to zero whereas oxygen consumption in normal cells returns to normal and the cells remain undamaged (38). This study brought the first indications that, in contrast to its starting substances thiotepa (a well known cytostatic agent) and greater celandine alkaloids, NSC631570 is in fact only toxic against cancer cells and not against normal cells.
The second indication was provided by clinical use, where NSC631570 caused no noteworthy side-effects (21). It improved patients’ general condition as well as their immune status which had previously been impaired by chemotherapy (22).
The third indication was provided by a study at the University of Miami, based upon which the therapeutic index of NSC631570 was calculated to be 1250 (26). This is unusually high for an anti-cancer preparation. The therapeutic index is the ratio of the toxic dose to the therapeutic dose and reflects the level of safety of a medicine. The therapeutic index of conventional cytostatic preparations is in the range of 1. 4-1.8 meaning that an overdose can have fatal consequences. There is no risk of an overdosage with NSC631570 on account of its very high therapeutic index of 1250.
The development of NSC631570 was a trail-blazing discovery. The preparation has shown that the problem can be solved and has changed our ideas about healthy and cancer cells.
The presentation at the congress was greeted both by scepticism and great interest. Many reputed research institutes such as the National Cancer Institute (USA), EORTC, the University of Miami, the Rochester University (USA), and University of Tübingen (Germany) began to test the preparation to better explain its unique properties and anticancer potential. In the NCI test model, in contrast to conventional cytostatic preparations which caused the growth inhibition only in some cancer cell lines, like thiotepa inhibited the growth of MLI-09 (non-small lung cancer) and UOK-57LN (renal cancer), NSC631570 killed all 60 tested cancer cell lines (190), which represent the eight important human tumours, including the cell lines which were resistant to the strongest cytostatic drug at the time, cisplatin.
This generated still more interest in scientific circles. Leading scientists examined NSC631570, each group with the method available. Thanks to this variety of experimentation the fine mechanisms of action of NSC631570 at different levels could be deciphered: firstly at cellular level with oxygen consumption (effect on mitochondria; 38), then on the level of chromosomes (effect on DNS and RNS; 58, 63), cell organelles and molecules (43, 62). These experiments produced extremely interesting results and not only confirmed the selective effect of NSC631570 but thoroughly destroyed all doubt on the matter. This means that NSC631570 can differentiate between healthy and malignant cells, a feature not yet managed by other anti-cancer drugs. Interest in NSC631570 is growing and research is continuing (261-266).
Researchers at the University of Natural Resources and Life Sciences, Vienna compared NSC631570’s inhibitive effect on the reproduction of malignant and normal cells. In order to achieve 50% growth inhibition a tenfold concentration of NSC 631570 had to be administered to normal endothelial cells in comparison to a human osteosarcoma cell line. Laser scanning microscopy showed a high capacity to absorb NSC631570 in malignant cells while absorption in normal cells was considerable lower under the same experimental conditions (36).
In a study of the effect of NSC631570 on K-562 erythroleucemia cells it was found that the preparation causes bimodal cell death. At lower NSC631570 concentrations malignant cells die as a result of apoptosis, at higher concentrations the formation of microtubules is prevented und polyploidy occurs (62).
In 1998 a group around Anne Panzer (University of Pretoria, South Africa) proved the selective effect of NSC631570 on molecular level. Tests on human cervical carcinoma cells HeLa, squamous cell carcinoma WHCO5 and normal equine lung cell lines demonstrated that NSC631570 is selectively toxic against cancer cells. It causes a metaphase block which is characterised by an abnormal distribution of chromosomes and the formation of micronuclei and results in apoptosis. Normal cells are not influenced in the process (139).
In 2000 in a study of cell proliferation after absorption of BrdU in the cell lines AsPC1, BxPC3, MiaPaCa2, Jurkat and THP-1 and the cell cycle phases – with the help of Giemsa staining, researchers from Ulm found that 10 µg/ml NSC631570 causes a clear accumulation of cancer cells in phase G2/M. Interestingly no difference was seen in the rate of apoptosis in normal peripheral mononuclear cells treated with NSC631570 compared to those untreated. The blastogenic response of mitogen-stimulated lymphocytes was even significantly increased. The authors showed that NSC631570 blocks pancreas cancer cells in the prophase by inhibiting tubulin polymerisation (181). This study confirmed that NSC631570 exerts no influence on normal cells.
Also in 2000 researchers at Rochester University (USA) examined the effect of NSC 631570 on concentrations of cyclins and cyclin-dependent kinases in epidermoid carcinoma cell lines ME180 and A431 as well as the prostate cancer cell line LNCaP. Changes were found in the concentrations of mitotic cyclins A and B1 as well as CDK1 and CDK2. The researchers also observed increased expression of the CDK-inhibitor p27 in both cell lines, which led to the accumulation of cancer cells but not normal cells in the G2/M phase (147, 149).
In another study from 2000 entitled „NSC631570TM, a semisynthetic Chelidonium majus alkaloid derivative, acts by inhibition of tubulin polymerization in normal and malignant cell lines“ (Cancer Letters 160 (2000) 149-157)  researchers from South Africa found that NSC631570 inhibits the polymerisation of tubulin (185). The authors used human fibroblasts as a normal cell line.

Normal human fibroblasts were later used in the tests by the scientists of the Eberhard-Karls-University Tuebingen (Germany) as well as of the Instituto Nacional de Cancerologia (Mexiko City, Mexiko), also. Both research teams could not find any toxic effects of NSC631570 on these normal cell lines. Moreover, A. Panzer et al noted in their article: „Both NSC631570TM and chelidonine had weak activity in this system…“ (185).

Drug Concentration

Fig. 1. Effect of different NSC631570 concentrations on G2/M cell population. Cells were treated with the indicated concentrations for 24 h and analyzed for DNA content by flow cytometry. The data presented are the percentage of NeoK (normal human keratinocytes), A431 and ME180 (epidermoid carcinoma cells) cell populations in G2/M phase. From Roublevskaia et al, 2000 (147).

In 2002 scientists at the Eberhard-Karls-University Tuebingen (Germany) examined the effect of NSC 631570, alone or combined with radiation (1-10 Gy), on cell survival, the modification of the cell cycle and the induction of apoptosis in the exponentially growing human tumour cell lines MDA-MB-231 (breast cancer), PA-TU-8902 (pancreatic cancer), CCL-221 (colonic cancer), U-138MG (glioblastoma) and the human skin fibroblasts HSF1, HSF2 and lung fibroblasts CCD32-LU. Without radiation NSC 631570 had a time and dose dependent cytotoxic effect which was more pronounced in cancer cells than in normal cells. Combined with radiation NSC 631570 resulted in increased cytotoxicity against colonic cancer and glioblastoma cell lines but not against breast cancer and pancreatic cancer cell lines. By means of flow cytometry it was shown that NSC 631570 modulated the toxic effect of radiation on these human cancer cell lines by causing their accumulation in the G2/M phase of the cell cycle. Its protective effect on normal human fibroblasts speaks in favour of its use in combined radio-chemotherapy (184).

In 2005 the ability of NSC631570 to induce apoptosis was studied at the University Hospital Tuebingen (Germany) on a Jurkat lymphoma model. NSC631570 proved itself to be a strong inducer of apoptosis. More detailed investigations showed that it caused the depolarisation of mitochondrial membranes and as a result the activation of caspases (246).

Cell lines

Fig. 2. Survival of irradiated cells with and without NSC631570 treatment. R – irradiation, 2 Gy; R + U – irradiation, 2 Gy and treatment with NSC631570. Cell lines: CCL-221 - colorectal carcinoma, U-138MG - glioblastoma, HSF1 und HSF2 - normal fibroblasts. After Cordes et al, 2002 (184).

In 2006 researchers at the Instituto Nacional de Cancerologia (Mexico City, Mexico) found that NSC631570 triggers apoptosis in a series of cancer cell lines (human cervical carcinoma HeLa, HeKB, HeKS32, HeBcll3, HeNFR and HelKK, colonic cancer SW480, kidney cancer HEK293, osteosarcoma MG 63) by activating the intrinsic cell death pathway. Interestingly the non-transformed fibroblast cell line hTERT was not sensitive to this drug (255).
At the University of Pisa (Italy) the cytotoxic effect of NSC 631570 was examined on two primary pancreatic cancer cell lines (PPTCC), fibroblasts from ductal pancreatic cancer tissue samples (F-PDAC) and an immortalised ductal epithelial pancreas cell line (HPNE). Cytotoxicity was established by means of CellTiter 96 kit. The modulation of NSC 631570 absorption in the medium was determined with the help of the fluorescence of NSC 631570 under UV light. The cytotoxic effect of NSC 631570 on pancreatic cancer cell lines was considerably higher than on the fibroblasts and epithelial cells (20% as against 80% living cells). In addition the fluorescence test showed that PPTCC cells absorbed more of the preparation than F-PDAC and HPNE cells. These results show a selective effect of NSC 631570 on the pancreatic cancer cells, which indicates different transport systems or a higher rate of metabolism of the preparation in the PDAC cells (265).

As can be seen, many aspects of the effect of NSC631570 on cancer and normal cells have so far been studied. Nevertheless, the possibility exists that these effects are merely the results of an as yet unknown process which NSC631570 induces in cancer cells but not in normal cells. As NSC631570 killed in the tests almost all cancer cell lines, it can be suggested that a factor occurs when a normal cell becomes a cancer cell. This factor is affected with NSC631570. If this phenomenon can be deciphered it could provide very important indicators for the significant difference between normal cells and cancer cells, point towards the real cause of cancer and open up completely new perspectives for the development of new anti-cancer drugs, not only for treatment but also for use in cancer prevention.

Cellular dencity

Fig. 3. Effects of NSC631570 on cervical carcinoma cell line HeLa and on the normal fibroblasts hTERT expressed as percentage of living cells after 48 h incubation with NSC631570 40 µg/ml. From Mendoza et al, 2006 (255).

These studies on the selective effect of NSC631570 clearly revealed cancer cells can be killed without any damage to the health tissues.

 

Affinity of NSC 631570 to the cancer cells

It is well known all living cells have a membrane potential of about -60 to –100mV. The negative sign of the membrane potential indicates that the inside surface of the cell membrane is relatively more negative than the than the immediate exterior surface of the cell membrane.
As far back as 1938 Dr. Paul Gerhardt Seeger originated the idea that destruction or inactivation of enzymes, like cytochrome oxidase, in the respiratory chain of the mitochondria was involved in the development of cancer.
1948 G.R. Mider and co-workers revealed the cancer cells are more negatively charged than normal cells.
During the production of NSC631570 celandine alkaloids become positively charged. This explains the high affinity of NSC631570 to the cancer cells.
Researchers at the University of Natural Resources and Life Sciences, Vienna (Austria) revealed human osteosarcoma cells to have a high capacity to absorb NSC631570 while absorption in normal endothelial cells was considerable lower under the same experimental conditions (36).
This selective uptake of NSC631570 in the cancer cells has been confirmed due to its unique property to autofluorescence under UV light (5).
A study examined how NSC 631570 affects the electrokinetic potential (EKP) of malignant and benign cells. The EKP of the Ehrlich’s carcinoma cells dropped after incubation with NSC 631570. The EKP decrease of normal cells was less pronounced (221).
The cytotoxic effect of NSC 631570 was examined on two primary pancreatic cancer cell lines (PPTCC), fibroblasts from ductal pancreatic cancer tissue samples (F-PDAC) and an immortalised ductal epithelial pancreas cell line (HPNE). Cytotoxicity was established by means of CellTiter 96 kit.
In the tests with primary pancreatic cancer cell lines the modulation of NSC 631570 absorption in the medium was determined with the help of the fluorescence of NSC 631570 under UV light. The fluorescence test showed that carcinoma cells absorbed more of the preparation than fibroblasts and normal epithelial cells (265).
The selective accumulation of NSC 631570 in the cancer cells explains its favourable safety profile and high therapeutic index of 1250.

 

Immune modulating properties

Unusual for an anticancer agent NSC 631570 possesses some distinct immune properties (24, 44). It was Prof. Andrejs Liepins of the St. John’s Memorial University, St. John’s, Canada who first pointed to this interesting fact. In the work with the C57BL/6 mice he revealed NSC631570 to be an effective biological response modifier (BRM). After incubation with NSC631570 the lytic activity of the splenic lymphocytes from the alloimmunised mice increased up to 48 fold (fig. 4). On the day 18 this increase was the highest and then faded slightly (fig. 5). The lytic activity of the interleukin-2 treated spleen cells and of the peritoneal exudate lymphocytes increased as well (17).
In several immune target-effector systems NSC 631570 significantly amplified the malignotoxic activity of macrophages (231), lymphocytes and NK cells (47), and stimulates dendritic cells maturation in vitro (258). While the parameters like B-lymphocytes count, immune globulin concentrations, complement and acute phase proteins did not changed significantly, it can be postulated NSC 631570 modulates the cellular part of the immune system whereas the humoral part remains unaffected.
Human lymphocytes as well as guinea pig lymphocytes were activated more pronounced when incubated with NSC631570 than with phytohemagglutinin. In rats NSC 631570 caused a clear increase of macrophages with NK-activity (49).

It was revealed in the tests on CBA mice and Wistar rats that NSC 631570 stimulates macrophages. As marker of this activity the enzyme chitotriosidase, a part of the native immunity was used (168).

Cytolytic Activity

Fig. 4. The effect of NSC631570 on the cytolytic activity of spleen (SL) and peritoneal (PL) lymphocytes from alloimmunized mice. From Liepins et al, 1992 (17).

Days after alloimmunisation

Fig. 5. The effect of 1.18 µM NSC631570 on the cytolytic activity of spleen cells harvested at various time intervals after autoimmunization. From Liepins et al, 1992 (17).

In a study on intact and thymus-ectomised mice NSC 631570 augmented the endocrine function of thymus. After NSC 631570 administration, increased production of substances with thymosin-like activity was detected. Repeated administration of NSC 631570 caused a 2fold rise of T-cells in blood, a 4.5fold rise of large granulocytes and increase of the NK-activity of splenocytes. The production of interferon and antibodies after the antigen administration was increased as well (180).
The effect of well-known immune modulators interferon-gamma, NSC 631570 and pokeweed mitogen on the selective uptake of technetium-99m(99mTc)-labelled tumor necrosis factor (TNF) was studied in the intramuscular implanted murine embryonic carcinoma. The highest absolute tumor uptake of 99mTc- TNF was achieved when NSC 631570 was used, followed by IFN-γ and pokeweed mitogen (fig. 6; 104).
In the experiments on BALB/c and F1 (BALB/c x C57BL/6J) mice it was revealed that NSC 631570 inhibits the allergic sensitization of animals against ovalbumin, expressed in the weakened IgE-reaction and decreased histamine release. The incubation of ovalbumin with NSC 631570 induced decreased antigenicity of this protein (84).

Absolute tumor uptake

Fig. 6. Absolute (% administered dose/g) tumor uptake of -TNF-α in mice BALB/c 1,5 h after administration. After Thakur et al, 1992 (104).

The immune modulating effect of NSC 631570 was studied in several studies in mice. Repeated subcutaneous injections of NSC 631570 to mice infected with the twofold LD50 of E. coli, S. aureus, or influenza virus increased the survival rate of the animals significantly (60, 87, 89).
When human lymphocytes were incubated with phytohemagglutinin (PHA) and NSC 631570, increased absorption of 3H-thymidin in the cells was observed. The authors point out the strong synergetic effect of NSC 631570 and phytohemagglutinin (76).
By means of the cell proliferation assay the mitogenic effects of PHA and NSC 631570 on human peripheral blood mononuclear cells (PBMC) were studied. It was revealed even a short pretreatment of the PBMC with NSC 631570 has a strong synergetic effect on the PHA-mitogenesis. Consequently, the cell stimulation parameters were much higher after combined stimulation than after using PHA alone (65).
In experiments with murine (CC57 Black/6) macrophages and rabbit G-actin, the effects of NSC 631570 and sanguinarine on phagosome-lysosome membrane fusion and actin cytoskeleton were studied. The most stimulating effect on the phagosome-lysosome fusion exerted sanguinarine at 10 µmol and NSC 631570 at 5 µmol. At the same dose NSC 631570 doubled the content of fibrillary actin in murine peritoneal macrophages. Moreover, NSC 631570 and sanguinarine induced the polymerisation of rabbit globular actin. These effects were dose-dependent. The authors suggest sanguinarine and NSC 631570 can alter intracellular membrane transport (231).
The publications mentioned here point out that NSC631570 due to its many beneficial immune properties can be categorized as one of the best immune modifier.

 

The angiogenic properties

Another important feature of NSC631570 is the inhibition of the formation of the new blood vessels supplying a tumor. Due to these antiangiogenic properties NSC631570 administered before surgery brings about better demarcation of the tumor from surrounding tissue and the tumor encapsulation. This alleviates the surgical removal of tumors what has been confirmed in breast cancer studies (68-73, 114). In tests in vitro, NSC631570 inhibited in a dose-dependent manner the proliferation of human endothelial cells without exerting cytotoxic effect. The angiogenesis inhibition was observed on the capillary formation model (136). This inhibition of the neoangiogenesis prevents the metastasis formation as well.

Total capillar tube length

Fig. 7. The length of the total capillary tubes after the incubation with NSC631570 in given concentrations. 1 – HUVEC were incubated with NSC631570 for 4 h. 2 – HUVEC were preincubated with NSC631570 for 4h and then incubated for 2 h in fresh medium without NSC631570. HUVEC – human umbilical vein endothelial cells. After Koshelnick et al, 1998 (136).

EFFICACY: the in vivo studies

THE IN VIVO STUDIES

The anticancer effect of the intravenous administration of NSC 631570 (4 µg/day) was demonstrated in the tests on BALB/c mice with the implanted rapidly proliferating mammary adenocarcinoma D1 DMBA-3. Three routes of administration were employed for NSC 631570, e.g. intravenous, intraperitoneal and subcutaneous. Intravenous administration was proven to be the most effective and inhibited the tumor growth significantly. The authors evaluated also the role of immune factors in the effect of NSC 631570 and revealed this agent can restore the cytotoxic effect of macrophages (26, 43).

Later this mode of action was confirmed in a study on CBA mice. Once again the role of macrophages in the antitumor effect of NSC 631570 was stressed by the authors of the study (120).

In an experimental study NSC 631570 inhibited the growth of the primary melanoma B-16 and its metastases in mice (107).

In the tests with mice C57BL6 NSC 631570 inhibited the growth and metastazing of the primary implanted Lewis’ carcinoma compared to the control group (219). This effect of NSC 631570 was confirmed in another study. Here too, the intravenous administration was the most effective (254).

In a study on 18 mice CBA, the mixture of 0.1 mg NSC 631570 and 10000 carcinoma cells Krebs-2 was injected intramuscularly. In the control group only tumor cells were injected. After 11 days a significant inhibition of the tumor growth was established in the group treated with NSC 631570. Similar effect was achieved also in mice with implanted hepatoma cells (118).

In mice with implanted hepatoma HA-1, NSC 631570 increased the concentration of procathepsin B in the ascitic fluid whereas the concentration of cathepsin B decreased. Moreover, the concentration of the macrophageal p-hexosaminidase increased. This indicates an inflow of macrophages into the peritoneum (119).

The anticancer effect of NSC 631570 was also confirmed on Wistar rats with implanted sarcoma-45 (122).

Two different forms of Ehrlich’s carcinoma cells were transplanted intraperitoneally and subcutaneously to mice. NSC 631570 was administered intraperitoneally for six days. The effect of the drug on the tumors was estimated by the indexes of the tumor growth inhibition (TGI), total number of tumor cells in ascitic fluid, number of viable tumor cells and average life span of experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. The number of circulating phagocytes was estimated by flow cytometry using FITC-labelled S. aureus. Intraperitoneal administration of NSC 631570 in mice with ascitic form of Ehrlich’s carcinoma resulted in moderate tumor growth inhibition, but was accompanied by acute local inflammation and caused reduction of life span of experimental animals. In the mice bearing the solid variant of Ehrlich’s carcinoma the treatment with NSC 631570 led to significant TGI and slight increase of life span. The treatment caused restitution of the number of circulating phagocytes in peripheral blood in the both groups of animals. The authors concluded the antitumor effect of NSC 631570 was mediated by its direct proapoptotic action as well as by the interactions with the murine immune competent cells (262).

 

The Effect on the Growth of Melanoma B16 in Mice

The effect of NSC-631570 alone or in combination with pathogen-associated molecules (PAM) on the growth of low- and high-metastasizing melanoma B16 in mice was studied. NSC-631570 was administered intravenously and PAM intramuscularly to tumor-bearing mice seven times every third day, starting from the second day after the transplantation of tumor cells. The effect of monotherapy and combined therapy on tumor growth was evaluated by the indices of tumor growth inhibition in experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. TAP1 and TAP2 expression was evaluated by RT-PCR. The metabolic activity of phagocytes was determined by NBT-test, phagocytosis was tested by flow cytometry, and arginase activity was estimated by colorimetric determination of urea. Combined therapy and monotherapy with NSC-631570 resulted in significant inhibition of tumor growth in melanoma-bearing mice. Monotherapy with Ukrain was more effective in mice with high-metastasizing tumors. The therapeutic efficacy of NSC-631570 used in combination with PAM was more expressed in mice with low-metastasizing melanoma. Authors concluded the effectiveness of monotherapy and combined therapy with NSC-631570 in the treatment of melanoma B16 depends on the biological properties of the tumor and the immune state of the animal (263).

 

The Effect on Cystein Proteases

In mice with implanted LS-lymphosarcoma and HA-1 hepatoma, the concentrations of cystatin C, cathepsin B and cathepsin L were estimated after the treatment with cyclophosphamide and NSC 631570. An increased concentration of cystatin C was found only in hepatomas treated with NSC 631570. The concentrations of cathepsin B and cathepsin L were increased in LS lymphosarcoma. These results indicate apoptosis as the mechanism of action of NSC 631570 in these tumor models (169).

On the same tumor models the effect of NSC 631570 on cathepsin D was studied. Cathepsin D is an important lysosomal protease, modulating apoptosis induced by interferon-gamma and TNF-alpha. NSC 631570 combined with cyclophosphamide increased cathepsin D activity in mice with LS-lymphosarcoma. NSC 631570 alone did not exert this effect (170).

Cystatin C is a well known extracellular protease inhibitor and has been used as a possible marker of the tumor growth and efficacy of the anticancer therapy (204). The using NSC 631570 in mice brought about a 4fold increase of cystatin C in the tumor tissue (171).

In the tests on male mice CBA/C57BI/6J the serum concentration of cystatin C after the implantation of Lewis’ adenocarcinoma was only half as high as in the control group. The combined treatment with NSC 631570 and cyclophosphamide normalised the cystatin values (218).

On the experimental mice tumors LS-lymphosarcoma, hepatoma HA-1 and Lewis’ adenocarcinoma, the effect of NSC 631570 on the cystatin A (stefin A) was studied. The treatment with NSC 631570 caused the increase of stefin A in the liver of animals with Lewis’ adenocarcinoma, but not in the tumor itself. In other tumor models the concentration of cystatin A both in the liver and tumor tissue did not change. In LS-lymphosarcoma and hepatoma HA-1 the serum concentration of cystatin A increased in the course of the treatment with NSC 631570 (220).

The treatment of the hepatoma HA-1 implanted mice with NSC 631570 caused the deceleration of the tumor growth and increased the survival of the animals. The macrophages count increased whereas the number of tumor cells in the ascitic fluid diminished. NSC 631570 did not affect the activity of cathepsin B and cathepsin L (199).

EFFICACY: antiviral properties

ANTIVIRAL PROPERTIES

The researchers from St. Petersburg, Russia have used the antiviral properties of NSC 631570 in their works with viral hepatitis C (HVC). They reveal NSC 631570, at optimal dosage, has brought about the elimination of the virus from the blood in 40 (80%) of 56 patients (165).

In their further study these researchers compared the effects of NSC 631570 at various dosages with the recombinant human interferon-alpha-2b (IFN) in 75 HVC patients. The best results were achieved when NSC 631570 was administered at a single dose of 1 mg (203).

Pilot studies evaluating the effect of NSC 631570 on HIV virus and related diseases were also performed (12). The authors noted, for example, improved immune values after the therapy (103).

The antiviral properties of NSC 631570 were confirmed in the in vivo experiments (42, 51, 52, 88, 90).

THE INHIBITION OF THE TUMORAL ANGIOGENESIS

NSC 631570 inhibits the formation of the new blood vessels supplying a tumor. Due to these antiangiogenic properties NSC 631570 administered before surgery brings about better demarcation of the tumor from surrounding tissue and the tumor encapsulation. This alleviates the surgical removal of tumors what has been confirmed in breast cancer studies (68-73, 114). It is recommended to reduce the tumor burden 7-10 days after the start of the therapy with NSC 631570.

In tests in vitro, NSC 631570 inhibited dose-dependent the proliferation of human endothelial cells without exerting cytotoxic effect. The angiogenesis inhibition was observed on the capillary formation model. The tumor angiogenesis is the formation of new blood vessels supplying a growing tumor with nutrients. Angiogenesis is of critical importance for the tumor growth (136).

Inhibition of the capillary sprouting by NSC 631570 in the endothelial spheroids model

Hemmung der Sprossen durch Ukrain

Pancreatic adenocarcinoma, preoperative treatment with Ukrain, total 100 mg, 10 days prior to surgery (pancreatico-duodenectomy). Formation of a capsule (A) around the tumour. Tumour cells do not infiltrate the capsule. Massive round cell infiltration (B) of the tumour–capsule border area. H-e, x100.

Bauchspeicheldrüsen-Adenokarzinom

Pancreatic adenocarcinoma, preoperative treatment with Ukrain, total 100 mg, 10 days prior to surgery. Necrosis of tumour tissue (A), inhibition of new vessels formation (defect in capillary wall, defect in endothelial covering, B). H-e, x200.

Pancreatisches Adenokarzinom

Pancreatic adenocarcinoma, preoperative treatment with Ukrain, total 100 mg, 10 days prior to surgery. Nuclear changes: Chromatin dispersion (A) and fragmentation (B), hydropic cytoplasm degeneration (C). “Iron” hematoxylin – van Gieson, x500.

Pancreatisches Adenokarzinom



THE IN VITRO STUDIES

Clinical efficacy of NSC 631570 is not coincidental or even ‘spontaneous remission’ but rather a consequence of its mechanisms of action confirmed in various in vitro and in vivo studies. NSC 631570 has been tested on more than 100 cancer cell lines so far.Among others, NSC 631570 was tested at the National Cancer Institute (Bethesda, Maryland, USA) on 60 cell lines representing eight important human malignant tumors: brain tumors, ovarian, small cell and non-small lung cancer, colon cancer, kidney cancer, leukaemia and malignant melanoma. NSC 631570 exerted toxic effects against all these cell lines (40, 190). Compared to 5-fluorouracil (5-FU) and gemcitabine, two standard cytotoxic agents in the treatment of digestive tract tumors, NSC 631570 achieved better results and not only inhibited the cell growth but reduced the cell mass, also.

Vergleich der Toxizität von Ukrain (NSC 631570) und 5-Fluorouracil (NSC 19893)

The cytotoxic effect of NSC 631570 on the cancer cell lines M-HeLa and Hep-2/0-6-5 war more pronounced than the effect of a synthetic agent Oliphen (61). NSC 631570 inhibited the growth of four Ewing’s sarcoma cell lines dose- and time-dependent. In this experiment the effect of NSC 631570 was more pronounced than this of thiotepa (243, discussed in 244). In the tests on Ehrlich’s carcinoma cells and lympholeucemia P-388, the researchers revealed that the sensitivity of the cancer cells to NSC 631570 depends highly on the cell cycle phase, the first sensitivity peak being at the end of G1 phase and the second - in G2 phase (148). The effects of glucose, succinat, pH value and increased temperature on the efficacy of NSC 631570 against cancer cells were investigated in vitro. Glucose reduced the cytotoxic effect of NSC 631570, but succinat intensified it. The most pronounced effect was at pH 7.3-8.0. The temperature up to 41.5 °C did not impact the effect of NSC 631570 (117).

Results of the Ukrain (NSC 631570) study at the National Cancer Institute, Bethesda, Maryland, USA. The effects of NSC 631570 on the 60 various human cancer cell lines

THE IN VITRO STUDIES

The Selective Effect of NSC 631570

In comparative studies NSC 631570 was tested on 18 malignant and 12 non-malignant cell lines at identical conditions (36, 38, 63, 143, 147, 149, 181, 184, 190, 245, 255). These experiments eliminated all doubts on the selective effect of NSC 631570. Numerous studies have confirmed NSC 631570 to be the first and only anticancer agent being toxic against cancer but not normal cells. This explains also its good tolerability in clinical use.

First indications on the selective effect of NSC 631570 on the cancer cells provided an early study in 1976 of the Bundesstaatlichen Anstalt für Experimentell-Pharmakologische und Balneologische Untersuchungen (Vienna, Austria). This study revealed different oxygen consumption by normal liver cells and Ehrlich’s tumor ascitic cells after the incubation with NSC 631570 was revealed (38).

About at the same time, the researchers from the Vienna University of Agriculture compared the inhibiting effect of NSC 631570 on the proliferation of malignant and normal cells. For 50% growth inhibition of normal endothelial cells, the NSC 631570 concentration had to be tenfold higher than for the same growth inhibition of human osteosarcoma cell line (36). 

The different influences of Ukrain on oxygen consumption in healthy cells and cancer cells

Einfluss von Ukrain auf den Sauerstoffverbrauch in Zellen

Die Aufnahme von Ukrain in Melanom-Zellen, verglichen mit normalen Zellen (in vitro)

Ukrain in Melanoma cells

Hohenwarter O. et al. Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain, Drugs under Experimental Research, 1992 (36).

This selective effect of NSC 631570 on cancer cells has been confirmed in numerous studies at the renowned universities and research facilities.

In the study from European Organisation for Research and Treatment of Cancer (EORTC), human tumor xenografts (HTX) were implanted into nude mice. These tumor cells were later incubated with NSC 631570 at various concentrations. Following HTX were used: colon cancer CXF 1103/11, stomach cancer GXF 217/17, lung cancer LXFL 529/14, breast cancer MAXF 401/13, melanoma MEXF 276/10, and ovarian cancer OVXF 899/9. NSC 631570 was active against OVXF 899/9 at 10 µg/ml and in all colonies tested at 100 µg/ml with the T/C-ratio (‘test to control’) 1/135 in OVXF, 8/109 in CXF, 10/98 in GXF, 15/187 in LXFL, 34/133 in MAXF, and 10/122 in MEXF (64, 189).

In 1998, at the 89th Annual Meeting of the American Association for Cancer Research in New Orleans, USA, researchers from the University of Pretoria, South Africa presented their work on the selective effect of NSC 631570 on various cancer cell lines. The authors concluded ‘that Ukrain is selectively toxic to malignant cells by causing a metaphase block which is characterised by abnormal chromosomal distribution, and results in the formation of micronuclei and in apoptosis’ (139). In 2000 the same group discovered NSC 631570 to inhibit the tubulin polymerisation. In this paper they denied the selective mode of action of NSC 631570 on cancer cells (140).

Researchers from Eberhard-Karls-University Tubingen, Germany, investigated the effects of NSC 631570 on cell survival, alteration of the cell cycle and induction of apoptosis without and in combination with ionising radiation (IR) at a dose of 1-10 Gy. The tests were performed on the exponentially growing human tumor cells MDA-MB-231 (breast), PA-TU-8902 (pancreas), CCL-221 (colon cancer), U-138MG (glioblastoma), and human skin and lung fibroblasts HSF1, HSF2 and CCD32-LU. Without IR, NSC 631570 exerted a time- and dose-dependant cytotoxic effect, more pronounced against the cancer cells. Flow cytometry revealed NSC 631570 to modulate radiation toxicity against human cancer cell lines and to protect normal cells from radiation. The combination of NSC 631570 plus IR gave enhanced toxicity in CCL-221 and U-138MG cells with their accumulation in the G2/M phase of the cell cycle, but not in MDA-MB-231 and PA-TU-8902 cells. A radio protective effect was found in normal human skin and lung fibroblasts. The authors suggest a reasonable use of NSC 631570 in combined radiochemotherapy (184).

The cytotoxic effects of NSC 631570 were evaluated in two primary pancreatic cancer cell lines (PPTCC), fibroblasts derived from pancreatic ductal adenocarcinoma specimens (F-PDAC), and an immortalized epithelial ductal pancreatic cell line (HPNE). Cytotoxicity was assessed by the CellTiter 96 kit based on the cellular metabolism of the tetrazolium compound XTT, which is reduced by living cells to yield a soluble formazan product in the presence of the electron coupling agent phenazine methosulfate, while the modulation of NSC 631570 uptake in the medium was studied using the fluorescence of NSC 631570 with the AlphaDigiDoc software by UV light excitation. Cytotoxic effects of NSC 631570 in PPTCCs were significantly higher than those observed in F-PDAC and HPNE cells (20% vs. 80% alive cells). Furthermore, the ULA-DC test revealed that PPTCCs cells consumed more drug than F-PDAC and HPNE cells. These data demonstrated the selective effect of Ukrain in PPTCCs, which may be related to a different transport system or higher metabolism of the drug in PDAC, and warrant further investigations in order to support the possible role of Ukrain in PDAC treatment (265).

Induction of the Apoptosis in Cancer Cells

In the study on the erythroleucemia cells K-562, it was revealed NSC 631570 to bring about the bimodal death of cancer cells. At lower concentrations of NSC 631570, cancer cells die in as a consequence of apoptosis. At higher concentrations, the formation of microtubules is inhibited and polyploidy occurs (56, 62).

The researchers from the Rochester University, USA revealed NSC 631570 causes the accumulation of prostate cancer cells as well as epidermoid carcinoma cells in the G2/M phase, however, not of normal cells (149, 150).

In the tests on human cervix carcinoma cells HeLa, squamous carcinoma cells WHCO5, normal kidney cell line Graham 293, and transformed kidney cell line Vero from African green monkey the researchers of the University of Pretoria, South Africa revealed NSC 631570 ‘is selectively toxic to malignant cells by causing a metaphase block which is characterised by abnormal chromosomal distribution, and results in the formation of micronuclei and in apoptosis’ (139).

The scientists of the Eberhard Karl University (Tübingen, Germany) investigated the effect of NSC 631570 on the cell survival, the cell cycle modification and the apoptosis induction alone and combined with radiation (IR). They discovered NSC 631570 combined with IR increased the toxicity against the cell lines CCL-221 and U-138MG. The normal human skin and lung fibroblasts were protected from the damaging effects of IR (184).

Ukrain’s (NSC 631570) deadly action against cancer cells - Induction of apoptosis

Apoptose

Estimating the cell proliferation according to the BrdU uptake in the cell lines AsPC1, BxPC3, MiaPaCa2, Jurkat, and THP-1 and the cell cycle phases by means of Giemsa staining, the authors established NSC 631570 at a dose of 10 µg/ml brought about a considerable accumulation of cancer cells in the G2/M phase after 24 h incubation. The apoptosis rate in the peripheral mononuclears was similar at the same incubation conditions. Moreover, the mitogene stimulated lymphocytes showed increased blastogen reaction (181).

The apoptosis induction by NSC 631570 has been also confirmed in the in vitro tests on the Chinese hamster ovarian cells. In this experiment the effects of NSC 631570 and etoposide were synergistic (167).

 

Inhibition of the Tubulin Polymerisation

In the experiments with cow brain tubulin, the scientists from the Pretoria Universioty (South Africa) discovered NSC 631570 to inhibit the tubulin polymerisation (146).

Later on, the researchers from the University of Ulm revealed in the tests with pancreas cancer lines AsPC1, BxPC3, Capan1, MiaPaCa2, and Panc1 NSC 631570 brings about a dose dependant cell cycle arrest in the G2/M phase. Moreover, NSC 631570 stabilizes the tubulin monomers in cancer cells and consequently inhibits the formation of microtubules (143).

 

Activation of Mitochondrial Caspases

Caspases, or cysteine-aspartic proteases are a family of enzymes, which play essential roles in apoptosis (programmed cell death) hence in cancer therapy.

In the tests on the Jurkat lymphoma model, NSC 631570 has been proven to be a strong apoptosis inductor. Profound research showed NSC 631570 brought about the depolarisation of mitochondrial membranes and consequently the activation of caspases (246). 

There are two types of apoptotic caspases: initiator (apical) caspases and effector (executioner) caspases. Initiator caspases CASP8 and CASP10 induce the death factor dependant or extrinsic apoptosis pathway, whereas effector caspase CASP9 mediates the intrinsic or mitochondrial apoptosis. In the tests at the Nacional de Cancerologia, Mexico City, Mexico NSC 631570 revealed that Ukrain induced apoptosis in a panel of cancer cell lines (cervical cancer HeLa, HeKB, HeKS32, HeBcl3, HeNFR and HeIKK, human colon cancer SW480, human renal carcinoma HEK293, human osteosarcoma MG-63) by activating the intrinsic cell death pathway. Interestingly, non-transformed fibroblasts hTERT were insensitive to the drug (255).

 

The Effect on Cyclins and Cyclin-Dependant Kinases

Cyclins are a family of proteins which control the progression of cells through the cell cycle by activating cyclin-dependent kinase (Cdk) enzymes. A cyclin forms a complex with Cdk. Complex formation results in activation of the Cdk active site. When concentrations in the cell are low, cyclins dissociate from Cdk, thus inhibiting enzymatic activity; this probably occurs due to a protein chain of the Cdk blocking the active site upon cyclin dissociation. There are several different cyclins which are active in different parts of the cell cycle and which cause the Cdk to phosphorylate different substrates. Increasing concentration of cyclin A triggers the cell into the G2 phase, whereas cyclin B is essential for the mitosis initiation.

The researchers from the Rochester University explored the effect of NSC 631570 on cyclins and Cdk in the epidermoid cancer cell lines ME180 and A431 as well as in the prostate carcinoma line LNCaP. They found changes in the concentrations of mitotic cyclin A and B1 as well as Cdk1 and Cdk2. Increased expression of Cdk inhibitor p27 has been also observed in both cancer cell lines. This can promote accumulation of the cells in the G2/M phase (147, 149).

 

The Effect on the Expression of hENT1 and dCK

The interactions between NSC 631570 and the molecular determinant expressions involved in the metabolism of gemcitabine, such as hENT1 and dCK were evaluated in in vitro studies on pancreatic ductal adenocarcinoma cells (PDAC). Two ATCC cell lines (PL45 and MiaPaCa-2) and 2 Primary Cell Cultures obtained from PDAC patients underwent surgical resections (PPTCC78 and PPTCC109) were used. Cells were treated with Ukrain at IC50 concentration levels for 48h. All the amplifications were carried out with normalization of gene expression against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping control gene, and the quantitation of gene expression was performed. NSC 631570 positively modulated the expression of hENT1 mRNA in all PDAC cell cultures treated with IC50 (p<0.001). The analysis revealed a mean increase of 2.8 fold (p=0.001) compared to untreated control cells. In PL45 and MiaPaCa-2 cells NSC 631570 positively affects mRNA expression of dCK gene as well (264).
Based on the previous clinical data the NSC 631570-gemcitabine combination appears a promising regimen and the results of this study provided the experimental basis for the further clinical testing of the NSC 631570-gemcitabine schedule in PDAC patients (Funel et al, ‘Molecular mechanisms underlying the synergistic interaction of the novel anticancer drug Ukrain with gemcitabine in preclinical models of pancreatic cancer’, 44th Annual Pancreas Club Meeting, New Orleans, USA 2010).

 

The Effect on the DNA and Protein Synthesis

The effect of NSC 631570 on the DNA and protein synthesis in cancer cells was studied in vitro on the cell lines HeLa, Yoshida, mice lymphoma, mice myeloma, human WiDr tumor, EB, EsB, YAC-1, und P815. NSC 631570 (1-100 µg/ml) has been proven to inhibit in dose-dependant way the DNA, RNA and protein synthesis in these cell lines. At the same test conditions, the inhibition in normal cells (human tonsil cells and guinea pig liver cells) was much lesser distinct (58, 63).

Among many possible mechanisms of action of NSC 631570, its effect on the endonucleases of the rat liver and on topoisomerases was studied (topoisomerases play an important part in the DNA metabolism). In these experiments NSC 631570 inhibited the metal-dependant endonucleases and the topoisomerase I as well (166).

 

The Effect on the Proteins Involved into the Remodelling of Extracellular Matrix

An Italian research team from the University of Milan used RT-PCR, Western blot analysis and SDS-zymography to evaluate the effect of NSC 631570 on the expression of genes and proteins involved into the remodelling of the extracellular matrix. This mechanism plays an important role in the tumor invasion of human glioblastoma cells. There was a significant dose-dependant decrease of the proliferation of glioblastoma cells and a trend to the down-regulation of secreted protein acidic cysteine rich (SPARC). This study provided theoretical reasons for the using NSC 631570 in glioblastoma. The authors concluded: NSC 631570 ‘may be a useful therapeutic tool for brain tumors’ (245). This has been proved in clinical use (see “Brain tumors”).

In their further work with glioblastoma cells the Italian researchers revealed NSC 631570 to increase the expression of the glial fibrillary acidic protein (GFAP). The connexion 43 expression was not modulated by NSC 631570. These results ‘support the possible potential of NSC 631570 for the therapy of brain tumors’ (250).

This Italian team investigated also whether NSC 631570 is able to modulate the in vitro expression of some proteins involved in tumor progression on three clear cell type renal carcinoma cell lines (ccRCC). CAKI‐1, CAKI‐2 and ACHN clear cell carcinoma (ccRCC) cells were treated with three doses of NSC 631570 (5, 10, and 20 μM) or left untreated, and cultured for 48 h in duplicate. SPARC protein levels were assayed in Western blot, MMP‐2 and MMP‐9 protein levels and activity were determined by SDS‐zymography in cell culture supernatants, and the distribution of the different cell cycle phases was calculated using FACS analysis. The results suggest that NSC 631570 modulates two major aspects involved in tumorigenesis of RCC cancer cells, that are extracellular matrix remodelling and cell proliferation. The tendency to MMP‐2 and MMP‐9 down‐regulation in UK‐treated cells suggests that UK may decrease RCC cell invasion. Moreover, SPARC protein down‐regulation in supernatants point to an inhibition elicited by UK also on extracellular matrix remodelling in the tumor microenvironment, possibly rendering the tumor microenvironment less permissive for tumor invasion. At the same time, SPARC intracellular protein levels up‐regulation in ccRCC cells suggest that NSC 631570 may be involved in the inhibition of cell proliferation by cell cycle inhibition, as shown by FACS analysis. (Pettinari et al, ‘Matrix metalloproteinases activity and SPARC expression are targeted by Ukrain administration in renal cell carcinoma’, presentation at the symposium ‘Targeting Cancer Invasion and Metastasis’, Miami, USA, 2010).

The effect of NSC 631570 on the modulation of some of the key markers of tumor progression in pancreatic carcinoma was investigated on three cell lines HPAF-II, PL45, and HPAC. The cell lines were treated with NSC 631570 (5, 10 and 20 µM) for 48 h, or left untreated. Secreted protein acidic and rich in cysteine (SPARC) mRNA levels were assessed by real-time PCR. Matrix metalloproteinases (MMP)-2 and -9 activities was analyzed by SDS zymography; SPARC protein levels in cell lysates and supernatants were determined by Western blot. Cell cycle was determined by flow cytometric analysis, and invasion by matrigel invasion assay. NSC 631570 down-regulated MMP-2 and MMP-9, suggesting that this agent may decrease pancreatic cancer cell invasion, as confirmed by the matrigel invasion assay. SPARC protein downregulation in supernatants points to an inhibition NSC 631570 of extracellular matrix remodelling in the tumor microenvironment. At the same time, SPARC mRNA and cellular protein level up-regulation suggests that NSC 631570 can affect cell proliferation by cell cycle inhibition, showing a cell cycle G2/M arrest in UK-treated cells (266).

 

The Effect on the Heat Shock Proteins

In the tests with human lymphoma cells the effect of NSC 631570 on the lines U-937 and U-937/hsp70 was determinated. These cell lines differ in the expression of the heat shock protein 70 (Hsp70). The line U-937/hsp70 turned out to be more resistant against the effect of NSC 631570. The authors attribute this to the protective effect of Hsp70 (200).

 

The Effect on the Electrokinetic Potential

A study examined how NSC 631570 affects the electrokinetic (EKP) potential of malignant and benign cells. The EKP of the Ehrlich’s carcinoma cells dropped after incubation with NSC 631570. The EKP decrease of normal cells was less pronounced (221).

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