Pyran copolymer

Not only PVNO [1] but also the so-called pyrane copolymer stimulated the study of the physiological properties of synthetic polymers. This cyclic copolymers [5] from divinyl ether and maleic anhydride, first time described by Regelson (38), induces the formation of interferon. 

It should be pointed out that the interferon inducers presented in Table 2 are widely different in activity, polyribonucleotide duplexes [such as poly(I) poly(C)] being superior to most other polynucleotides, especially those in which the 2 —OH group has been replaced by a 2 -H, 2 -F, 2 —Cl, 2 -N3, 2 -0-CH3 or 2 -0-CO-CH3 group (see Table 7). The interferon inducers listed in Table 2 also differ in the kinetics of interferon production poly-carboxylates (e.g. pyran copolymer, PAA, PMAA, COAM) and fluorenone... 

C—C C—C—C- backbone PAA, PMAA, pyran copolymer) (-C-C-O-C-O-backbone COAM, COAP, COCEL)... 

A second structural feature for interferon induction by polycarboxylates is the presence of negative charges. They may be placed in either alternate (PAA) or adjacent (pyran copolymer) position but should occur in a regular and dense sequence. The polyanionic character of the polymer appears to be a prerequisite for interferon induction, antiviral activity and antibacterial activity, since uncharged polymers such as dextran, polyacrylamide, non-carboxylated polyethylene analogues of pyran copolymers and incompletely oxidized amylose were devoid of any of these activities48,49,51,107). 

Various synthetic substances are able to stimulate interferon production, some of them only in vivo (polycarboxylates pyran copolymer, polyacrylic acid, chlorite-oxidized oxyamylose low molecular weight compounds tilorone,... 

Breslow DS, Edwards El, Newburg NR. Divinyl ether-maleic anhydride (Pyran) copolymer used to demonstrate the effect of molecular weight on biological activity. Nature 1973 246 160-162. 

Interaction of Synthetic Polymers with Cell Membranes and Model Membrane Systems Pyran Copolymer ... 

We have Investigated the interaction of pyran copolymer with erythrocyte ghosts, with Intact erythrocytes and with liposomes of pure dlpalmitoyl-phosphatldylchollne (DPPC). Particular attention was given to the importance of divalent metals (especially Ca2+) in promoting the association of pyran copolymer with cell and lipid surfaces. High sensitivity differential scanning calorimetry shows ... 

The 1 2 copolymer of dlvlnyl ether and maleic anhydride ("pyran copolymer," I) exhibits a broad range of biological activity, and has been discussed elsewhere in this volume and in earlier reviews (JL, 2) Of particular interest is the polymer s antitumor activity after initial clinical testing and then withdrawal from clinical use due to severe toxicity, the drug is again under evaluation as an agent for the treatment of human cancer. ... 

In this paper, we examine the Interactions of pyran copolymer with model biomembranes of two kinds 1) the human red blood cell membrane (or red cell "ghost") and 11) multilamellar suspensions (liposomes) of dlpalmltoylphosphatldylchollne (DFPC), a pure synthetic phospholipid. Each of these systems offers advantages In studies of polymer-cell surface Interaction The red cell membrane, idille complex. Is still the most readily Isolated and best understood of the membranes of nonnal human cells, and Its molecular architecture Is, In a general way at least, typical of such membranes. The pure phospholipids provide a much simpler biomembrane model, with the prospect of yielding more complete Interpretation of experimental observations. 

The results described In this paper support the suggestion that divalent metal Ions promote the Interaction of pyran copolymer with membrane and lipid surfaces. 

Pyran Copolymer. Pyran copolymer used In calorimetric studies of red cell ghosts was NSC-46015, a gift of Dr. David S. Breslow of Hercules, Inc. For studies of pyran-llpld mixtures, the polymer was prepared by radical copolymerization of maleic anhydride and dlvlnyl ether according to the technique of Breslow (1), using 9/1 acetone/tetrahydrofuran mixture as solvent, and azoblslsobutyronltrlle as Initiator. The Inherent viscosity of the sample was 0.189 dl/g (0.5 g/100 ml In 0.05H NaCl, 30 C). 

Polymer-lipid mixtures were prepared by hydrating the dry lipid (L-a-phosphatldylchollne, dipalmitoyl Sigma Chemical Co.) in 50 mM Tris (tris(hydroxymethyl)amlnomethane) buffer, pH 7.4 containing pyran copolymer at a concentration of 1 mg/ml. The final lipid concentration was also 1 mg/ml. Ca2+ was added In the form of CaCl2- Thermal transitions of lipid samples were recorded at a heating rate of 12 C/hr. 

Figure 1 shows heat capacity profiles of human erythrocyte ghosts Incubated with Increasing concentrations of pyran copolymer In 310 Imosm phosphate buffer. The five endothermic transitions of the control sample (bottom curve) are labeled A, B2j C and D, In accord with earlier work by Brandts and coworkers (13-16) and from this laboratory (17-20). The A transition Is assigned quite securely as a partial denaturation of spectrin, the major cytoskeletal protein on the erythrocyte membrane (13). Bj. and B2 appear to Involve proteins and lipids (protein Bands IV.1 and... 

Pyran copolymer at a concentration of 3.7 mg/ml causes a marked change In the appearance of the heat capacity profile. In fact, each transition Is affected, with the possible exception of... 

In trying to characterize this Interaction more fully, we determined the reversibility of the pyran-lnduced perturbation of the heat capacity profile. Figure 2 shows profiles for control ghosts (the lower curve) and for ghosts Incubated with 3.7 mg/ml of pyran and then washed with fresh buffer 3 times - a treatment which reverses completely any effects of pH, Ionic strength or divalent Ions (14). The upper curve shows that the effect of pyran copolymer Is partially, but not completely, reversible under these conditions. In that the B2 and C transitions remain shifted. A small loss In amplitude of Bx may also be significant. This Is consistent with our quantitative binding experiments (discussed below) In which we have observed that a small but measurable amount of pyran remains associated with whole cells after a comparable treatment with fresh buffer. 

Effects of Divalent Ions. Because of the known affinity of pyran copolymer for divalent Ions (6-9), It seemed plausible that... 

Figure 2. Reversibility of pyran-induced perturbation of heat capacity profile. Key lower curve, control profile with ghosts suspended in 310 imosm sodium phosphate and upper curve, ghosts incubated with 3.7 mg/mL pyran copolymer, then washed three times with fresh 310 imosm phosphate buffer.

The Importance of divalent Ions shows up most clearly In quantitative binding experiments using C-labeled pyran copolymer. In these experiments, whole ei throcytes - not Isolated membranes - were Incubated at room temperature for 10 minutes with the desired concentration of pyran copolymer In buffer. The cells were then sedimented by centrifugation, washed twice with fresh buffer, disrupted with detergent, and the radioactivity detennined by liquid scintillation counting. 

Complexation with Ca2+ seems to cause pyran copolymer to become "sticky," and to adhere to the red cell surface, but the meaning of this result Is obscured somewhat by the fact that, at higher concentrations - higher than 60 y,g/ml - significant radioactivity Is sedimented In the absence of cells, suggesting flocculation of pyran/Ca microspheres as observed by Flel and coworkers. It Is possible that we are seeing only a hetero-flocculation of microspheres plus red cells, and that the association of pyran with the membrane Is nonspecific. 

Still, the question of structural perturbation of the membrane by pyran copolymer in the presence of physiological concentrations of divalent ions is an important one, so we applied calorimetry to this problem. Figure 5 shows as the top curve the heat capacity profile for ghosts in the presence of 5 nM Ca2+. 

Addition of pyran copolymer (at a concentration of 1 mg/ml) to the hydration buffer causes little, if any, perturbation of the transition behavior - perhaps a slight increase in transition half-width - whereas the addition of the same amount of pyran in the presence of 5 mM Ca causes each transition to shift up in temperature - the main transition by 1.2 C, and the pre-transition by 4 C. The main transition is also broadened, with the halfwidth Increased from 0.37 C to 0.62 C. Ca2+ alone cannot reproduce this effect in the presence of Ca2+ alone each transition is shifted by not more than 0.5 C. We feel that this result suggests a Ca +.promoted association of pyran copolymer with the lipid surface. The greater perturbation of the "head-group"... 

Figure 5. Heat capacity profiles of erythrocyte ghosts suspended in isotonic cacodylate buffer ed pH 7.4. The CtF and pyran copolymer were added as shown.

Figure 6. Thermal transition behavior of DPPC in multilamellar suspension in SOmM. Tris at pH 7.4. Key lower curve, DPPC I mg/mL middle curve, DPPC 1 mg/mL + pyran copolymer 1 mg/mC and upper curve, DPPC 1 mg/mC + pyran copolymer I mg/mL + CaCl, 5otM.

Ca at concentrations typical for divalent ions in human plasma, promotes the association of pyran copolymer with a pure phospholipid (dipalmitoylphosphatidylcholine) and with intact erythrocytes. This may suggest a more general mediation by divalent ions of the cell surface interactions and pharmacological properties of pyran copolymer. 

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