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Part 9: Miscellaneous Complications and Pathophysiologic Mechanisms |
Department of Nephrology and Dialysis,1 Ospedale A. Manzoni, Lecco, and Department of Hypertension and Preventive Nephrology,2 IRCCS Multimedica, Sesto San Giovanni, Milan, Italy
Correspondence to: F. Locatelli, Divisione di Nefrologia e Dialisi, Ospedale A. Manzoni, Via Dell'Eremo 11, Lecco 23900 Italy. nefrologia{at}ospedale.lecco.it
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ABSTRACT |
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 TOP ABSTRACT EPIDEMIOLOGY OF PRCA IN...POSSIBLE CAUSES AND...CONCLUSIONSREFERENCES |
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Starting in 1998, the number of pure red-cell aplasia (PRCA) cases in
patients treated with recombinant human erythropoietin (rHuEPO) increased
dramatically. Most cases were observed in patients treated with epoetin alfa
produced outside the United States. The peak was observed in 2002; since then,
the PRCA incidence has declined.
Many factors are likely to have contributed to this upsurge. The
molecular structure of the various epoetins and patient characteristics do not
seem to play a major role. The route of administration holds some importance,
because most PRCA patients received rHuEPO subcutaneously. The peak of PRCA
cases overlapped with the removal of human serum albumin from the Eprex
formulation (Janssen-Pharmaceutica NV, Beerse, Belgium), for which polysorbate
80 and glycine were substituted. Polysorbate 80 may have increased the
immunogenicity of Eprex by eliciting the formation of epoetin-containing
micelles or by interacting with leachates released by the uncoated rubber
stoppers of prefilled syringes. Compared with the previous formulation, the
polysorbate 80 formulation has lower stability, making it more susceptible to
stress conditions such as insufficient attention to the cold chain. This
situation could facilitate protein denaturation or aggregate
formation.
Uncoated rubber stoppers were replaced with coated stoppers, and the
cold chain was reinforced; the Eprex formulation has remained
unchanged.
Even though the incidence of PRCA returned to very low levels,
discriminating the cause–effect relationship of a single action is
difficult, given that all occurred with a similar chronology, and that PRCA
develops after a relatively long exposure period. Careful observation of
future trends of new PRCA cases is thus mandatory.
KEY WORDS: Pure red-cell aplasia; anti-erythropoietin antibodies; erythropoiesis-stimulating agents; anemia; chronic kidney disease; immunogenicity; cold chain.
Recombinant human erythropoietin (rHuEPO) is a well established treatment for renal anemia. This product of molecular genetic technology has been used since the late 1980s and has an excellent therapeutic index, given its selective and potent effect on erythropoiesis and the occurrence of only mild-to-moderate adverse events.
As in the cases of other biopharmaceuticals generated by expression of DNA cell lines, immunogenicity was an early recognized risk (1). In humans, development of epoetin-induced antibodies can generate pure red-cell aplasia (PRCA), a rare condition defined as severe anemia secondary to the virtual absence of red blood cell precursors in the bone marrow. In this disease, epoetin-induced antibodies neutralize all the exogenous rHuEPO and cross-react with endogenous erythropoietin (EPO). As a result, serum EPO is undetectable and erythropoiesis become ineffective. The main features of this condition are severe anemia, low reticulocyte count, normal platelet and granulocyte counts, and bone marrow smears exhibiting an almost complete absence of red-cell precursors without any other remarkable signs.
Despite the widespread use of rHuEPO, the development of neutralizing antibodies against it remained a very rare complication for many years. Only 3 case reports were published before 1998 (2–4). Since then, the number of reported cases has increased dramatically (5,6). Most of the cases occurred in patients treated with Eprex (Janssen-Pharmaceutica NV, Beerse, Belgium), the epoetin alfa produced outside of the United States.
This marked peak in PRCA incidence opened the immunogenicity scenario to deep investigations into possible causes of the increase. Several putative causes were identified and a number of practical actions were instituted accordingly. As a result, the number of PRCA cases has progressively decreased over the last few years.
Does this mean that the mystery has been completely revealed and that PRCA is no longer something to worry about?
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EPIDEMIOLOGY OF PRCA IN PATIENTS TREATED WITH ERYTHROPOIETIN |
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TOPABSTRACTEPIDEMIOLOGY OF PRCA IN...POSSIBLE CAUSES AND...CONCLUSIONSREFERENCES |
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In 2003, the European Renal Association–European Dialysis and Transplant Association (7) collected official data on PRCA cases from the various brands of erythropoiesis-stimulating agents (ESAs). Confirming FDA data, they found that the annual incidence of PRCA was much higher for Eprex than for all the other epoetins: 5 cases of PRCA in patients treated solely with epoetin beta and 4 cases treated with Epogen [that is, epoetin alfa produced in the United States (Amgen, Thousand Oaks, CA, U.S.A.)] were described at that time; 3 more cases treated with epoetin beta and 3 cases treated with darbepoetin alfa were considered unlikely to be associated with those ESAs (7).
By 30 November 2005, Johnson & Johnson had confirmed 215 cases of antibody-positive PRCA: 189 in patients exposed only to Eprex, and 21 in patients exposed to another ESA in addition to Eprex (8). Looking at the distribution of the incidence rate of PRCA cases in patients exposed to epoetin alfa, the maximum peak was observed in 2002, with a reporting rate of 4.5 cases per 10,000 patient–years (8). Since then, the incidence has fallen dramatically, with a reporting rate of 2 cases per 10,000 patient–years in 2003, 0.5 cases per 10,000 patient–years in 2004, and just 0.3 cases per 10,000 patient–years in 2005 (to 30 November).
Bennett et al. (9) reviewed the reports of all PRCA cases associated with the use of the epoetin alfa received by the FDA between January 1988 and April 2004. In addition, data about epoetin beta were obtained from the manufacturer. Confirming Johnson & Johnson official data (8), the number of reported cases associated with Eprex increased annually, reached a peak between 2001 and 2002 and began to decrease thereafter. In nearly all cases, patients had received epoetin administered by the subcutaneous route. Interestingly, nearly half of the cases were reported from France, Canada, Spain, and the United Kingdom. Conversely, just 14 cases were reported from Germany and Italy. The interpretation of these data is not obvious, given that subcutaneous administration is common in Italy, but not in Germany.
Taken together, the data clearly suggested that the rise in the incidence of PRCA cases was associated mainly with subcutaneous epoetin alfa use outside the United States, but that after 2002, the number of reported cases dropped dramatically.
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POSSIBLE CAUSES AND IMMUNOGENICITY RISK FACTORS |
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TOPABSTRACTEPIDEMIOLOGY OF PRCA IN...POSSIBLE CAUSES AND...CONCLUSIONSREFERENCES |
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Many factors are likely to contribute to increased immunogenicity (10). Patient characteristics, dose, duration of therapy, and route of administration are certainly important. The formulation, the structure of the protein, and inappropriate handling of the drug may also influence immune response.
Patient Characteristics: Except for 2 patients with myelodysplastic syndrome (11), and despite the heavy use of rHuEPO in the oncology setting, all PRCA cases associated with the drug were reported solely in patients with chronic kidney disease (CKD). Patients with cancer are probably less likely to develop an immune response because of reduction in immunocompetency, the treatment itself, and a lesser duration of exposure to the drug.
Besides CKD, no other clinical feature has emerged as being associated with an increased risk of developing PRCA. In particular, the distribution of cases by treatment modality revealed that more hemodialysis patients were affected by this complication than were peritoneal dialysis patients or patients in the conservative phase of treatment (9,12); however, that finding reflects only the much higher number of hemodialysis patients treated with rHuEPO. No correlation was found with cause of kidney failure, age, or sex (6).
In general, patients receiving a certain drug for longer time may be more likely to develop an immune response against it. However, the median duration of treatment before PRCA diagnosis is quite variable, ranging from 1 month to 5 years (6).
Route of Administration: Immunogenicity is a property of given molecules, but the subcutaneous route is more likely than the intravenous to trigger immunogenicity because skin has a highly developed immune system. In skin, various cell types contribute to antigen presentation and immune response. Prolonged exposure of those cells to epoetin after subcutaneous administration could possibly increase immunogenicity (13). The subcutaneous route also increases the possibility of self-administration, making home storage more common and increasing the risk of inappropriate handling or storage of the drug.
It is fact that most of the patients who developed PRCA received rHuEPO subcutaneously and that the rise in the incidence of PRCA cases was preceded by a major shift from intravenous to subcutaneous administration, especially outside the United States. The different reporting rates of PRCA cases secondary to Eprex administration across various countries seem to support a role for increased immunogenicity by the subcutaneous route. However, that situation is not invariably true, given that, in some countries (such as Italy), most patients received Eprex subcutaneously, but PRCA was nearly absent (9).
Starting in December 2002, regulatory authorities contraindicated the subcutaneous use of Eprex in CKD patients in Europe and strongly discouraged such use in Canada and Australia. From the viewpoint of chronology, those steps were followed by a marked decrease in the observed incidence of new PRCA cases since 2003, suggesting that the route of administration plays an important role in increased immunogenicity with rHuEPO. However, this explanation cannot be considered the sole answer, given that the incidence of PRCA following subcutaneous administration of ESAs other than Eprex has remained unchanged over the intervening years.
Molecular Structure of Different ESAs: Human EPO is a hydrophobic protein of 165 amino acids stabilized by three N-glycans and one O-linked sugar chain. The carbohydrate content is essential to stability and plays some important roles in the activity and biosynthesis of the molecule. In addition, protein glycosylation can modulate conformation or proper exposure of peptidic epitopes because of intramolecular carbohydrate–protein interactions. This modulation may mask or enhance the immune reaction (14). Most of the antibodies against rHuEPO studied so far (3,5) show enhanced binding to partially or totally deglycosylated antigen, suggesting recognition of peptidic epitopes.
Currently, four different ESAs are available on the market: epoetin alfa, epoetin beta, epoetin omega (only in Eastern Europe), and darbepoetin alfa. The first three molecules have the same amino acid composition as EPO does, but they differ slightly in their carbohydrate content. That difference can hardly provide a reason for varying immunogenicity (15,16) considering that the glycate content of epoetin alfa and beta has not been modified in recent years.
Darbepoetin alfa differs from EPO at five positions in the amino acid sequence and contains 5 N-linked carbohydrate chains instead of 3 (17). As a result, it has a larger molecular weight, sialic acid content, and negative charge than EPO does. These differences in the amino acid sequence and carbohydrate content could theoretically be immunogenic. However, the locations of the amino acid substitutions at or proximal to the site of the carbohydrate addition probably hides them from immune surveillance (17). Factually speaking, the occurrence of PRCA following exposure to darbepoetin alfa is very rare.
In addition, new agents under clinical development and the imminent launch of biosimilar epoetins leave open the issue of unknown risk of immunogenicity.
Drug Formulation and Downstream Processing: The drug rHuEPO is poorly water-soluble; excipients are required to enhance its dispersion or to inhibit precipitation once the solution is mixed with water. Excipients vary from one ESA to the next, possibly explaining the various degrees of immunogenicity.
In 1998, the formulation of Eprex was modified in Europe because of the fear of "mad cow" disease. Human serum albumin (HSA) was removed from the initial formulation and polysorbate 80 and glycine were substituted. Since its registration in 1990, epoetin beta has contained polysorbate 20. Darbepoetin alfa contains polysorbate 80, but at a lower concentration than that found in Eprex. Polysorbates are a heterogeneous mixture of partial fatty acid esters of sorbitol-derived cyclic ethers (sorbitans and sorbides) condensed with ethylene oxide. They are commonly used in pharmaceuticals, cosmetics, and foods as solubilizers, stabilizers, and emulsifiers.
Like other surfactants, polysorbates tend to form micelles after a given critical concentration is reached in the solution. Starting from the observation that Eprex contains a polysorbate 80 concentration (0.03% weight:volume) far above its critical micelle concentration (CMC), but that epoetin beta contains a polysorbate 20 concentration only slightly above its CMC (0.01% weight:volume), Hermeling et al. (18) tested the hypothesis that micelles could be a trapping point for oligomers or monomers of erythropoietin. Increased immunogenicity could be a result of the presence of multiple epitopes exposed on the micellar surface (19). Subcutaneous administration may elicit further immunogenicity in this small fraction of micellar epoetin, because, after subcutaneous injection, the micelles may initially stay intact and encounter immune cells before they are diluted by body fluids.
In line with the foregoing hypothesis, Hermeling and colleagues found that Eprex contains not only monomeric epoetin but also a quantity of the protein eluting in high-molecular-weight fractions on gel permeation chromatography. For epoetin beta, minute amounts of epoetin are eluted before the main peak only after addition of polysorbate 20 to the eluent (18). The protein structure of micelle-associated epoetin was not confirmed by spectroscopic techniques, probably because of the very low content of these fractions as compared with free epoetin (18).
Researchers working for the manufacturer repeated the chromatographic studies using various Eprex preparations and did not confirm an association of erythropoietin with polysorbate 80 micelles (20). Instead, the manufacturer said that increased immunogenicity to Eprex may be caused by an interaction between the uncoated rubber stoppers previously used in prefilled syringes of Eprex and the stabilizer polysorbate 80 (8). That interaction results in the presence of organic compounds called leachates that could act as adjuvants; their concentration in the syringes is variable and increases with time and exposure to heat (21). In mice, leachates failed to show any significant effect on the immunogenicity of Eprex, but they yielded a positive, concentration-dependent antibody response in the same animal model immunized with ovalbumin, supporting the hypothesis of adjuvant properties (22). However, the appropriateness of this experimental model has been questioned, because ovalbumin is a foreign protein and acts as a vaccine, invoking an immune response that differs from the response associated with activation of autoreactive B cells (23).
As a precautionary measure, Johnson & Johnson retired prefilled syringes with uncoated rubber stoppers in July 2003 and instead introduced Teflon-coated stoppers in all prefilled syringes, preventing interaction with the stabilizer. Leachates are not present in other epoetin products, all of which have coated stoppers.
Drug Handling and Storage: The HSA-free formulation of epoetin alfa may be more susceptible to denaturation or to formation of aggregates under stress conditions, leading to increased immunogenicity. These stress conditions include drastic changes in temperature, prolonged exposure to bright light, and excessive shaking of the vial or syringe. Thus, inadequate handling and storage of the drug may have played an important role in the PRCA upsurge. This possibility is particularly true for subcutaneous use, because of the increased possibility of self-administration outside the hospital setting. Inadequate handling and storage may be also one of the explanations for national discrepancies in the PRCA incidence. In France, which had one of the highest reporting rates, a patient must go to the pharmacy in person to obtain the product, creating a subsequently greater risk of disruption in the cold chain. In Germany and Italy, where the number of PRCA cases was lower, rHuEPO is routinely administered by dialysis staff.
Tighter controls to minimize exposure to higher temperatures during manufacture and shipping and preceding patient use were introduced by the manufacturer after July 2002. In particular, it was emphasized that Eprex should be stored at between 2°C and 8°C at all times from manufacture to reaching the patient. Detailed instructions and recommendations were given for receipt of the product in its protective, insulated cool box or for delivery by refrigerated truck and storage in cold rooms and refrigerators. Guidance was also given for transferring Eprex to other hospitals or to outpatients in an appropriately insulated container.
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CONCLUSIONS |
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TOPABSTRACTEPIDEMIOLOGY OF PRCA IN...POSSIBLE CAUSES AND...CONCLUSIONSREFERENCES |
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Starting in 2003, the number of reported PRCA cases dropped dramatically. This change may have been caused by the shift in administration route, reinforcement of the product's cold chain, or elimination of uncoated rubber syringe stoppers. However, it is difficult to discriminate the cause-and-effect relationship of a single action, given that all have a similar chronology and that PRCA develops after a relatively long exposure period. Polysorbate 80, which has been hypothesized to favor micelle formation, is still present in the Eprex formulation.
Even if the incidence of PRCA has returned to very low levels, the mystery cannot be considered to be completely solved given that the mechanisms leading to the increased immunogenicity are still not understood in full. Careful observation of future trends in new PRCA cases is mandatory, especially if Eprex is again to be administered by subcutaneous injection. The possible imminent launch of biosimilar epoetins requires similar consideration (24).
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REFERENCES |
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TOPABSTRACTEPIDEMIOLOGY OF PRCA IN...POSSIBLE CAUSES AND...CONCLUSIONSREFERENCES |
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