Search

The furan or pyran related hetero cycles play basic role in structural units of nucleic acids (NA) and polysaccharides (PS), significantly predetermining their functional specifics. Some of such properties, in great relevancy for medicine, can be imitated through mimicry of polymers synthetic. Particularly, a formation of similar cycloisomeric chains is possible in process of free-radical cyclocopolymerization of divinyl ether (DVE) and maleic anhydride (MA). The products yielded (DVEMA) of general formula [DVE(MA)-alt-MA]n become precursors for a broad family of water-soluble derivatives capable of wide spectrum of bioactivity, including induction of interferon, immune-stimulated and direct antiviral protection. In this connection, the knowledge: what is content of different heterocyclic isomers in backbone of the preparations and what their partial contributions in promotion of the certain bioactivities observed, are in great importance. Available experimental data (NMR, IR, etc.), controversial for interpretations, didn't elucidate a required estimation of the DVEMA isomerism. The current work represents an independent exploration of the problem via quantum chemistry-based analysis of kinetic (activation barriers) and thermodynamic (enthalpies) priorities in competition between variable isomerism within the chain synthesis. The system is considered in maximal range of hypothetically allowable variations of two levels for double regioselective bifurcations: there are four competitive ways, each of which involves a sequence of four type elementary reactions for a diverse-isomeric formation of chain units. A genesis of six chiral centers (62 stereoisomers permitted) per every of the four part ways was accounted in view for up to 256 isomeric variations in total. The required time-minimized but precisely accurate computations were conducted via B3LYP/6-31G(d), M06-2X/6-311+G(d), M06-2X/6-31+G(2df,p) techniques, which were preselected through model test-systems. As a result, the mechanisms, crucial points and factors for the process-permitted regulation of isomeric content of DVEMA were studied in details. The narrow enough set of most probable enantiomers within highly competitive 5-exo- and 6-endo- ring closing sub-ways was revealed. The results obtained are very actual for an adequate modeling (docking / molecular dynamics) of DVEMA derivatives in their interactions with biopolymer targets, in search for purposed advancement of current background in design and synthesis of highly effective agents for combined antiviral protection (against HIV, flu, herpes, and other infections).Geterotsikly, rodstvennye furanu ili piranu, igraiut bazovuiu rol' v strukturnykh zven'iakh nukleinovykh kislot (NK) i polisakharidov (PS), v sushchestvennoĭ mere opredeliaia ikh funktsional'nye osobennosti. Riad takikh osobennosteĭ, imeiushchikh prakticheskoe znachenie dlia meditsiny, udaetsia imitirovat' s pomoshch'iu mimikrii polimerov sinteticheskikh. Formirovanie skhodnykh tsiklo-izomernykh struktur vozmozhno, v chastnosti, v protsesse svobodno-radikal'noĭ tsiklosopolimerizatsii divinilovogo éfira (DVÉ) i maleinovogo angidrida (MA). Obrazuiushchiesia produkty (DVÉMA) obshcheĭ formuly [DVÉ(MA)-cher-MA]n sluzhat prekursorami obshirnogo semeĭstva vodorastvorimykh proizvodnykh s shirokim spektrom biologicheskoĭ aktivnosti, vkliuchaia induktsiiu interferona, immunostimuliruiushchuiu i priamuiu protivovirusnuiu zashchitu. V sviazi s étim vazhno znat', kakovo sootnoshenie razlichnykh geterotsiklicheskikh izomerov v ostove étikh soedineniĭ i kakova ikh rol' v realizatsii tekh ili inykh vidov nabliudaemoĭ aktivnosti. Imeiushchiesia éksperimental'nye dannye (IaMR, IK i dr.), vvidu neodnoznachnosti ikh interpretatsii, ne pozvoliali priĭti k iasnoĭ otsenke izomernogo sostava DVÉMA. Poétomu v dannoĭ rabote provedeno nezavisimoe kvantovo-khimicheskoe issledovanie s analizom kineticheskikh (énergiia aktivatsii) i termodinamicheskikh (éntal'piia) prioritetov konkurentnoĭ izomerizatsii tsepi v protsesse sinteza. Rassmotrena sistema reaktsiĭ v maksimal'nom diapazone gipoteticheski dopustimykh variatsiĭ dvukh urovneĭ parno-razvetvlennoĭ regioselektivnosti: chetyre konkuriruiushchikh marshruta v posledovatel'nostiakh chetyrekh tipov élementarnykh reaktsiĭ formirovaniia zven'ev tsepi, razlichnykh po strukturnoĭ izomerii. Po kazhdomu iz marshrutov uchten poshagovyĭ genezis shesti khiral'nykh tsentrov (64 stereoizomerov) – do 256 izomernykh variatsiĭ summarno. Ékonomichnyĭ rezhim tochnykh raschetov obespechivalsia metodami B3LYP/6-31G(d), M06-2X/6-311+G(d), M06-2X/6-31+G(2df,p), proshedshimi predvaritel'nuiu selektsiiu na model'nykh test-sistemakh. V rezul'tate detal'no izuchen mekhanizm, kriticheskie tochki i faktory vozmozhnoĭ reguliatsii izomernogo sostava DVÉMA. Ustanovlen dostatochno uzkiĭ interval naibolee veroiatnykh énantiomerov v riadu vysoko konkurentnykh sub-marshrutov 5-ékzo- i 6-éndo- tsiklizatsii. Poluchennye rezul'taty neobkhodimy dlia adekvatnogo modelirovaniia (doking/molekuliarnaia dinamika) proizvodnykh DVÉMA vo vzaimodeĭstviiakh s biopolimernymi misheniami v tseliakh razvitiia imeiushchegosia zadela po dizaĭnu i sintezu vysokoéffektivnykh agentov kombinirovannoĭ protivovirusnoĭ zashchity (ot VICh, grippa, gerpesa i dr. infektsiĭ).

To endow chitosan with solubility in the whole pH-range without loss of functionality of the amino groups, the cationic polysaccharide was exhaustively alkylated yielding N-[(2-hydroxy-3-trimethylammonium) propyl]chitosan chloride (QCht). Each alkylated unit of QCht contained both quaternary amino group and secondary amino group. Recently we demonstrated that QCht forms with nucleic acids of soluble polyelectrolyte complexes stable at physiological conditions and capable of cell transfection in vitro . In the current work, the anionic counterpart of QCht was hydrolyzed copolymer of divinyl ether and maleic anhydride (DIVEMA) which is known to possess some anti-tumor and immune stimulating activity and use as a drug carrier in anti-tumor drug delivery systems. According to the potentiometry data and ζ-potential measurements, almost all carboxylic groups of DIVEMA were able to form ion pairs with QCht. In aqueous and water–salt solutions, formation of either soluble or insoluble complexes was controlled by pH, ionic strength, a ratio of the oppositely charged groups, and degree of polymerization of the chains following general rules revealed on studying polyelectrolyte complexes of polycarboxylic acids. These findings evidence plausible advantages of the complex formation as the non-covalent modification that imparts to both polyelectrolytes of the ability for reversible soluble–insoluble transformation under enzyme-friendly conditions.

Copolymer of divinyl ether and maleic anhydride (DIVEMA) is known to possess some anti-tumor and immune-stimulating activity and use as a drug carrier in anti-tumor drug delivery systems. Samples of DIVEMA of different degrees of polymerization were synthesized and characterized. Interaction of the hydrolyzed water-soluble DIVEMA polyanions with poly(N-ethyl-4-vinylpyridinium) cations (PEVP) has been studied. According to the potentiometry data, almost all carboxylic groups of the polyanions were able to form ion pairs with PEVP. In aqueous and water–salt solutions, formation of either soluble or insoluble polyelectrolyte complexes occurred depending on pH, ratio of the oppositely charged groups, and degree of polymerization of PEVP and/or DIVEMA. The phase separations followed general rules revealed by studying mixtures of PEVP and polycarboxylic acids. However in the case of DIVEMA, a significant broadening of the region for insoluble complexes at the expense of the region of soluble complexes was established. The data obtained demonstrate plausible advantages of the complex formation as the non-covalent modification of the polymeric carrier that endow DIVEMA with the ability for reversible soluble–insoluble transformation, in particular at physiological pH and ionic strength.

Highly reactive hydroxyl radical (OH) is generated during reaction of Cu,Zn-SOD (SOD)1 with H202, its own enzymatic reaction product (SOD plus 0;-). During inactivation of Cu,Zn-SOD with H202, Cu2+ liberated from the SOD catalyzes the formation of OH from H202. The OH thus generated will react with a,-protease inhibitor (a,-PI) if it is available in the milieu. To circumvent OH generation, we prepared eight different polymer-SOD conjugates and compared their stability against H202 treatment. Furthermore, the effect of each polymer-SOD conjugate on a,-PI in the presence of H202 was tested. Most polymer-SOD conjugates, except poly(styrene-co-maleic acid butylate; SMA)-SOD and poly(ethylene glycol; PEG)-SOD, showed higher stability than native SOD against inactivation by H202. The residual activity of divinylether maleic acid/anhydride copolymer (pyran copolymer)-conjugated SOD was 87% of the initial activity after incubation with 0.1 mM H202 for 2 h, whereas that of native SOD was 49%. Antiprotease activity of ca,-PI was completely abrogated when the inhibitor was treated with native SOD and 3.0 mM H202, but there was no change with pyran copolymer-SOD after 3 h of incubation with H202. Our results suggest that pyran copolymer captured loosely bound Cu2+ during the reaction of SOD with H202, thus resulting in suppression of both 0OH-mediated inactivation of SOD and suppression of activity of a,-PI. PEG showed little Cu2+-binding activity; no appreciable protection of SOD and a,-PI against OH generation was observed when Cu,Zn-SOD was conjugated with PEG. Circular dichroism and electron spin resonance studies of Cu,Zn-SOD treated with H202 indicated that the peptide conformation as well as the copper ligand of Cu,Zn-SOD can be stabilized not only by pyran copolymer per se but also by conjugation of Cu,Zn-SOD with some copolymers such as pyran.

Protein kinases are important enzymes in solid tumour and leukaemia pathologies. Their structures are well understood at the atomic level and their key role in the progression of certain cancers makes them valuable targets for anti-cancer therapy. Through medicinal chemical approaches, we developed an efficient preparative stereospecific synthesis of N12, N13 pyran-bridged indolocarbazoles that opens access to functional diversity within this previously challenging series. We focused upon the indolocarbazole class of chemical inhibitors, which includes S27888, an inhibitor we previously identified. We used biochemical and cell-based assays to identify small molecule inhibitors of Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase that is activated when cancer cells are treated with genotoxic agents. These compounds show a promising inhibitory profile on Chk1. Furthermore, these compounds are active against FLT3, which is a tyrosine kinase that is frequently activated in human leukaemias. These data suggest that this chemical class may provide a source of therapeutic compounds for a broad range of human cancers.

To enhance the therapeutic usefulness of antitumor cytokines in vivo, we synthesized bioconjugated tumor necrosis factor-alpha (TNF-alpha) with divinyl ether and maleic anhydride copolymer (DIVEMA), which has intrinsic antitumor activity as a synthetic biological response modifier. The degree of modification could be controlled by the addition of 2,3-dimethylmaleic anhydride (DMMAn), which binds to amino groups of TNF-alpha by changing the pH. In addition, the specific activity of DIVEMA-TNF-alpha was hardly decreased in vitro. DIVEMA-TNF-alpha showed a marked antitumor effect compared to native TNF-alpha without any side effects such as sudden death, body-weight reduction, and decrease in platelet count on mice bearing solid tumors. These results suggest that DIVEMA is a useful polymeric modifier for-bioconjugation of TNF-alpha in order to increase its antitumor activity, and multifunctionally bioconjugated TNF-alpha may be a potentiated antitumor agent for therapeutic use.

Neocarzinostatin (NCS) was conjugated with divinyl ether-maleic acid anhydride copolymer (pyran copolymer), and its therapeutic effect was compared with that of NCS. The conjugated NCS (pyran-NCS) with a molecular weight of about 23,000, exhibited in vitro cytotoxic activity against eight cell lines and bone marrow cells that was similar to the cytotoxic activity of NCS on a molar basis. Furthermore, both drugs had similar effects against a multidrug-resistant Chinese hamster ovary cell line (CHR C5) and its parent cell line (AUXB1) in vitro. However, pharmacological analysis showed that pyran-NCS had reduced accumulation in the spleen, and most important was three times less hematotoxic in vivo compared with NCS. Also, pyran-NCS had a 1.7-fold higher 50% lethal dose (LD50). Antitumor activity of pyran-NCS and NCS was tested against two different forms of Meth A tumor. In a solid tumor model, pyran-NCS and NCS suppressed tumor growth at three-fourths of the LD50 to 12.8 and 19.0% of the control tumor as evaluated on day 28, respectively (P less than 0.025). In an ascitic tumor model, the percentage increase in the median life span caused by pyran-NCS and NCS was more than 400 and 150% on day 60, respectively. Pyran-NCS is more effective than NCS because the reduced acute toxicity permits an increased drug dosage.

Many naturally occurring polyanionic materials are known, each of which possesses its own set of physiological properties. Recently, a variety of synthetic polyanions have become available, and their physiological properties have been compared with the naturally occurring polyanions. Probably the most widely investigated synthetic polyanion is the 1:2 regularly alternating cyclo-copolymer (DIVEMA) (Eq. 1) of divinyl ether (DVE) and maleic anhydride (MA), first discovered in these laboratories in 1951 [1], This material has been investigated extensively, both from the standpoint of its chemical structure [2] and its antitumor activity [3]. Although certain aspects of its structure remain to be determined [4], it has been shown to possess a wide spectrum of biological activity [5] and is an interferon inducer [3]. Many structural modifications of DIVEMA have been synthesized which also possess antitumor activity and interferon-inducing capability [6].

Natural and synthetic polyanions are known to have a broad spectrum of physiologic and pharmacologic properties. Some polyanions can activate in vitro nucleic acid biosynthesis in isolated nuclei, and induce varying degrees of nuclear swelling and chromatin decondensation. This in vitro phenomenon has an in vivo analog in gene activation. Pyran, a copolymer of divinyl ether and maleic anhydride, is a particularly interesting polyanion because of its antitumor, antiviral, and immunostimulating activities. Although the mechanism of its activities is unknown, its polyanionic character suggests that it may be considered as a model “gene regulator.” The present investigation focused upon the interaction of Pyran with nuclei isolated from normal mouse spleens. Small angle light scattering was used to monitor nuclear swelling, and the conditions required to demonstrate swelling were established. It was shown that Pyran-induced nuclear swelling results from the ability of this polyanion to form a complex with Mg 2+ . It was also shown by nephelometric titration methods and small angle light scattering that the interaction of Pyran and Mg 2+ resulted in the precipitation of “microspheres.” These results have led to a working hypothesis relating to the possible molecular mechanism of the pharmacologic action of Pyran copolymer.

The effect of four immunomodulators (BCG, Corynebacterium parvum, pyran copolymer, and levamisole) on the cellular arm of antibody-dependent cellular cytotoxicity (ADCC) was investigated in mice with 51Cr-labeled chicken erythrocytes employed as targets. All these drugs, except levamisole, stimulated the effector cells of ADCC in the spleen, but the kinetics of their effect differed. Stimulation of the effector cells of ADCC peaked on day 15 after injection of BCG and C. parvum and on day 7 after injection of pyran, which was less efficient in this respect than the two bacterial immunostimulants. The increase in ADCC activity caused by BCG and C. parvum was eliminated by treatment with carbonyl iron of the splenocyte suspensions.

The degree of inhibition of mammalian DNA-dependent RNA polymerases I and II and Moloney leukemia virus RNA-dependent DNA polymerase by pyran copolymer was dependent on the concentration of the divalent cation cofactor in the reaction mixture. Inhibition was completely blocked by an excess of divalent cations. It was concluded that pyran inhibited these enzymes by complexing with the essential divalent cation cofactor.

SummaryData are presented which show that protection of mice by endotoxin against viral infection is not mediated by interferon while that of poly(I:C) and pyran can be at least partially attributed to interferon induction. The results also suggest that circulating interferon does not protect against a virus administered intraperitoneally.

A weakly immunogenic, 3-methylcholanthrene-induced, subcutaneous fibrosarcoma syngeneic to inbred C3H/HeJ mice was used. Pyran copolymer was injected either directly into the tumor, ip, or iv as soon as tumors appeared or when tumors were 8 mm in diameter. One, three, or five doses of pyran copolymer at 10 or 20 mg/kg/dose were injected, with multiple doses being given every other day. Pyran copolymer injected intratumorally once, three times, or five times significantly retarded tumor growth and prolonged the survival times of the hosts. Of the other routes and doses, only pyran copolymer given three times iv significantly retarded tumor growth, but none of these significantly prolonged the survival times of the hosts. Pyran copolymer alone did not induce any complete regression of tumor. Local tumor irradiation with a single exposure to 2,000 rads of X-ray induced complete regressions in some mice, but a higher percentage of tumor cure was observed when tumor irradiation was followed by pyran copolymer treatment.

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of 125I-labelled PVP and colloidal 198Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [14C]DIVEMA and 125I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.

The toxicology of synthetic polyanions relates to molecular weight. This developed from experience with pyran copolymer (divinyl ether maleic anhydride copolymer), which was tested clinically as an antitumor agent and found to be too toxic for continued clinical trial. Synthetic polyanions of low molecular weight are potent stimuli to macrophage phagocytic function and possess antitumor activity. Increased host resistance to pathogens including bacteria, fungi, viruses, and trypanosomes is seen. In contrast to lower-molecular-weight fractions, higher-molecular-weight samples inhibit macrophage function, and apparently increased molecular weight > 15,000 is necessary for antiviral and immunologic stimulating capacity. One possible mechanism for the action of polyanions may relate to redistribution of calcium ions within the cell and increasing cyclic GMP activity. This is a redefinition for the role of polyanions in sol–gel alteration in cytoplasm. The killing effect of the polyanion-activated macrophage on tumor cells resembles that of the polyanion on the nucleus. Polyanions may be potent controllers of the stability of DNA within the nucleus and can act to derepress cells to turn on protein synthesis or, alternatively, inhibit enzymes involved in virus synthesis and control of RNA or DNA polymerase. Thus, they are cogent tools in an understanding of the physiology of nuclear interaction. Biological use for these agents can be developed not only in clinical approaches to inhibition of tumor or viral infection, but to eliminate plutonium, and provide selective enzyme inhibition and protein interaction which allows for isolation, neutralization, and characterization of blood and tissue protein and glycoprotein fractions vital for a wide range of pathophysiology.

A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro . However, following the administration of pyran in vivo , mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.

Pyran copolymer is an antitumor and antiviral drug with immunostimulating activity. Its ability to form soluble complexes and coacervates with divalent cations is thought by us to be an important factor in the mechanism of these activities. The molecular weight (MW) of the polymer has been identified as a modifier of its biologic activities. This investigation focused upon the effect of MW on the autocomplex coacervation of Pyran and divalent cations. Two whole polymers and three fractions were studied. Turbidity onset, a measure of coacervation, was recorded as a function of pH, and was found to decrease with increasing MW of the fractions. The same order of MW dependence was determined from differential small-angle light scattering measurements of the coacervate suspensions, using Sloan plot analyses. The average diameter of the suspended particles was apparently independent of the MW of Pyran.

A series of copolymers related in structure to the 1:2 alternating cyclocopolymer of divinyl ether and maleic anhydride (DIVEMA) have been shown to possess antitumor properties. The synthesis and structures of these copolymers are discussed, and their effectiveness as antitumor agents is presented. Certain of the copolymers have been prepared in controlled molecular weight ranges using chain transfer agents, and the resultant copolymers finally fractionated via use of solvent-nonsolvent systems. These samples of narrow molecular weight distribution have been evaluated for their antitumor properties and have been found to be quite effective.

A series of Maleic anhydride divinyl ether (MVE) polyanions, synthesized with molecular weight ranging from 12500 to 52600, were found capable of enhancing macrophage tumoricidal activity of MBL-2 leukemia cells. These agents also augmented Natural Killer cell activity against the YAC lymphoma and M109 adenocarcinoma cell lines.

Several interferon inducers administered iv or per os to mice enhanced the spreading of peritoneal macrophages freshly plated on a glass surface. Spreading was scored with a microscope after the monolayers were incubated in serum-containing medium. The effective agents were a ribopolynucleotide (Poly rI:Poly rC), 1 a bacterial lipopolysaccharide (LPS), Newcastle disease virus (NDV), tilorone, cycloheximide or pyran copolymer. However, while induction of interferon and enhanced macrophage spreading appeared to be associated, we failed to obtain significant correlation coefficients between peak serum interferon titers and % of spread macrophages after the administration of NDV, tilorone or cycloheximide. Spreading was also increased by the macrophage activator C. parvum or by intraperitoneal administration of thioglycollate broth. Lethal preirradiation did not influence the spreading enhancement by LPS, but markedly reduced the spreading that followed the extraperitoneal administration of NDV, tilorone, cycloheximide or C. parvum , or the intraperitoneal injection of thioglycollate broth. The effect of NDV on macrophages was inhibited by preliminary splenectomy. Splenectomy, however, did not influence the macrophage response to the other agents tested. The results are compatible with a direct effect of LPS on the macrophages and with an indirect, possibly lymphocyte-mediated effect of NDV. Spreading enhancement by NDV was found to be mouse-strain dependent. The results provide models for the investigation of cellular or humoral mechanisms that underlie macrophage activation.

The effect of pyran copolymer, injected into mice bearing the M109 Madison lung carcinoma, on serum concentrations of lysozyme, β-glucuronidase, and N-acetyl-β, D-glucosaminidase was studied and compared with that of other immunoadjuvants. Increases in lysozyme levels ranging from 50 to 100% were observed after injection of pyran, BCG and Bru-Pel; increases in the levels of the other enzymes were less consistent. Other immunoadjuvants were less effective in raising serum concentrations of lysosomal enzymes. The findings were correlated with the results of previous studies on macrophage activation and antineoplastic action produced by these immunoadjuvants and suggest that serum levels of lysozyme can serve as indices of these effects.

Mouse resistance to foot-and-mouth disease virus (FMDV) was induced by intraperitoneal injections of pyran copolymer. A biphasic pattern of protection occurred with greatest resistance 4 and 48 hr after injection of this polyanion. Viremia was not detectable in pretreated mice challenge-exposed with FMDV. Incubation of virus with pyran did not alter viral infectivity in mice or tissue culture. Serum interferon was demonstrated 1 and 2 days after pyran administration.

Both pyran copolymer and the Burroughs-Wellcome vaccine of Corynebacterium parvum were able to elicit a migratory response by human neutrophils or monocytes when incubated with serum. However, in the absence of serum, neither agent elicited such a response. The generation of the chemotactic factor by pyran was dependent on heat-labile components in the serum, while suggested involvement of the complement sequence. Conversely, the migratory response of C. parvum in heated serum was equivalent to that in normal serum. No specificity for either monocytes or neutrophils was observed.

DIVINYL ether and maleic anhydride form a 1:2 copolymer, which is reported to have the structure shown here1. It is commonly known as pyran copolymer, although we prefer to call it DIVEMA, an acronym for divinyl ethermaleic anhydride. It has been designated as NSC 46015 by the National Cancer Institute.

Summary Guinea-pigs given pyran copolymer intraperitoneally were protected to some degree against challenge with foot-and-mouth disease virus. Cattle or pigs given pyran intraperitoneally and exposed to contact infection with foot-and-mouth disease showed little or no resistance. No interferon was detected in the samples of serum tested. Administration of pyran led to loss of weight, and toxic lesions were found in the peritoneal cavity in all species examined.

A NUMBER of synthetic polyanions stimulate interferon production1–7 and these materials are active against infection with viruses and also with parasites of a more complex nature8–12. Mice inoculated intraperitoneally with pyran, a random copolymer of divinyl ether and maleic acid anhydride, are resistant to an ordinarily lethal challenge of mengo virus7. Such antiviral resistance persists for as long as 60 days in spite of the absence of measurable levels of interferon in the sera of the mice after 4 to 6 days7,13. These polyanions modify a number of host defence mechanisms including delayed hypersensitivity14, antibody response15 and the number of circulating white blood cells16. In view of the suggestion that a common mechanism of host resistance to intracellular pathogens may exist17, we were interested to determine if the long term resistance against viral challenge observed following administration of such polymers may also be extended to intracellular bacterial infection. In these studies, three anionic polymers*, pyran copolymer, polyacrylic acid and the double stranded synthetic ribonucleic acid poly I·poly C, each conferred resistance to Listeria monocytogenes infection in mice.

The ability of pyran copolymer to interact with the classical and alternative pathways of complement was assessed in human and C4-deficient guinea pig serum. Pyran induced a dose-dependent inhibition of hemolytic activity in both serum systems. Immuno-electrophoretic analysis of pyran-treated human serum revealed that C3 was not cleaved. Factor B was altered into a more anionic mobility which was not similar to biologically cleaved Ba or Bb fragments. Pyran-treated serum was unable to lyse antibody-coated erythrocytes (EA) or EA coated with C1 and C4 and EA coated with C1, C4 and C2. Pre-treatment of serum with ethylenediaminetetraacetic acid did not prevent inhibition of hemolytic activity by pyran. Cobra venom factor did not cleave C3 in the presence of pyran. These data indicate that pyran does not activate complement by standard mechanisms but does inhibit one or more of its components.

Mice inoculated with both L1210 murine tumour vaccine and pyran copolymer were more resistant to L1210 than those inoculated with either of these agents alone. Rabbit anti-mouse thymocyte globulin and silica reduced the augmented resistance of these mice, suggesting the involvement of activated anti-tumour T cells and macrophages in the augmented resistance. We studied the activation of these two cells separately and examined the possible contribution of pyran copolymer-induced peritoneal cells to the augmented resistance to an inoculation of live tumour. Pyran copolymer-induced peritoneal cells endowed the tumour vaccine-primed mice, but not unprimed mice, with resistance to implanted L1210 and, among those peritoneal cell populations, macrophages but not T cells were responsible for this effect since the activity was associated with a cell population which was (1) adherent to nylon wool columns, (2) sensitive to silica and (3) insensitive to anti-Thy 1.2 antibody plus complement. The pyran copolymer-induced peritoneal cells had very little antiproliferative activity when tested against L1210 in vitro and mice inoculated with these peritoneal cells did not survive a challenge of live L1210 cells much longer (

The polyanion, pyran copolymer, is formed by the polymerization of divinyl ether and maleic anhydride. This agent has numerous immunomodulatory effects (Shopp and Munson 1984) including induction of interferon, complement, enhanced antiviral activity, antineoplastic activity and priming of macrophages. Effects on the reticuloendothelial system (RES) are often biphasic with a suppression of RES activity followed by an enhancement of the RES (Munson et al. 1981).

Synthetic polyanions have been shown to alter host resistance to infection. The anticryptococcal effect of pyran copolymer was assessed in vivo and in vitro. Pretreatment with pyran copolymer significantly extended mean survival in mice lethally infected with Cryptococcus neoformans when compared to untreated animals (p less than 0.01). The anticryptococcal effect of peritoneal exudate cells (PEC) elicited by 10% thioglycollate or pyran copolymer (25 mg/kg) was assessed in vitro. Initial percent phagocytosis of both encapsulated and non-encapsulated isolates of C. neoformans was greatest in the pyran elicited PEC. Significant killing of C. neoformans in vitro was observed only in pyran-activated PEC cultures combined with non-encapsulated cells of C. neoformans, although pyran PEC did inhibit initial growth of phagocytized encapsulated yeast cells. The protection of pyran copolymer pretreated mice from infection with C. neoformans, but the absence of significant killing of encapsulated yeast in vitro suggest a complex mechanism of host defense which may involve an activation of the reticuloendothelial system by pyran copolymer.

The pathogenicity of influenza virus infection in the mice involves, at least in part, overreaction of the immune responses of the host rather than a direct effect of virus multiplication. Xanthine oxidase, which is responsible for the generation of oxygen free radicals, was elevated in serum and lung tissue of mice infected with influenza virus. To test the theory that oxygen-free radicals are involved in pathogenesis, free radicals were removed by injecting superoxide dismutase (SOD), a specific superoxide radical scavenger, which was conjugated with a pyran copolymer. The conjugate protected mice against a potentially lethal influenza virus infection if administered 5 to 8 days after infection. These findings indicate that oxygen radicals are important in the pathogenesis of influenza virus infection, and that a polymer-conjugated SOD has therapeutic potential for this virus infection and other diseases associated with free radicals.

Both pyran copolymer and BCG augmented specific macrophage-mediated cytotoxicity against allogeneic MBL-2 leukemia cells in a synergistic fashion. Similarly, the local passive transfer of peritoneal exudates (PE) from MBL-2 alloimmune mice treated with pyran or BCG protected against MBL-2 tumor development in the syngeneic host, whereas exudates from immunized or adjuvant-treated animals alone were ineffective. Tumor neutralization by alloimmune-activated PE was specific for the immunizing cell type and required intimate cell contact with target cells. Both adherent and nonadherent PE cells were required for optimal therapeutic effect. Long-term survivors demonstrated resistance to subsequent tumor challenge.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), respectively, were covalently attached to a cross-linked copolymerisate of maleinic anhydride and butanediol-divinylether. Based on the coupling procedure reported by Brummer et al. (Brummer, W., Hennrich, N., Klockow, M., Lang, H. and Orth, H.D. (1972) Eur. J. Biochem. 25, 129–135), a simple method is described which requires only 24 h for completion and provides a sufficient yield. Although a polyanionic carrier was used the K m and k 2 values as well as the substrate and pH optima of the bound acetylcholinesterase and bound cholinesterase did not differ considerably from the corresponding values of the free enzymes. Bound acetylcholinesterase and to some extent also bound cholinesterase did not lose any enzymatic activity after storage in saline at 4°C for 140 days.

Intraperitoneal administration of pyran copolymer (pyran-2-succinic anhydride-4,5-dicarboxytetrahydro-6-methylanhydride polymer) protected C3H/HeN male mice against tumor development after intradermal challenge with cells of the transplantable, methylcholanthrene-induced, syngeneic fibrosarcoma 1038. Peritoneal cells from pyran-treated but not from normal animals suppressed tumor development in local passive-transfer experiments. Adherent peritoneal cells from pyran-treated mice were cytotoxic in vitro to several syngeneic murine tumor cell lines including 1038.

Pyran copolymer is a polyanionic drug with a broad spectrum of biologic activities. The present study focuses on the physicochemical interactions of pyran with divalent cations. Potentiometric titration curves of pyran are typical of the general polyelectrolyte behavior of high charge density polycarboxylates and show no apparent conformational transitions. The interaction of pyran with divalent cations follows an order of affinity for site binding, Mn 2+ > Ca 2+ > Mg 2+ , that is common also to four other polycarboxylates tested. The titration data indicate the formation of a soluble chelate complex, for which pyran, of the five polymers, shows the greatest propensity. Specific conditions in terms of pH and degree of neutralization are found under which complex formation is preceded by the precipitation of microspheres. However, only pyran and polyitaconic acid precipitate under conditions which are closest to physiologic. The experimental results suggest a mechanism for microsphere formation that is consistent with a hypothesized mechanism of pharmacologie action of pyran-divalent cation complexes.

Pyrans are co-polymers of divinyl ether and maleic anhydride. Four pyrans of various molecular weights more potently inhibited terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) from a human cell line of acute lymphoblastic leukemia origin (Molt-4) than they did DNA polymerases alpha, beta and gamma from these cells and DNA polymerase from simian sarcoma virus. For example, the concentrations of one pyran required for 50% inhibition of terminal deoxynucleotidyltransferase, DNA polymerases alpha, beta and gamma and viral DNA polymerase were 0.9, 110, 125, 35 and 47 microgram/ml respectively. Quantitatively similar results were obtained with the other pyrans. Inhibition of these enzymes by pyran was dependent on the concentrations of both the bivalent cation and template/primer or initiator in assay mixtures, but not on the concentrations of the substrate (deoxyribonucleoside 5′-triphosphate), enzyme, or bovine serum albumin. These results suggested that pyran inhibited these enzymes by complexing bivalent cations, which caused a decreased affinity of template/primer or initiator for each enzyme and a decrease in enzyme activity.

Corynebacterium parvum (C. parvum), Bacillus Calmette-Guerin (BCG) and Pyran copolymer (Pyran) are capable of significantly reducing thymocyte suppressive activity as assessed by the capacity of C3H thymus cells to inhibit the local graft vs host reaction (GVHR) mounted by C3H spleen cells in immunosuppressed H-2 histoincompatible hosts. This effect, which is dose related, is already evident 2 days after the injection of C. parvum or BCG and 4 days after administration of Pyran; it lasts 1–3 weeks, depending on the immunomodulator and is accompanied by a significant reduction of thymus cellularity. Evaluation of GVHR kinetics shows that thymus suppressive activity is inhibited by these agents at all phases of the reaction. No reduction of thymocyte suppressive activity is observed in adrenalectomized mice treated with C. parvum or Pyran.

Treatment of older mice with pyran copolymer, a known interferon-inducer, was found to result in a rapid boosting of cell-mediated cytolytic activity against YAC-1 tumor target cells. The effector cells were characterized as being non-adherent and were presumed to be natural killer (NK) cells. Augmentation occurred in various lymphoid organs and was detectable 2-3 days after drug treatment. Differences in the levels of boosted activity among the lymphoid organs resulted when the route of administration was varied. The degree of augmentation was largely independent of the dose of pyran, but did vary among different strains of mice. Augmentation, moreover, was followed by a rapid decline by 5-7 days.

The regularly alternating 1:2 cyclocopolymer of divinyl ether and maleic anhydride, often referred to as ‘Pyran’ copolymer, has been found to possess interesting anti-tumor properties and to be an interferon inducer. A variety of copolymers of this and similar types have been evaluated by the Drug Research and Development Branch of the National Institutes of Health. The most significant results of this evaluation study will be reported. It is the purpose of this paper to review some of the mechanistic aspects of cyclocopolymerization in general and to relate these theories to the structure of pyran copolymer, which was the first cyclocopolymer discovered. Those 1,4-dienes which readily undergo cyclocopolymerization with suitable alkenes also form charge-transfer complexes with the same alkenes. These results are consistent with participation of the 1:1 charge-transfer complex of divinyl ether and maleic anhydride in this system in a copolymerization process with a second molecule of maleic anhydride to form the cyclocopolymer. Previous studies have indicated the copolymer to have a rather broad molecular weight distribution. In view of the extensive interest in the physiological properties of this material, further studies on the molecular weight and molecular weight distribution, including use of gel permeation chromatographic methods of fractionation have been undertaken and reported. Attempts have been made to fractionate pyran cyclocopolymer as the polyanion by GPC on Deactivated Porasil columns without success. Sucessful fractionation was attained on the dimethyl ester of the cyclocopolymer on Styragel columns, and comparisons were made between the copolymer prepared in this manner and the copolymer prepared by cyclocopolymerization of divinyl ether and dimethylfumarate. This paper reviews some of the results of these studies.

N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid, and MVE-2, a maleic anhydride-divinyl ether copolymer, were both effective inhibitors of mammary carcinogenesis induced in Sprague-Dawley rats by N-methyl-N-nitrosourea and of urinary bladder carcinogenesis induced in C57BL/6 x DBA/2F1 mice by N-butyl-N-(4-hydroxybutyl)-nitrosamine. However, combined administration of 4-HPR and MVE-2 was no more effective in cancer inhibition than was either agent alone. Retinoids and maleic anhydridedivinyl ethers may exhibit a mechanistic or metabolic antagonism which precludes an additive or synergistic interaction in inhibiting chemical carcinogenesis.

Intraperitoneal treatment with the interferon inducer, maleic anhydride-divinyl ether copolymer (MVE), has previously been demonstrated to effectively reduce metastatic growth in the lungs and prolong survival times of BALB/c mice bearing the syngeneic Madison lung (M109) carcinoma. Resistance to lung metastasis formation induced by MVE appears to result from an activation of alveolar macrophage function. Since E-type prostaglandins (PGE) suppress the cytotoxic activity of activated macrophages, we sought to determine the effect of indomethacin, a prostaglandin synthetase inhibitor, on the antimetastatic activity of MVE. An artificial metastasis model was developed in which single-cell suspensions of the M109 tumor were injected i.v. into BALB/c mice. A 52,600 molecular weight fraction of MVE (MVE-5) was administered i.p. at 20 mg/kg two days prior to tumor inoculation. MVE-5 treatment produced greater than 80 percent reduction in macroscopic lung lesion formation at Day 15 and Day 19 after tumor inoculation and a resultant 45 percent increase in lifespan. Chronic administration of indomethacin in the drinking water at 10 micrograms/ml potentiated the MVE-5 antitumor induced macrophage activation in vivo. In the absence of any evidence for an interaction between indomethacin and circulating M109 cells, it was felt that the potentiating effect could be best explained in terms of interference with PGE-mediated feedback inhibition of macrophage functional activity.

The immunostimulator pyran copolymer and the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have been studied to determine their effect on T and B lymphocytes from BALB/c mice bearing a syngeneic tumor, Madison lung 109 carcinoma. Indirect immunofluorescent and mitogenic assays revealed that the tumor-bearing controls usually had T and B cell populations lower than those observed for the normal controls. The tumor-bearing group treated with BCNU generally had T and B cell levels lower than the tumor controls. The administration of pyran resulted in an increase of T cells and a temporary increase in the B cell population. Although this increase in the B cell population of tumor-bearing mice treated with pyran is higher than that seen for the untreated tumor controls it was never higher than in the normal control mice. The experimental results indicate that pyran appears to reconstitute the depressed T cell population and has a transitory effect on the B cell population resulting from tumor burden.

Pyran Copolymer (Divema, NSC46015*), a synthetic polyanion, divinyl ether maleic anhydride copolymer, has achieved clinical trial because of antitumor activity and its role as an anti-viral agent. Most of the previous biologic and clinical work with this agent has been done with pyran copolymer of an average molecular weight of 18000. (NSC46015).

Several factors have been shown to be involved in the antiviral protection provided by synthetic polymers. A large molecule with a high density of free anionic groups, not readily degradable or eliminated, seems essential for interferon stimulation. One of the carboxylate copolymers induced prolonged protection against lethal Mengo virus infection in the mouse. More transient protection was conferred by a nucleotide homopolymer pair. The nucleotide homopolymer had a broad range of reactive cell types, whereas only peritoneal macrophages responded with interferon production to the carboxylate copolymer (when complexed to cations). The spleen appears to play only a limited role in interferon production induced by the carboxylate copolymer. Factors other than interferon may be responsible for part of the antiviral protection observed with the carboxylate copolymer.

SummaryPyran copolymer, administered with sheep red blood cells to mice, enhances the early rate of appearance of antibody-forming spleen cells. Although the cytotoxicity and pyrogenicity of pyran and bacterial endotoxins are similar, pyran, in contrast to endotoxin, does not trigger a nonspecific initiation of antibody formation; it only enhances specific responses. The major effect of pyran appears to be on macrophages.

Small angle light scattering techniques were used to determine the effects of selected polyanions on isolated mouse spleen nuclei. The light scattering measurements made it possible to distinguish nuclear swelling from lysis and/or aggregation. Heparin and dextran sulfate were shown to induce nuclear swelling. Chondroitin sulfate, although structurally similar to heparin, had no observable effect on the nuclei. Polystyrene sulfonate and pyran copolymer caused nuclear aggregation and lysis, respectively.

The mechanism of resistance to foot-and-mouth disease virus (FMDV) which occurs 2 days after divinyl ether-maleic only anhydride (pyran copolymer) treatment but diminishes by the 7th day was investigated. Adsorption of virus by minced infant mouse spleen, muscle, kidney, heart or liver was not affected either at 2 days or 7 days after treatment of the mice with pyran. However,in vitro multiplication of FMDV in minced kidney was affected by pyran at both times. The resistance of mice 2 days after treatment was related to absence of virus in muscle, kidney, and spleen tissues. Seven days after pyran administration, however, FMDV was recovered from muscle and spleen but not kidney. Interferon was recovered from various tissues 2 days but not 7 days after mice were injected with pyran. Other unknown polymer-induced antiviral factors as well as interferon may have been related to the resistance mechanism.

Pyran copolymer, a known immunostimulator, was found to be a potent inhibitor of purified DNA polymerase (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) isolated from avian myeloblastosis virus. Unlike other inhibitors, pyran showed unique features of inhibition. It interacts with the polymerase at a region other than the template site. The inhibitory effect was overcome only by excess enzyme and not affected by excess template. The degree of inhibition was not template specific for the templates tested: 70S RNA from avian myeloblastosis virus, synthetic hybrid poly(rA)·oligo(dT) 10 , synthetic copolymer poly(dA-dT), and activated calf-thymus DNA. The observed rate of inhibition by pyran was shown to vary with the different polymerases tested. Inhibition was shown with all oncornaviral polymerases and, to a lesser extent, with mammalian polymerases. However, two of the three bacterial polymerases, by contrast, showed a marked activation.