488 Novel immunosuppressives and xerwtransplantation 25 June 1997 -Poster presentations

P.5.23.04 Prevention and therapy of allograft rejection reactions and induction of long-term altograft survival by MNA 279 and MNA 715

J.K. Lindner ‘, N. Zantl 2, H.U. Schorlemmerl. ’ Hoechst Marion Roussel, DG-Rheumatology/immunology, Matburg, Germany 2Depattment of Surgery and Medicine, Technische U&emit& Mtinchen, Germany

Introduction: The malononitrilamides (MNA) 279 and 715 are denvatives of A77 1726, the active metabollte of Leflunomide. MNA 279 and 715 inhibit the pyrimidine synthesis pathway by interfering with the enzyme DHODH. By that way T and B cell proliferation, IgM and IgG synthesis are suppressed. Here we report the efficacy of NINA 279 and MNA 715 in the prevention and therapeutic treatment of allograft rejection reactions.

Material and Methods: Male inbred Lewis (RTl’) rats served as recipients and Brown Norway (BN) (RTl”) (10 to 14 weeks of age) rats were used as donors for hetemtopic cardiac transplantation. Graft rejection was taken as the complete cessation of heart beats and was confirmed by lapratomy. The MNAs 279 and 715 were dissolved in 1% CMC and given orally.

Reeutts: MNA 279 and MNA 715 were applied from day 0 to day 20 at dosages from 5 to 20 mg/k@day to test their efficacy on the prevention of allograft rejection reactions. A dose dependent increase of efficacy was seen by the application of both drugs (contml: 6.9 f 03,279: 5 rng&g 25.0 f 22.0, 10 ma/kg 42.3 f 16.2, 20 mg/kg 58.2 f 17.0; 715: 5 mgikg 14.6 f 3.0, 10 mg/kg 31 .O f 20.5, 20 mgikg 50.3 f 22.9). Within the 20 mgkg dosage groups more than 50% long term surviving grafts (~70 days) were observed. To investigate the immunological mechanism of cardiac acceptance in this model second-set (BN) and third-party (DA) skin transplantations are performed. Spleen cells from Lewis rats with long term surviving BN hearts were examined for their capacity to transfer donor specific tolerance to BN cardiac allografts in syngeneic Lewis hosts.

To test the efficacy of MNA 279 and MNA 715 in suppressing an ongoing ‘immune reaction, the beginning of drug application (15 mg/kg) was delayed to day 3 after heart transplantation (therapeutic treatment). Both substances showed a prolongation of allograft survival from 7.3 f 1 .l days in the control group up to 32.3 f 4.0 days in MNA 279 group.

Conclusion: MNA 279 and MNA 715 show a dose dependent increase of efficacy within a preventive treatment schedule. Both MNAs are effective in the treatment of ongoing rejection episodes. Long term surviving grafts are observed within the MNA 279 and MNA 715 monotherapy groups.

P.5.23.05 The effect of kinase inhibitor hyperlcin on normal and tumoral T-cells

C. Ursaciuc ‘, M. Rotaru ‘, P. de Wine *. ‘Dept. Immunology “V Sabes” Institute Bucharest, Romania, 2 Dept. Pharmacology; Catholic UniversiW Leuven, Belgium

_.

Intmductlon: The naphtodianthmne compound hypedcin (Hy) is known to show intracellular kinase-inhibition activity in tumour cells and consequentty antltumoral effect. Other charactedsttcs consist in antiviral and cytotoxic prop- erties.

In order to establish and compare the effects of Hy on normal and malignant T-lymphocytes, we realised two in vitro systems with mouse splenocytes and EL4 thymoma cells modulated with the kinase inhibitor.

Yatwlsls and Methods: Splenocytes from C57b/6 mice were tested by blast transformation test in response to in the presence of Hy 0.02 PM. ConA and IL-2 were added to the cultures simultaneously or after 24 hours from Hy addition. The cuttures were illuminated by fluorescence light at 24 hours from Hy addition. The degree of lymphocyte transformation was expressed as 3H-thymidine incorporation index.

Different Hy concentrations (between 20-0.002 FM) were added to EL4 cultures 0.1 ml/ml; after 24 h from Hy addition the cultures were illuminated 15 min. by fluorescent light and then m-cultured for another 24 h. After this, the cells were counted and checked for viability by tdpan blue exclusion.

Results: The inhibition of normal cells proliferation was dependent on the time of Hy contact with the cell, while the inhibition of EL4 tumour cells prolifer- ation was dose-dependent. Hy 0.02 PM inhibited ConA-stimulated lymphocyte transformation only if the mitogen was added after 24 hours from the Hy priming. An inhibition effect registered also in IL-2 stimulated cells in both situations of cttokine addition from the onset or after 24 hours of culture.

In the EL4 cell cultures Hy demonstrated a dose-dependent antiproliferative action, effective at doses between 0.4-0.004 &I.

Conofuslons: Kinase-inhibitor Hy showed an antipmliferative effect both in normal stimulated T-lymphocytes and tumoral thymocytes. The inhibition of normal T-cell transformation seems to invotve the signal transduction reactions in case of ConA a&Nation and respectively the membrane receptors in case of IL-2 activation. The antipmiiierative effect in tumour EL4 cells could be due to a dose-dependent intracellular Hy-induced enzyme inhibiiion.

P.5.23.08 Directed immunosuppression therapy in tracheal transplantation

A. Karpitzky ‘, S. Panko, V. Anytchkin, A. Oladko, G. Sapko, A. Lyzikov, V. Taller. Medical Institute of Vitebsk, Belams, ’ Research Institute for Radiation Medicine of Vitebsk, Selarus

Intmductlon: Existing methods of suppressing transplant rejection reactions based on general immunosuppression, are sufficiently effective in the trans- plantation of organs and tissues into the internal medium of the organism.

Materials and Methods: We have developed a method of allotransplan- tation of the trachea with omentopexia. followed by a special method of im- munosuppression therapy with specially developed prolonged action forms of Cyclospodn A. The experiments were carried out on 56 mongrel dogs in 3 series (1) replacement of circular tracheal defects by unconsewed allografts - 28 dogs (2) replacement of circular tracheal defects by unconserved allografts, along with omentopexia and the use of prolonged action forms of Cyclospodn A - 23 dogs; (3) replacement of circular tracheal defects by unconserved trans- plants with a specially developed protective covering along with omentopexia and directed transport of Cyclosporin A -15 dogs.

Results: The given method consists of allotransplantation of the trachea, followed by the insertion of a specially developed prolonged action microcap- sulated form of Cyclospodn A into the transferred omentum (series 2). This significantly reduces (upto 8.7%) the number of transplantation complications (lysis of carttlage rings, stenosis), which caused the death of all operated ani- mals from series 1 within 25 days of surgery. Likewise, the doses and frequency of insertion of the immunosuppressant are also reduced. The pmtectiie cover- ing of the tracheal allotransplant @ties 3) increases the selectivity of drugs of this group because of its impermeability to immunopasittve cells and prevents the rejection of the tracheal tube allotissue. Endcecopic, radiological and macro- scopic control of the tmcheobronchial tree in animals from series 2 and 3 shows that the transplants are viable, their carcass rlgidt and lumen are maintained.

Conclusion: Allotmnsplantatlon of the trachea in combination wtth omen- topexia and immunosuppression therapy with prolonged action forms of Cy- closponn A re-establishes the blood supply to the transplanted airway segment, prevents insufficiency of the tmcheo-transplant joints, causes long term inhibi- tion of rejection and maintains the viability of the allogmft.

P.5.23.07 Selenium influence on immunological reactions

RR. Tuhvatshin, V.A. Nasymv, R.A. Zulkameyev, G.B. Usenkulova. Central Research Laboratory Bishkek. Kjqyzstan

The aim of research work is to study selenatum nattium glutaminant (SNGA) and selenitum natrium glutaminant (SNGI) influence on indices of humoml and cells, immunity.

Subjects were 3 groups of white mouses. The first group accept selenatum natrtum glutaminant, the second - selenitum natrium glutaminant, and the 3rd control. 1st and 2nd group of white mouse use SNGA and SNGI 7 and 15 days in dose of 0.4 mg/kg.

Immunologic reaction was examined on the fifth day after peritoneal immuni- sation of sheep erythrocytes(SE) 2 x 10s cells by counting; (1) antitelgenemtion cells in spleen (AGC), (haemagglutininum serum (HS), (3) msettforming cells in spleen (RHS).

Resuh Group with selenit natrium glutaminant during 15 days the number of AGC increase to 467 f 58.3 (as control 180 f 44.9 (p = 0.01) and during 7 days - no changes, titre HS was excluded control in 2.3 times; (2) The selenat natrium glutaminant use dudng 15 days was increased AHC in 3 times (p = 0.01). and tttre of HS in 3.4 times.

We conclude that SNGA and SNGI in dose of 0.4 mg/kg activated anttbody- generation, by increasing the AHC number in spleen, and had poor influence on T activity of cell immunity.

1 P.5.23.08 1 Binary sample of platinum drugs and interferon conjugates

U. Plotnikov. Virus Leuk. Dept., Lab. of Biomodefs, Tomsk, Russia

At present progress on the field of drug antitumor and antivirus therapy is es- sentially related to the development and use of advanced preparations of new generation. We have worked out a new technology (know-how) of obtaining binary conjugates of the know cytostatic preparation of cisplatinum (cis-DDP) and promising platinum and 6-mercaptopurin complex of the common group of Pt-8-mp. As a carder, in the given case, we used human interferon-gamma. As test systems, we used leukemic K-562, HUT-102 cells, the reaction of lym- phocyte blastic transformation. Platinum content in conjugates was established by means of the neutron-activation analysis and chemical methods. The binary immunoconjugates we developed have shown a dose-dependent cytotoxic ac- tivity (60-95% of damaged). It should be noted that the above technology allows launching production of new cytostatic preparations on a large scale for scientific and clinic use. We think it expendent to epmloy the above immunological vector

25 June 1997 - Poster presentations Novel immunosuppressives and xenotransplantation 489

along with other proteins used in clinical practice. The above immunoconju- gate can be of cerlain interest in targeted antileukemic cytostatic and antivirus therapy and bone marrow cell transplantation.

1 P.5.23.09 1 Destabilase from medicinal leech influences upon vital processes in cells of blood

Y.N. Khomyakov ‘, V.V. Gusev’, I.A. Vasilenko 2, 0.1. Slinchenko. I.P. Baskova 3, L.L. Zavalova 4, S.N. Bereznoy 4, O.M. Laskina ’ . ’ State Research Center &r Applisd Microbic Obolensk. Russia, 2/nstitute of Reumatology of RAMS. Russia, 3 Biological Depariment of Moscow State lJniversi& Russia, 41nstitute of Bioorgsnic Chemistry RAS, Moscow, Russia

Introduction: Endc-e-(y-Glu)-Lys isopeptkiase also called destabilase from medicinal leech specifically splits isopeptids bonds. Due to its ability to dissolve the stabilized fibrin in vitro and thus to reverse the critical final step in the blood coagulation, this enzyme is a potential trombolytic agent. However, isopeptides bonds between side chains of Gln and Lys are very important in different vital processes such as a maturing of cells. So. it is very interesting to study the influence of destabilase uoon vital processes in normal human monocvtes and lymphocytes. To elucidate’this que&on and to evaluate qualitatively int&cellular dynamic processes was the objective of our study.

contribute to the fact that while PBL were labeled almost immediately (3-5 min), splenccytes 15-30 min and LNC 90 min. after i.v.. them was no binding of the conjugate to thymocytes even 24 hours after i.v. application.

Conclurlon: Antibody-targeted HPMA-conjugated drugs represent a very promising system for cancer or immunosuppressive therapy. Due to relatively low nonspecific toxicity against developing lymphocytes could help in bone mar- row transplantation to prevent graft-versus-host disease and at the same time allow the plufipotent stem cells to repopulate recipienrs bone marmw. In addi- tion, the fact that the specificity of HPMAconjugated antibodies is not abolished opens up the possibility of using such copo@nerdrug-antibody conjugates in organ-specific or cell-specific chemotherapy of cancer.

[P.5.23.11 1 Dynamics of immuno-allergologic Indices In allergodermatoses therapy by lodobromine solutlon of high mlnerallxetion

I.P. Koryukina ‘, L.A. Golovskaya’, V.V. Sidomv*, E.V. Zubov’, D.V. Sklorovz, T.P. Guseva 2. ’ Penn Medical Academy. Perm, Russia, * Ust-Katchka health resort, Penn, Russia

Materials and Methods: Highly purified destabilase preparations with spe- cific isopeptidolytic activity 0.1-0.6 nkat/mg were used. Human lymphocytes and monocytes were prepared from heparinized venous blood by standard method. As a test system we used a culture of cells from a hen’s embryo. For intraceilular dynamic processes investigation a laser computer-aided phase microscope with spatial resolution up to IO nm and real time resolution 0.03s in dynamic pro- cesses recording was used. Chemilurniniscent reactions of monocytes induced by destabilase were studied using chemiluminometer CLM-3.

Results: In preliminaty investigations we found the inhibition of growth of a culture of cells in our test system in the final concentration 27 @ml. To study the possible effect of the enzyme on the living blood cells we used laser computer -aided phase microscope. We performed different cross-section of cell phase image and measured intensity of the phase fluctuation at some points of the cell pmfile. The most matked quantitative and qualitative changes we obtained with concentration of enzyme about 2.7 &ml. Especially we should distinguish the periodic contractions of the cells. Researching chemiluminiscent reactions we determined that this phenomenon is accompanied by the excretion of an active oxygen.

The Penn region is one of the unfavoutable ecological regions of Russia. It favours the appearance of a large incidence of allergic diseases, allergoder- matoses being in the first ranks among them.

125 allergodermatosis patients were health-improved at the health resort in Ust-Katchka. On the whole pathologic process was localized in ulnar Hexions popliteal fussas. In 42% of the examined general eosinophilia was marked, in 45% - the gmwth of local eosinophilia in the secretion of the nose mucosa, in 51% - a significant increaSe of Ig E level, in 30% - the decrease of Ig A, T-suppressors.

A new method of treatment in the form of local application of bmminiodine solution of high mineralization has been used at the health resort for the first time since 1996. Against a background of the treatment clinical improvement was in 90% of the patients. Against a background of the decrease of Ig E contents (in 55%), lessening of general and local eosinophilia (in 61%).

Thus the use of local application of bmminiodlne solution of high mineralira- tion proved an effective method in allergodermatoses treatment.

P.5.23.12 In v&u lmmunomodulatory activity of thalidomide analog CC-3052

w&cludon: Our results demonstrate that highly putified destabilase influ- ences upon intracellular dynamic processes in the cells of human blood. Due to its abilkv to stimulate the excretion of active oxvoen we suooest that the enzvme

M. Guckian, J.B. Maniott, S. Cookson, M. Westby, G. Dalgleish. Division of Oncology, St Georges Hospital Medical School, Tooting, London, UK

has the-properties of immunomodulator. _- -I

P.5.23.10 Pharmacotdnetics and specificity of targeting by antibodies of HPMA conjugates of doxorublcin in vfifu and In vh

Marek &‘astn$, J. Stmhalm, K. Ulbtich. 6. &hove. Inst. of Microbio/~ ASCR, Department of lmmunologv and Gnotobiol~ ViderM.4 Prague, Czech Rep, Inst. of Macmnwlecular Chemim ASCR, Heyrovsk$ sq., Prague, Czech Rep

Introduction: Thalidomide is an effective immunomodulatory agent with well recoanised inhibitow effects on LPS-stimulated TNFu production bv monocvtes. Herewe report thaia more soluble thalidomide anal& CC-30521 exhibits sig- nificantly greater immunomodulatoty activity than thalidomide at log-fold lower concentmtions.

Materlals and Methods: Nonal PBMC were cultured for 3 days with 1 @g/ml PHA in the presence of 0, 1, 10 or 100 PM thalidomide or CC-3052, and pmliieration was estimated by 3H-thymidine incorporation. NK activity was assessed in the presence of thalidomide or CC-3052 by measudng 5’Cr release from K562 tamet cells. Cvtokine oroduction was assessed in 18 hour culture supematants Gy ELISA. _ ’

Rwub: Analog CC-3052 reduces PHA-induced proliferation by 33% at 10 tntroduotkm: It has been previously demonstrated that antibody-targeted HPMA copolymers containing doxorubicin can have greater antitumour effect in tivo in animal models than the drug in free form. The present studies are first to charactelize the binding kinetics of antibody-targeted HPMA conjugates of doxorubicin (DOX) and to confirm the specificity of targeting by monoclonal antibodies in vitro as well as in viw using flow cytometry analysis.Materlals and Methods: Flow cytometry (FC) was used for the phatmacokinetic experi- ments. In vittu experiments - isolated cells were incubated with anti-thymocyte serum (ATS)-targeted HPMA conjugates of DOX for 1, 5, 30 min, 1. 2, 4, 8 and 24 hours. In viva experiments - mice were i.v. injected via tail vein and blood samples were taken at different time intervals. To visualize the presence of the conjugates on the cell surface, the cells were incubated with secondary, FITC-labeled anti-IgG antibody and analysed by FC. In order to phenotype the cell popu-lation binding the anti-CD4 targeted DOX with high affinity, the cells were incubated with phycoetythrin-labeled mAbs (anti-CD3, -CD8, -CD4, -1-A) and analysed by two-color FC.

Resuh In Vito analyses have shown the presence of ATS-targeted conju- gate on the surface of peripheral blood leukocytes (PBL), splenocytes, lymph node cells (LNC) and thymocytes. Different results emerged from in vita stud- ies. ATS-targeted DOX was detected on PBL. splenocytes and LNC 1.5 hour after i.v.. However, even 24 hours after i.v. administration the conjugate was not detectable on the surface of thymocytes, although in Vito studies have shown strong binding of the conjugate to isolated thymocytes. Similar results were obtained with anti-CD4 targeted doxorubicin. We proposed that the bloobthy- mus barrier could be the factor which prevents the entrance of the conjugates into the thymus. Discontinuous endothelium in the spleen, continuous in lymph nodes, and basement-membrane-supported endothelium in the thymus could

PM concentration, compared to 4% inhibition by 10 PM thalidomide (n = 4). Dose-dependent decreases in TNFa and IFNy are also measured in pmlifer- ation assay supematants. Thalidomide does not affect NK activity at concen- trations up to 100 MM, whereas analog CC-3052 exhibits a dose-dependent reduction in NK activity at equimolar concentrations (n = 3). Analysis of super- natants from NK assays show a dceedependent decrease in TNFIY production and increased IL-10 production within the same dose range, suggesting that their regulation is closely interdependent. The reduced proliferation and NK ac- tivity may be only partially abrogated by addition of recombinant TNFa, IL-12 or indomethacin, while PGE2 causes further suppression. Neutralizing anti-TGFb antibodies had no significant effect on drug-induced immunosuppression.

Conclurlon: Although the precise mechanism of action is unknown, thalido- mide analog CC-3052 is likely to act via a CAMP regulated pathway, as features of its immunomodulatory activity am consistent with the activity of other CAMP- elevating drugs in vitro. While the superior ability of thalidomide analogs to suppress pro-inflammatory cytokines TNFa and IFNy and to concomitantly ele- vate the synthesis of the monocyte deactivating cytokine IL-10 may have clinical applications, the associated reduction in NK activity may be an undesirable side effect of thalidomide-based drugs in polytherapeutic approaches to treatment of many immune-mediated diseases.