CONCEPTO DE AMPARO ADHESIVO

A compound with a high binding constant as well as a high degree of specificity for iron is required to remove iron in vivo effectively and safely. Iron(III) possesses an extremely high charge density due to a spherically symmetrical tripositive charge of radius 0.65 “A; therefore, it binds tightly to atoms bearing a highly negative charge density, particularly to oxygen species such as carboxylates, catecholates and HPO. Iron(III) is most stable when coordinated with 6 oxygen atoms. These may be supplied by three chelator molecules donating two oxygen atoms each (three bidentate chelators), or two chelator molecules providing three oxygen atoms (two tridentate chelators) or one molecule donating six oxygen atoms (a hexadentate chelator) (Singh et al., 1995).

The stabihty of the metal complex is influenced by the number of covalently linked arms on the chelator molecule; consequently, hexadentate ligands are more stable than bidentate ligands and also have greater binding power at low concentrations (>20 |iM). Comparatively, bidentate-hgand complexes (2:1 and 1:1) partially dissociate at low concentrations and these generate hydroxyl radicals. For example, the coordination of iron by a bidentate ligand (L) occurs as follows:

[Fe-L]'+ [Fe-Lî]^ Fe-L, 1:1 complex 2:1 complex 3:1 complex

The stability (affinity) constant for iron(III) is given by FFe-Ln^^l [Fe’^][L]" where n = 3 in this case.

The affinity of bidentate ligands for iron(III) reflects the pK^ values (the pH at which half is dissociated) of the two chelating oxygen atoms-the higher the affinity, the higher the pK^ values. The stability of iron(III) can be enhanced over that observed with bidentate chelators using oligodentate structures. Unfortunately, tridentate and tetradentate ligands have an undesirable tendency to form polymeric iron complexes. By contrast, only one hexadentate molecule is needed to coordinate one iron atom. Thus, the overall stabihty constant is given by the same equation where n = 1 for hexadentate, n = 2 for tridentate and n = 3 for bidentate. Most microbial siderophores are hexadentate, which do not suffer from the disadvantage of iron precipitation, and apparently are more stable with iron complexes under dilute conditions.

Complexing agents such as ethylenediamine tetraacetic acid (EDTA) should have high affinities for metal ion binding and so reduce the free aqueous metal ion concentration substantially. However, such nonselective ligands tend to bind not only Ca^"^ but also some

cations including Mn^^, as well as Fe^^. In contrast, microbial siderophores have a high affinity and selectivity for Fe^ and have been used as prototypes or models for therapeutic iron chelating agents.

1.5.3.2 Physical properties

The pK^ values of any titratable group, for example, the pH at which half is dissociated, must be determined in order to calculate the proportion of chelator which possesses zero net charge at physiological pH. The value can be determined either spectrophotometrically or potentiometrically.

An oral iron chelator can cross biological membranes enabling it to be absorbed from the intestinal tract and also to enter cells of a range of tissues including liver and heart. Most drugs diffuse through the hydrophobic region of the cell membrane, and uncharged drugs permeate more rapidly than charged molecules into the cells. So neutral oral chelators are more active and likely to cross epithelial cells of the intestine than charged molecules, and should be able to penetrate into the cytoplasm of cells. To achieve oral activity, the chelator must be resistant to the acidity of the stomach and enzymatic cleavage; therefore, esters, amides, Schiff base ligands and hydroxamates should be avoided. When intracellular iron has been chelated by the ligand, the iron-hgand complex should move across the cell membrane easily.

The ability of hydrophobic free ligand to cross membranes must be controlled to avoid its penetrating the blood-brain barrier, causing adverse effects. Both the free ligand and iron- ligand complex should be water soluble and possess no charged groups to be able to circulate in the plasma and finally be excreted by the kidneys in liganded form. One physical parameter, the partition coefficient (Kpan) between an organic phase (usually n- octanol) and buffered water pH 7.4, is measured to predict the ease with which molecules penetrate membranes. A value of 1 denotes approximately equal solubility in lipid and aqueous phases. For example, a Kpait for a chelator in the range 0.2-1.0 is close to the ideal for a combination of oral activity, ability to permeate hepatocytes and lack of acute toxicity. A combination of high concentration of hydrophilic chelator with low concentration of more lipophilic chelator is an alternative way to achieve maximum chelation with low toxicity (Porter et al., 1989).

Previous studies indicated the relation between Kpart value and MW of compound with permeability coefficient for the blood brain barrier in rat. Penetration of low MW molecules ^ is strongly dependent on Kpart values. Most hexadentate ligands (MW~400-1000 D) are i' •

predicted to penetrate the blood-brain barrier inefficiently, and bidentate ligands (M W -150- 250 D) will penetrate relatively easily (Table 1.3).

Table 1.3 The relative advantages and disadvantages of bidentate and hexadentate ligands (Hider et al., 1994).

Bidentate ligands Hexadentate ligands

1. Molecular weight 150-250 1. Molecular weight 400-1000 2. High oral bioavailability 2. Low oral bioavailability

3. Penetrate blood-brain barrier easily 3. Penetrate blood-brain barrier slowly 4. Possible^redistribution of iron 4. Unlikely redistribution of iron 5. Affinity for iron is concentration-dependent 5. Affinity for iron is concentration

independent

1 6. Form 2:1 and 1:1 complexes which are 6. Only form 1:1 complexes which are potentially toxic generally non-toxic

1.5.3.3 K inetic stability

Kinetic stability indicates how frequently a ligand (L) dissociates from an iron-ligand (Fe- L) complex and exchanges with another ligand molecule (L*). Fe-L complexes have a range of kinetic stabilities depending on the design of clinically useful iron chelators. If an Fe-L complex has low kinetic stability, iron will be dissociated easily and possibly distributed throughout the body. Thus, a kinetically stable ligand molecule should be used for chelation therapy because the chelated iron remains tightly bound to the ligand.

FeL) + L* ---> FeLjL* + L

Hexadentate ligands generally have an even higher kinetic stability than bidentate ligands. Fe-L complex should be excreted rapidly in the faeces or urine with no redistribution of iron from non-toxic sites such as liver to more harmful ones such as the heart. It has been shown that hexadentate ligands are more stable than bidentates. Hexadetate ligands also

have greater binding power at low concentrations (<20 pM) and therefore are less likely to dissociate (Porter et al., 1989).

The use of the stability constant alone does not fully reflect this concentration dependent chelator effect. The use of a term called the pM, defined as -log [Fe^^] under defined conditions (e.g. when pH = 7.4, ligand^,^^^) = 10 |iM and = 1 pM), is in many ways more useful (Hider et al., 1996; Motekaitis and Martell, 1991). Compounds with high pM values under these conditions (i.e. a low concentration of free metal) will have advantages over compounds with low pM values, even if the stability constant for the chelator in question appears less favourable. This is because the concentration of free iron is given by this term at physiologically relevant free iron concentrations and chnically achievable chelator concentrations. The importance of this concept can be illustrated by contrasting the pM for the bidentate acetohydroxamic acid (pM = 12.5) with that of DFO (pM = 26.3) (Motekaitis and Martell, 1991). Thus, although the difference in the stability constant is relatively small (28.3 for hydroxamic acid vs 30.99 for DFO), the difference in the pM value is large, a feature resulting from the higher denticity of DFO. For the competing reactions, it is possible to define an effective stabihty constant (K^^f) which gives an accurate measure of the stability of a metal complex over a wide range of pH and in the presence of interfering metal ions.