Targeting of naproxen covalently linked to HSA to sinusoidal cell types of the liver.

The kinetic behaviour of a naproxen human serum albumin conjugate (Nap23-HSA) was investigated in rats and in isolated perfused rat livers (IPRL), as compared to its active metabolite naproxen-lysine (Nap-lysine) and free naproxen. Through covalently linking the anti-inflammatory drug naproxen to HSA, this drug can be selectively delivered to non parenchymal cells of the liver. Liver endothelial and Kupffer cells play an important role in the pathogenesis of inflammatory liver diseases. Targeting naproxen to these cells might increase its efficacy and reduce the side effects. The altered kinetic properties of Nap23-HSA, after i.v. injection of 22 mg x kg(-1), as compared to an equimolar amount of the uncoupled drug, were demonstrated in vivo by a decrease in the steady state volume of distribution (41 +/- 5 vs. 134 +/- 19 ml x kg(-1)), a decrease in its clearance (0.48 +/- 0.05 vs. 0.63 +/- 0.1 ml x min(-1) x kg(-1)), a shorter plasma half life (60 +/- 11 vs. 152 +/- 44 min) and a sustained biliary excretion. Liver targeting of Nap23-HSA was clearly demonstrated: drug content of the liver 180 min after injection was about 30 times higher for Nap23-HSA as compared to naproxen itself. The IPRL experiments showed that the Vmax of hepatic removal of the conjugate was 40 microg x min(-1) x g liver(-1). With doses below receptor saturation a rapid removal of the conjugate (t1/2 = 6 min) from the perfusion medium was found. In conclusion, this study demonstrates the saturable uptake of Nap23-HSA and its lysosomal degradation in both in vivo and IPRL experiments. Covalently linked naproxen is released as Nap-lysine. This active metabolite accumulates in Kupffer and endothelial cells in which it reaches therapeutic concentrations. Release from these cells leads to rapid uptake by hepatocytes and carrier mediated excretion into bile. Levels of Nap-lysine in bile and plasma reflect the slowest step in its generation: the proteolytic release in endothelial and Kupffer cells.

Targeting naproxen to non-parenchymal liver cells protects against endotoxin induced liver damage.

Non-steroidal anti-inflammatory drugs (NSAID's) could be of value in the treatment of liver disease; however, their use in this situation is limited by renal side effects. Therefore, we explored whether naproxen covalently bound to human serum albumin NAP-HSA) was able to reduce toxicity in an acute model of liver disease induced by endotoxin in rats pretreated with Corynebacterium parvum. In the isolated perfused liver of such animals endotoxin induced cholestasis (0.62 +/- 0.05 vs. 0.24 +/- 0.09 microliter.min-1.g liver-1; p < 0.05), increased vascular resistance (11300 +/- 400 vs. 311000 +/- 2000 dyn.s.cm-5; p < 0.05) and alanine aminotransferase release (22 +/- 9 vs. 149 +/- IU/l; p < 0.05). At the highest dose tested (22 mg/kg, corresponding to 6.0 mumoles naproxen), NAP-HSA normalized ALT release (21 +/- 10 IU/l: p < 0.05) while an equimolar amount of non-targeted naproxen was only partially effective (56 +/- 19 IU/l). A conventional dose of naproxen similarly prevented transaminase release. Cholestasis and increased vascular resistance were also prevented by NAP-HSA. Drug targeting by linking drugs to proteins is a potentially useful approach to maximizing drug effect while minimizing adverse events; this could be particularly useful for compounds with potentially serious adverse effects in patients with chronic liver disease such as the nonsteroidal anti-inflammatory agents used in the present study.

Drug-targeting to the kidney: renal delivery and degradation of a naproxen-lysozyme conjugate in vivo.

A renal-specific controlled release of an active drug may enable a reduction of the required dose and may provide a reduction of extra-renal toxicity. To achieve renal specific targeting of the NSAID naproxen, the low-molecular-weight protein (LMWP) lysozyme was employed as carrier since it is mainly taken up and catabolized in the proximal tubules of the kidney. A conjugate was synthesized with an average coupling degree of 2 mol naproxen per 1 mol lysozyme in which the drug was directly coupled to the protein via a peptide bond. First, we investigated whether naproxen conjugation affects the renal disposition of lysozyme. As native lysozyme, the conjugate was predominantly and rapidly (within 20 min) taken up by the kidney. The subsequent decrease in renal content reflecting the renal degradation of the conjugated lysozyme molecules appeared also to be similar to that of native lysozyme with a half life of four hours. Second, the effect of lysozyme conjugation on the body distribution of naproxen was studied. An important observation with regard to the aimed reduction in extra-renal side effects was that no detectable amounts of free naproxen were present in the plasma after administration of conjugate. Conjugation of naproxen to lysozyme resulted in a pronounced (70-fold) increase of naproxen accumulation in the kidney. In agreement with the protein disposition study, the conjugate was rapidly taken up by the kidney and subsequently degraded. In conclusion, renal selective targeting of the NSAID naproxen can be obtained by conjugation with the LMWP lysozyme. This concept of drug delivery to the kidney has the potential to improve drug efficacy and safety.

Binding of the substrate analogue perseitol to phosphorylated and unphosphorylated enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

Enzyme IImtl catalyzes the concomitant transport and phosphorylation of the hexitol mannitol. Here we demonstrate that the heptitol perseitol is not phosphorylated and not transported by the enzyme. However, the enzyme binds perseitol with an affinity comparable to the affinity for mannitol. Apparent affinities of the phosphorylated enzyme for perseitol were inferred from the inhibition by perseitol of mannitol phosphorylation and uptake. Apparent affinities of the unphosphorylated enzyme follow from the inhibition of mannitol binding to the enzyme. Mechanistic interpretations of the apparent inhibition constants are discussed, and it is concluded that phosphorylation of the cytoplasmic domain of enzyme IImtl has little effect on the affinity of the membrane-bound domain of the enzyme for perseitol.

Heat-labile enterotoxin crystal forms with variable A/B5 orientation. Analysis of conformational flexibility.

A new native crystal form of heat-labile enterotoxin (LT) has two AB5 complexes in the asymmetric unit with different orientations of the A subunit with respect to the B pentamer. Comparison with other crystal forms of LT shows that there is considerable conformational freedom for orientating the A subunit with respect to the B pentamer. The rotations of A in different crystal forms do not follow one specific axis, but most of them share a hinge point, close to the main interaction area between A and B5. Analysis of the two high-resolution structures available shows that these rotations cause very little change in the actual interactions between A and B5.

X-ray studies reveal lanthanide binding sites at the A/B5 interface of E. coli heat labile enterotoxin.

The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin. One of the interface sites is conserved in the very similar cholera toxin sequence. These sites may be potential calcium binding sites. Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.

Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli.

Examination of the structure of Escherichia coli heat-labile enterotoxin in the AB5 complex at a resolution of 2.3A reveals that the doughnut-shaped B pentamer binds the enzymatic A subunit using a hairpin of the A2 fragment, through a highly charged central pore. Putative ganglioside GM1-binding sites on the B subunits are more than 20A removed from the membrane-crossing A1 subunit. This ADP-ribosylating (A1) fragment of the toxin has structural homology with the catalytic region of exotoxin A and hence also to diphtheria toxin.