Speciation 98: Abstracts

Many complexing agents pharmacologically inactive by themselves display antiinflammatory properties when associated with copper. ^1,2 Among these, DIPS has been the most extensively studied and may be considered as the reference copper-potentiating ligand in animal models of inflammation. In spite of this large body of pharmacological data, the interpretation of the role of copper(II)-DIPS interactions in the antiinflammatory activity of copper(II)-DIPS complexes or mixtures at the molecular level is still a matter of conjecture. Superoxide dismutase mimetic activity was originally suggested as a possible underlying process, but induction of lysyl oxidase, modulation of prostaglandin syntheses, stabilisation of lysosomal membrane and modulation of histaminic activity, T-lymphocyte responses and enzymatic activities were also advanced.¹ More recently, down regulation of nitric oxide synthase has been proposed,³ and catalase- and peroxidase-mimetic activities have been characterised for Cu(II)₂(3,5-DIPS)₄ in vitro.⁴

Another possibility for DIPS to potentiate copper inflammatory properties is that DIPS corresponds to the recently defined notion of ^.OH inactivating ligand (OIL).⁵ The first requirement for a potential OIL to be active is that it can significantly mobilise copper at the inflammatory site. Salicylate, the reference non-steroidal antiinflammatory drug (NSAID), was recently investigated in this context using computer-aided speciation and found to bind a negligible fraction of copper(II) at any pH in vivo. Therefore, salicylate is not a potential OIL.⁶ In contrast, anthranilate, an inactive copper-potentiating ligand, is expected to bind increasing fractions of copper(II) as the pH decreases (i.e., as inflammation grows) and is thus a potential OIL.⁷ These preliminary results suggest that the copper-induced extra activity of NSAIDs is independent of any Cu(II)-NSAID interaction, but that the potentiation of copper by inactive substances is effectively due to the interactions of these with copper(II) at inflammatory sites.

Given the position of DIPS among copper-potentiating substances, its capacity to mobilise copper(II) under inflammatory conditions in vivo is of a particular importance. This question was addressed in the present work. First, formation constants for copper(II)-DIPS complexes (binary and ternary with histidine, the predominant ligand of copper(II) in blood plasma) were determined at 37 ^oC in aqueous NaCl 0.15 mol dm^-3. On account of the poor solubility of DIPS and copper(II)-DIPS complexes in water, a series of determinations was carried out in water-ethanol mixtures from which constants relative to the aqueous medium were then extrapolated by linear regression. Glass-electrode potentiometry was used for these determinations, the identity of the identified complexes being confirmed by electronic absorption and circular dichroism spectroscopies.

Based on our latest results on the Cu(II)-histidine-DIPS ternary system, speciation simulations show that, like salicylate and anthranilate, DIPS is unable to bind plasma copper(II) significantly. However, a clear distinction among the three ligands appears under inflammatory conditions: contrary to salicylate, DIPS binds increasing fractions of copper as the pH decreases (i.e., as inflammation grows), a behaviour which is comparable to that of anthranilate and makes DIPS a potential OIL. Although DIPS binds proton and Cu(II) in a way similar to salicylate, its two isopropyl substituents, which apparently favour the formation of acidic ternary species, make DIPS more similar to anthranilate in terms of effective copper mobilisation. The high lipophilicity of DIPS and its complexes, which facilitates their solubility in membranes, is an additional factor in favour of DIPS high pharmacological activity. Preliminary reactivity experiments tend to corroborate these findings.

References

1. J.R.J. Sorenson, in Metal Ions in Biological Systems, Vol. 14, H. Sigel, Ed., Marcel Dekker, New York, 1982, p. 77.
2. J.R.J. Sorenson, Prog. Med. Chem. 26, 437 (1989).
3. J.G.L. Baquial and J.R.J. Sorenson, J. Inorg. Biochem. 60, 133 (1995).
4. G.A. Reed and C. Madhu, in Biology of Copper Complexes, J.R.J. Sorenson, Ed., Humana Press, Clifton, New Jersey, 1987, p. 287.
5. G. Berthon, Agents Actions 39, 210 (1993).
6. V. Brumas, B. Brumas and G. Berthon, J. Inorg. Biochem. 57, 191 (1995).
7. H. Miche, V. Brumas and G. Berthon, J. Inorg. Biochem. 68, 27 (1997).

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