Therapeutic use of transferrin to modulate anemia and conditions of iron toxicity.

As the main iron transporter, transferrin delivers iron to target tissues like the bone marrow for erythropoiesis. Also, by binding free iron, transferrin prevents formation of reactive oxygen species. Transferrin deficiency due to congenital hypotransferrinemia is characterized by anemia as well as oxidative stress related to toxic free iron. Transferrin supplementation may be beneficial in two ways. First, transferrin can correct anemia by modulating the amount of iron that is available for erythropoiesis. This is obvious for patients that suffer from hypotransferrinemia, but may also have beneficial effects for ß-thalassemia patients. Second, under conditions of iron overload, transferrin reduces oxidative stress by binding free iron in the circulation and in tissues. Hereby, transferrin protects the host against the reactive oxygen species that can be formed as a consequence of free iron. This beneficial effect is shown in hematological patients undergoing chemotherapy and stem cell transplantation. Transferrin may also be beneficial in lung injury, ischemia-reperfusion injury and hypomyelination. This review summarizes the preclinical and clinical data on the efficacy of exogenous transferrin administration to modulate certain forms of anemia and to prevent the toxic effects of free iron. Thereby, we show that transferrin has promising therapeutic potential in a wide variety of conditions.

Redox-active iron released during machine perfusion predicts viability of ischemically injured deceased donor kidneys.

Redox-active iron, catalyzing the generation of reactive oxygen species, has been implicated in experimental renal ischemia-reperfusion injury. However, in clinical transplantation, it is unknown whether redox-active iron is involved in the pathophysiology of ischemic injury of non-heart-beating (NHB) donor kidneys. We measured redox-active iron concentrations in perfusate samples of 231 deceased donor kidneys that were preserved by machine pulsatile perfusion at our institution between May 1998 and November 2002 using the bleomycin detectable iron assay. During machine pulsatile perfusion, redox-active iron was released into the preservation solution. Ischemically injured NHB donor kidneys had significantly higher perfusate redox-active iron concentrations than heart-beating (HB) donor kidneys that were not subjected to warm ischemia (3.9 +/- 1.1 vs. 2.8 +/- 1.0 micromol/L, p = 0.001). Moreover, redox-active iron concentration was an independent predictor of post-transplant graft viability (odds ratio 1.68, p = 0.01) and added predictive value to currently available donor and graft characteristics. This was particularly evident in uncontrolled NHB donor kidneys for which there is the greatest uncertainty about transplant outcomes. Therefore, perfusate redox-active iron concentration shows promise as a novel viability marker of NHB donor kidneys.

A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance.

BACKGROUND AND OBJECTIVES: The increasing demand for intravenous immunoglobulin (IVIG) necessitates the development of improved plasma fractionation methods, providing higher immunoglobulin G (IgG) recovery. Here, we describe a new IVIG production process resulting in a high yield of IgG and effective reduction of physico-chemically resistant viruses. MATERIALS AND METHODS: IgG was purified from Cohn fraction II+III by caprylic acid treatment, polyethylene glycol precipitation, anion-exchange chromatography, nanofiltration and ultrafiltration. Stability of the purified IgG was studied in different formulations. Virus reduction was studied with two viruses: bovine viral diarrhoea virus, assessed by an infectivity assay; and human parvovirus B19, assessed by polymerase chain reaction. RESULTS: The combination of caprylic acid treatment with polyethylene glycol precipitation and a single anion-exchange chromatography yielded polymer-free, pure IgG. The purified IgG could be filtered through a small pore-size virus filter (Millipore V-NFP) with high throughput and excellent yield. The formulated product was stable as a 100 g/l IgG solution. Bovine viral diarrhoea virus was effectively inactivated by the caprylic acid treatment, and parvovirus B19 was effectively removed in the polyethylene glycol precipitation and nanofiltration stages, the total reduction of parvovirus being approximately 14 log10. CONCLUSIONS: The new process gives pure and stable IgG solution with an average yield of 4.8 g of IgG per kg of recovered plasma and has a very high capacity to remove even physico-chemically resistant viruses.

Viral safety of Nanogam, a new 15 nm-filtered liquid immunoglobulin product.

BACKGROUND AND OBJECTIVES: Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus-reducing steps into the manufacturing process. Here we present virus-elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam) that comprises two specific virus-reducing steps: a 15-nm filtration step combined with pepsin treatment at pH 4.4 (pH 4.4/15NF); and solvent-detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus-reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus-reducing steps were studied. MATERIALS AND METHODS: Selected process steps were studied with spiking experiments using a range of lipid enveloped (LE) and non-lipid-enveloped (NLE) viruses. The LE viruses used were bovine viral diarrhoea virus (BVDV), human immunodeficiency virus (HIV) and pseudorabies virus (PRV); the NLE viruses used were parvovirus B19 (B19), canine parvovirus (CPV) and encephalomyocarditis virus (EMC). After spiking, samples were collected and tested for residual infectivity, and the reduction factors were calculated. For B19, however, removal of B19 DNA was measured, not residual infectivity. To reveal the contribution of viral neutralization, bovine parvovirus (BPV) and hepatitis A virus (HAV) were used. RESULTS: For the pH 4.4/15NF step, complete reduction (> 6 log(10)) was demonstrated for all viruses, including B19, but not for CPV (> 3.4 but < or = 4.2 log(10)). Robustness studies of the pH 4.4/15NF step with CPV showed that pH was the dominant process parameter. SD treatment for 10 min resulted in complete inactivation (> 6 log(10)) of all LE viruses tested. Precipitation of Cohn fraction III resulted in the significant removal (3-4 log(10)) of both LE and NLE viruses. Virus-neutralization assays of final product revealed significant reduction (> or = 3 log(10)) of both BPV and HAV. CONCLUSIONS: The manufacturing process of Nanogam comprises two effective steps for the reduction of LE viruses and one for NLE viruses. In addition, the precipitation of Cohn fraction III and the presence of neutralizing antibodies contribute to the total virus-reducing capacity of Nanogam. The overall virus-reducing capacity was > 15 log(10) for LE viruses. For the NLE viruses B19, CPV and EMC, the overall virus-reducing capacities were > 10, > 7 and > 9 log(10), respectively. Including the contribution of immune neutralization, the overall virus-reducing capacity for B19 and HAV is estimated to be > 10 log(10).

Development of a pharmaceutical apotransferrin product for iron binding therapy.

High-dose chemotherapy of patients with haematological malignancies results in extracellular iron accumulation and appearance of non-transferrin-bound iron, which is thought to predispose the patients to septic infections and contribute to organ toxicity. We describe the development of a human plasma-derived apotransferrin product for iron binding therapy. The product is purified from Cohn fraction IV of human plasma by two ion exchange chromatography steps and ultrafiltration. The process comprises solvent detergent treatment as the main virus inactivation step and 15 nm virus filtration and polyethylene glycol precipitation as removal steps for physico-chemically resistant infectious agents. Product characterization by electrospray and MALDI-TOF mass spectrometry indicated no other chemical modifications than N-linked glycan chains and disulphide bonds, except minor oxidation. The purity of the product was more than 98%, main impurities being IgG, IgA and hemopexin. The product had intact iron binding capacity and native conformation. A stable liquid formulation for the finished product was developed. The product has proved safe and well tolerated in early clinical trials in iron binding therapy.

Non-transferrin-bound iron during allogeneic stem cell transplantation.

Hydroxyl radical formation catalysed by non-transferrin-bound iron (NTBI) might contribute to transplantation-related complications. The occurrence of NTBI in 10 adult allogeneic stem cell transplantation (SCT) patients was followed for 20 d. The transferrin saturation reached 99% on d -4 and remained > 80% thereafter. NTBI, measured as bleomycin-detectable iron, was detected for 6-18 d in all patients with a peak on d -4. High transferrin saturation levels were associated with the appearance of NTBI with a threshold at 80% saturation. Prevention of the potential deleterious effects of NTBI might reduce transplantation-related morbidity.

Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth.

The ability of Staphylococcus epidermidis strains to grow in the presence of human transferrin and varying amounts of ferric iron was studied. At initial bacterial densities up to 10(4) cfu ml(-1), none of the three strains grew when transferrin iron saturation was below the full saturation point, whereas the bacteria grew consistently when transferrin was fully iron-saturated and there was non-transferrin-bound iron in the medium. Precultivation of the bacteria under iron-restricted conditions to induce siderophore production did not abolish the growth dependence on non-transferrin-bound iron. At initial bacterial densities of 10(6) cfu ml(-1), the bacteria proliferated consistently also in the presence of partially saturated transferrin. The results indicate that at low bacterial densities, S. epidermidis cannot utilise transferrin-bound iron for growth and that its proliferation is dependent on non-transferrin-bound iron.

Catalytically active iron and bacterial growth in serum of haemodialysis patients after i.v. iron-saccharate administration.

BACKGROUND: I.v. iron is commonly administered to haemodialysis patients suffering from anaemia to improve their response to erythropoietin therapy. It has been unclear whether routinely used doses of i.v. iron preparations could result in iron release into plasma in amounts exceeding the iron binding capacity of transferrin. Here, we have studied the effect of 100 mg of iron saccharate given as an i.v. injection on transferrin saturation and the appearance of potentially harmful catalytically active iron. METHODS: We followed serum iron, transferrin and transferrin-saturation before and 5-210 min after administration of iron saccharate in 12 patients on chronic haemodialysis due to end-stage renal disease. We measured catalytically active iron by the bleomycin-detectable iron (BDI) assay and transferrin iron forms by urea gel electrophoresis, and studied iron-dependent growth of Staphylococcus epidermidis inoculated into the serum samples in vitro. RESULTS: The iron saccharate injection resulted in full transferrin saturation and appearance of BDI in the serum in seven out of the 12 patients. BDI appeared more often in patients with a low serum transferrin concentration, but it was not possible to identify patients at risk based on serum transferrin or ferritin level before i.v. iron. The average transferrin saturation and BDI level increased until the end of the follow-up time of 3.5 h. The appearance of BDI resulted in loss of the ability of patient serum to resist the growth of S. epidermidis, which was restored by adding iron-free apotransferrin to the serum. Iron saccharate, added to serum in vitro, released only little iron and promoted only slow bacterial growth, but caused falsely high transferrin saturation by one routinely used serum iron assay. CONCLUSIONS: The results indicate that 100 mg of iron saccharate often leads to transferrin oversaturation and the presence of catalytically active iron within 3.5 h after i.v. injection. As catalytically active iron is potentially toxic and may promote bacterial growth, it may be recommendable to use dosage regimens for i.v. iron that would not cause transferrin oversaturation.

Exogenous apotransferrin and exchange transfusions in hereditary iron overload disease.

OBJECTIVE: To investigate whether apotransferrin administration and exchange transfusion can improve outcome in patients with the recently described recessive congenital iron overload disease, presenting with intrauterine growth retardation, severe lactic acidosis, aminoaciduria, and hemosiderosis of the liver that so far has been treatment-resistant and lethal. METHODOLOGY: Because the patients have hypotransferrinemia, hyperferritinemia, increased transferrin saturation, and bleomycin detectable iron in plasma, we designed a treatment regime aiming at decreasing free iron and iron overload. The serum transferrrin concentration was increased to adult level (2-5 g/L) by intravenous apotransferrin administrations and thereafter exchange transfusion was performed. RESULTS: Two patients were treated. In patient 1, the transferrin saturation decreased from a baseline value of 100% and remained normal after the third exchange transfusion, and in patient 2, a reversible beneficial effect was seen on transferrin saturation and bleomycin-detectable iron. However, both infants died later of the disease, at 10 and 8 weeks of age, respectively. CONCLUSIONS: Exogenous apotransferrin administration proved to be safe and might deserve evaluation in other neonatal diseases with presence of free iron in plasma.