EFFECT OF SERUM DFO CONCENTRATION ON NTBI REMOVAL USING A BLOCKING STEP

4.3.1 R ationale

A consideration when choosing a therapeutic dose of DFO is to know what dose is required to minimise or eliminate plasma NTBI. Clearly other considerations such as the dose required to achieve iron balance and the maximum tolerable dose are of primary importance, but it is presently unknown whether the removal of NTBI is dependent on the plasma concentration of DFO. During iv administration of DFO, the kinetics of NTBI removal may not be dependent on interaction of DFO with NTBI in the plasma compartment alone. However, by examining the kinetics of interaction of DFO with NTBI in isolated serum, the direct interaction of DFO with this compartment can be examined. In this section the effect of the serum concentration of DFO on NTBI removal is examined.

4.3.2 Experimental design and methodology

The source of pooled semm was from Thai TM patients attending the Thalassaemia clinic at Maharaj Nakom Chiang Mai Hospital, Thailand and received regular blood transfusions to keep baseline Hb values above 10 g/dl and mean Hb value at 12 g/dl as in Section 4 .2 .

Each patient semm was made in ahquots and stored at -20 °C. Semm samples were transported on dry-ice to the Department of Haematology, UCL and stored at -20 °C until analysis. Sera (3-4 months old) of 5 Thai TM patients were pooled and used for the study. The experimental design was essentially as in Section 4.2.

DFO was added from a freshly prepared stock solution 10 mM in MOPS buffer pH 7.0 to pooled TM semm to obtain a final concentration of 10 pM or 100 pM and incubated for between 0 and 8 hours before the addition of aluminium (a final concentration of 200 pM) to block free metal binding sites on the DFO. NTA was then added to a final concentration of 80 mM, incubated at room temperature for 30 minutes before ultrafiltration and NTBI measurement by HPLC method as previously described in Section 2.1.2.

4.3.3 RESULTS

It can be seen in Figure 4.2 that the concentration of NTBI in pooled thalassaemic semm was reduced from 2.30+0.13 to 1.90+0.30 pM by 10 pM DFO at 1 hour (delta NTBI = 0.40 pM) with an initial rapid phase (K^ = 0.40 pM/hr) followed by a very slow phase during the subsequent 1-8 hours chelation period. NTBI could not be cleared completely at 10 pM DFO. In comparison, the rate of NTBI reduction was faster when semm was chelated with 100 pM DFO with an initial rapid phase (K^ = 0.80 p.M/hr) followed by a slow phase (Kp = 0.14 pM/hr). If the graph were to be extrapolated at 100 pM DFO, NTBI would be completely removed with 100 pM DFO by 16 hours. This indicates that DFO concentration at 10 pM is too small to remove aU NTBI at a discernible rate in the isolated serum compartment during the time course of this experiment. Nevertheless, it may be possible to remove NTBI in plasma compartment completely with continuous DFO chelation at 100 pM for 16 hours if the rate is linear beyond 8 hours.

CO 3.0 P o o l e d t h a l a s s a e m i c s e r u m □ +0 nM DFO ■ + 1 0 n M D F O * ■ +100 DFO* 2.5 - 2.0 - 0.5 - 0.0 0 1 2 3 4 5 6 7 8 Time (Hours)

Figure 4.2 Rate of removal of NTBI in pooled Thai thalassaemia major (TM) serum by DFO with the blocking step. In the assay 0.36 ml of pooled serum (3-4 months old, stored at -20 °C) from 5 Thai TM patients attending the Thalassaemia clinic at Maharaj Nakorn Chiang Mai Hospital, Thailand, to receive regular blood transfusions was incubated with 10 mM DFO solution to obtain the final concentrations of 10 and 100 |iM and incubated at 37 °C for 0, 0.5, 1, 2, 4 and 7 hours. At the end of incubation serum was duplicated for one without AP^, and the other with Af"^ blocking by adding 3.6 |l i1of 20

mM Af"^ solution (a final concentration of 200 |iM), incubated at room temperature for 1 hour. Finally, 40 |Lil of 800 mM NTA solution (80 mM at final concentration) was added,

incubated at room temperature for 30 minutes and centrifuged on filtration filters at 9,000 rpm (6,900g), 4 °C for 30 minutes. The ultrafiltrate was measured for NTBI concentration with the HPLC method as previously described in Section 2 .1 .2 . The results obtained from four independent experiments are shown as mean±SEM.

4.3.4 Discussion

These findings suggest that a small proportion of plasma NTBI can be rapidly chelated but a large component of NTBI species is relatively unavailable for DFO chelation.

consistent with the findings in Section 4.2. Due the relatively small number of points during the kinetic study, because of constraints with volumes of sera available, curve fitting is not precise and extrapolation has been used. Therefore, interpretation of the significance of the exact values of the fast and slow phases must be made with caution. The time necessary to remove NTBI from the serum compartment at 100 |xM was obtained by extrapolation, assuming linearity. This was necessary because incubation periods beyond 8 hours were not used as the sample collection and incubation system were not sterile and it was found that at 37 °C, incubation periods of 24 hours resulted in significant turbidity, suggesting bacterial contamination. Linearity may not be true beyond 8 hours. Indeed findings in subsequent experiments (see below) are compatible with there being effectively no further NTBI removal at 10-20 |iM DFO beyond 1-2 hours.

The slow phase of NTBI removal may represent oligomeric iron species which are nonspecifically bound to plasma proteins; possibly albumin, although this hypothesis requires confirmation. The finding that NTBI removal can be enhanced by increasing the DFO concentration in plasma suggests that in patients who fail to clear NTBI using standard doses of DFO, higher doses could in principle improve NTBI removal. However, it is clear that the second phase of NTBI removal from the plasma compartment is still slow, even at serum concentrations of DFO which exceed those which are safely achievable clinically.

In vivo, during the use of DFO, the kinetics might differ from these in vitro findings because in principle NTBI may constantly be being cleared by the liver and other tissues and NTBI is also constantly being formed by the breakdown of red cells in macrophages. In other words the in vivo situation is not an isolated plasma compartment model but is theoretically subject to influences affecting the rate of formation and clearance of NTBI. Therefore, the kinetics of NTBI removal and the concentration dependence may differ from the plasma compartment model explored in these experiments. If however in the clinical setting, the kinetics and concentration dependence of NTBI removal on plasma DFO are similar to the above findings, this would suggest that the plasma compartment is the key compartment determining the rate at which NTBI interacts with DFO.