Session 6: Development of Antioxidant Pharmaceuticals 511

6~13 USE OF A HIGH MOLECULAR WEIGHT IRON CHELATOR TO PREVENT OXIDATIVE DAMAGE IN REFERFUSION INJURY, BURN, AND OTHER SEVERE TRAUMA. B. E. Hedlund, I’. E. Hallaway, and P.R. Dragsten Biomedical Frontiers, Inc., 10th Ave. SE, Minneapolis, MN 55414.

The presence of free iron is known to greatly exacerbate oxidant injury in a variety of severe injuries. The classic example of this is reperfusion injury following &hernia in which iron is released from its cellular storage sites during anoxic stress, and catalyzes the generation of highly reactive hydroxyl radicals and lipid radicals when the oxygen supply is reestablished during reperfusion. It has been shown in animal models that removal of this free iron greatly reduces the tissue damage associated with this type of injury.

The only iron chelator currently used clinically is deferoxamine fDesferal@, DFO). It is unsuitable for use in such critical care indications due to its acute toxicity at high doses and short half life in plasma (5 to 10 minutes). However, by chemically conjugating DFO to polymeric colloids currently in clinical use for volume replacement, the iron binding characteristics of the resulting high molecular weight iron chelator are maintained unchanged, the plasma half life increased to 5 to 10 hours, and the toxicity reduced by several orders of magnitude compared to DFO.

A conjugate of deferoxamine with hydroxyethyl starch (HES-DFO) has favorable pharmacokinetics and markedly reduced toxicity as compared with DFO. It has shown considerable promise in preventing oxidative damage in several reperfusion injury models and in resuscitation from severe bum injury. In particular, following a 40% surface bum in sheep, subsequent intravenous infusion of HESDFO increased oxygen utilization, and prevented hemolysis and liver and lung tissue lipid peroxidation. It is currently being developed for use in resuscitation from severe bum and smoke inhalation injury.

635 IRON BINDING OF NITECAPONE AND ITS DERIVATIVES Teruyuki Kawabata and Lester Packer, Department of Molecular & Cell Biology, University of California, Berkeley, California 94720, USA.

Nitecapone(an inhibitor of catecohol-O-methyl trans- ferase) and its derivatives(OR1246 and entacapone) can chelate ferric iron in both water solution and organic solution(DMF, DMSO, methanol). Though Fe(III)- nitecapone and entacapone complexes did not show clear, visible absorption in the buffer solution, Fe(III)- OR1246 showed an absorbance around 5OOnm which is similar to Fe(III)-catecohol complex. The molar ratio of iron to OR1246 was l/3. By ESR spectra, all of the complexes showed ferric high-spin iron with maximal rhombicity. In organic solution, Fe(III)-nitecapone and its derivative complexes changed the absorbance in the visible region depending on the ratio of nitecapone to iron. In a higher ratio of nitecapone and its derivative to iron, the complexes showed absorbances around 500nm which was similar to the absorption in buffer, but in lower ratio of nitecapone and its derivative to iron, the complexes had a green color with an absorption around 700nm. The green complexes were 1:1 iron complexes of nitecapone and its derivative. ESR showed that they were in ferric high-spin state with maximal rhombicity by ESR. In addition the charactors of Fe(B)-nitecapone and its derivatives were studied in buffer(pH7.4). The ferrous iron was more easily oxidized in the buffer than Fe(B)-citrate and had a much lower activity while catalyzing conjugated diene formation from linoleic acd in SDS-dispersed system. We inferred that nitecapone and its derivative complexes could chelate iron with catecolatoferrate type at pH7.4 and decrease the activity of iron to induce lipid peroxidation.

IDENTIFICATION AND ANTIOXIDANT ACTIVITIES OF Mn(III)DESFEBRIOXAMINE B AND E COMPLEXES

6:14 Kevin FaulkneP, Irwin Fridovicha, Bob Steven& a Department of Biochemistry, Duke University Medical Center, Durham, NC 27710; b Department of Genetics and Metabolism, Duke University Medical Center, Durham, NC 27710

Our goal is to develop a low molecular weight Mn based

mimic of the MnSOD protein. The complex manganese

desfenioxamine B (MnDFB) has shown some promise, but there has

been debate as to its identity, and to its mode of action, We used

electrospray ionization mass specuometry @MS) to clearly identify

the MnDFB complex as a hexacoordinate Mn(lIl) complex, similar to

the Fe(III)desfcrrioxamine complex.

We have also studied a Mn desfetrioxamine E complex.

The desferrioxamine E ligand is a cyclic analog of the

desferrioxamine B ligand, and shows a higher affinity for the Mn(III)

ion in aqueous solution at pH 7.8. Using the cytochrome c assay

developed in this laboratory for SOD we have studied the SOD

activity of MnDFB, and the MnDFE complex. We compared the

lactate dcpcndence of the acuvities of these complexes and find that

whereas the activity of Mn(l1) is markedly enhanced by lactate that of

MnDFB is only weakly affected, and that of MnDFE hardly at all

affected by this &and. A search for a complex which is highly stable

and includes electrostatic attraction for Or.- continues.

SHORT-CHAIN HOMOLOGUES OF DIHYDRO- 6:16 LIPOIC ACID CAN PROTECT IRON-INDUCED LIPID PEROXIDATION IN THE AOUEOUS PHASE Teruyuki Kawabata and Lester Packe;, Department of Molecular & Cell Biology, University of California, Berkeley, California 94720, USA.

The effect of the reduced forms of alpha-lipoic acid fDHLA). its homoloeues and derivative on Fe(B)- citrate-induced lipid p&oxidation was studied in lipid dispersions and liposome systems. BisnorDHLA and tetranorDHLA were used as short carbon chain homologues and methylDHLA as a methylester derivative. In the dispersed system tetranorDHLA protected conjugated diene formation induced by Fe(II)- citrate depending on the concentration. In the presence of Fe(B)-citrate. the oxidation rate of tetranorDHLA was ~ , slower than the rate of DHLA. Oxygen consumption in the reaction mixture containing tetranorDHLA was much more rapid than for other homologues or derivative. However, in liposome systems, the iron- induced lipid peroxidation was not inhibited by tetranorDHLA,though the tendency of the oxidation and oxygen consumption rates were the same as in the dispersed system. TetranorDHLA inhibited Fe(II)- citrate-induced lipid peroxidation by accelerating the oxidation of ferrous iron to ferric iron in aqueous phase, while in the liposome system it was not enough to decrease the ferrous iron concentration in lipid phase. The more lipophilic homologues and derivatives with long carbon chain were not able to inhibit the lipid peroxidation in either dispersed or liposome systems because they could not effectively decrease ferrous iron, suggesting that only ferrous iron just at the reaction site determined the rate of lipid peroxidation.