Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Filing 54

DECLARATION re #53 Memorandum in Opposition to Motion of Michael R. Gottfried in Support of Amgen Inc.'s Opposition to Defendants' Motion to Dismiss for Lack of Subject Matter Jurisdiction and Failure to State A Claim for Which Releif May be Granted by Amgen Inc.. (Attachments: #1 Exhibit 1#2 Exhibit 2#3 Exhibit 3#4 Exhibit 4#5 Exhibit 5#6 Exhibit 6#7 Exhibit 7#8 Exhibit 8#9 Exhibit 9#10 Exhibit 10#11 Exhibit 11#12 Exhibit 12#13 Exhibit 13#14 Exhibit 14#15 Exhibit 15#16 Exhibit 16#17 Exhibit 17#18 Exhibit 18#19 Exhibit 19#20 Exhibit 20 (part 1 of 2)#21 Exhibit 20 (part 2 of 2))(Gottfried, Michael)

Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

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EXHIBIT 4

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REVIEW

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J NEPHROLPOOS; 16: 461-466

Pure red cell aplasia secondary to treatment with erythropoietin
Francesco Locatelli, Lucia Del Vecchio
Department of Nephrologyand Dialysis, A. Manzoni Hospital, Lecco - Italy

ABSTRACT:Pure red cell aplasia (PRCA) is a rare condition defined as severe anemia secondary to the virtual absence

of rcd blood cell precursors in the bone marrow. In the setting of patients treated with rHuEPO, the disease is generated by epoetin-inducedantibodiesthat neutrdise all the exogenousrHuEP0 and cross-react with endogenouserythropoietjn. As a result, serum erythropoietinlevels are undetectableand erythropoiesisbecomes ineffective. Only 4 cases of PRCA associated with rh-EPO have been reported before 1998. Thereafter, a sharp increase in the incidence of ti rare condihs tion has been reported, mainly associatedwith epoetin alpha use outside the United States. A number of possible mechanisms leading to PRCA development have been identified. Among these, modification of drug formukition and down stream processing probably has had a major role. Indeed, in 1998 the formulation of epoetin alpha in Europe was modified because of the fear of the "mad cow" syndrome. However, differencesin molecule structureand glycosylation among different epoetins can not be excluded.It should also be underlined that the rise in the incidence of PRCA caseshas been coincident with a major shift from intravenousto subcutaneousadministrationof rHuEPO. The abrupt rise i the incidence of PRCA cases observed in the last few years, deserves particular attention;however, we n have to balance its severity, but extreme rarity, w t the high number of chronickidney diseasepatients who die each year ih because of cardiovasculardisease that could p a r w be reduced by anemia treatment, Key words: Pure red cell aplarau, Anmza, Chronac kidney disease,Erythropoietin, Anti-erythropozetzn antibodies,Molecular rtructure

INTRODUCTION
Therapy with 1 ecombinant human ci ythropoietin (rWuEPO) is a well established treatment foi renal anemia This product of molecular genetic technology has been used for more than 15 years and has an excellent therapeutic index (a selective and potent effect on el ythropoiesis against only mild to moderate adverse events such as aggravation of hyperlension o r thrombotic complications) However, the inci easing incidence of acquired pure red cell aplasia (PRCA)in patients with chronic kidney disease (CKD) treated with rHuEPO in the last few years has given rise to a number of concerns about this pharmacological agent PRCA is a rai e condition defined as severe anemia secondary to the virtual absence of led blood cell precursors in the bone marrow In the setting of patients treated with rHuEPO, the disease is generated by epoetin-induced antibodies that neutralize all the exogenous rHuEPO and cross-react with endogenous ery-

thropieiin A a iesult, serum erythropoietin levels s are undetectable and ei ythropoiesis become ineffective. In this review we will try to summarize the main features of the disease, its incidence and prevalence and the possible causes of increased immunogenicity which have been identified so far
Pure red cell aplasia: Main features and diflmential diagnosis

Acquired PRCA is an immune-mediated disoider of erythropoiesis that can result in severe anemia, low reticulocyte counts (< 10,OOO/mmz), normal platelet and granulocyte counts, and bone marrow smears exhibit an almost complete absence of red cell piecursors without any other remarkable signs Ancibodies can iecognise either erythi-oid precursor cells or erythropoietin: in the first case seium erythxopoietin is high, in the second caSe it is lo~7 As a result of (1). almost complete interruption of erythropoiesis, the

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hemoglobin concentration declines very fast, at a rate corresponding to a red cell life span (nearly 0 1 g/dL/day, slightly less than lg/dL/week) (2); mean corpuscular volume is usually normal or slightly increased PRCA has been associated with a number of different conditions that are to be kept in mind in the evaluation of a suspected PRCA case associated with rHuEPO theiapy This raie disease has often been ieported in association with thymoma, myelodysplasia,B- and Tcell chronic lymphatic leukemia and chronic myeioid leukemia, autoimmune hemolytic anemia, lupus erythematosus systemicus, rheumatoid arthritis, and viral infections (in particular paivovirus 1319 and hepatitis B virus) In addition, several drugs can cause PRCA The commonest drugs described in the literature were anticonvulsants, antibiotics and anti-thyroid agents (3) Of interest, a numbei of drugs commonly used in nephrological clinical practice have been reported to rarely cause PRCA. Among these, allopurino1 ( 3 ) , azathioprine (4), mycopheriolate mofetil and (5) It is also worth noting that PRCA has been reported in patients with no underlying disease (6, 7) PRCA associated with treatment with rHuEPO has been described as the abrupt appearance of seveie anemia refractory to treatment The median age of the patients was 61 years, with a slight predominance of males (8) The median duration of tieatment with rHuEPO before diagnosis was seven months, ranging from one month to five years ( 8 ) N o correlation was found with cause of kidney failure, CKD treatment, age or gender as such, but there was a disproportionally higher incidence in males over 70 years However, it is of note that elderly male patients are very common in the ESRD popuiation and the Authors did not clarify whether they corrected the analysis taking into account this increased pi evalence The majority of the patients had relatively stable clinical conditions, hemoglobin levels and iHuEPO doses before the onset of the disease (9-12). Many of them were switched to treatment with another rHuEPO aftei the worsening of anemia, hindering the recognition of the leal causeeffect relationship between a given type of erythropoietin and PRCA For this reason, and also because anti-erythropoietin antibodies cross react with other erythropoietins, treatment with rHuEPO must be stopped immediately in suspected cases Indeed, dose increases are ineffective and expose patients to the risk of systemic reactions (11) All PRCA cases associated with rHuEPO have been solely repoited in CKD patients, in spite of the large use of this drug also in the setting of oncology Patients with cancer are probably less likely to develop the disease because of a decrease in immune-com462

petency, concomitant treatment and reduced time of exposure to the drug.
lnCdme ojpuw red cell aplasia associated With eythmpoietin treatment

Only 4 cases of PRCA associated with rHu-EPO have been repoited before 1998 (9,13-15) Between 1998 and 2000, French investigators identified new 13 PRCA patients after taking rHuEPO, 12 receiving epoetin alpha and 1 receiving epoetin beta (10). Shortly thereafter, the Food and Drug Administration reported 82 cases of PRCXfrom July 1997 to December 2001 (8) The majority of cases were observed in patients treated with EprexO, (epoetin alpha), which is a product of Johnson & Johnson outside the United States (8) The observed increase in the distribution of Eprex@, this time period can not account foi differin ences among the brands in the number of reported cases (8) Up to March 24, 2003 Johnson &Johnson confirmed 163 cases of antibody positive PRCA; 142 in patients exposed only to Eprex@ 21 in patients exand posed to anothei erythropoietic protein in addition to TEprex* Further 61 cases are under investigation (Johnson &Johnson press release) A stabilization in thr worldwide exposure-adjusted rate of antibodypositive PRCA case reports was observed in 2002 versus 2001 (Johnson &Johnson press release) Avery recent official editorial by the European Renal AssociationEuropean Dialysis Transplant Association (ERA-EDTA) (16) repoi trd 5 PRCA cases in patients solelj~ treated with epoetin beta and 4 cases treated with Epogen" (epoetin alpha produced by Amgen in the United States) In addition, three more cases tieated with epoetin beta and three cases treated with darbepoetin alpha were considered unlikely lo be associated with the disease (16) The incidence per 10,000 treated patient years was confirmed to be much higher for Eprex" (1.11)than foi epoetin beta (0 lZ), EpogenO (0 02) and darbepoetin alpha (0 5 ) (16) Possible additional cases, i e one patient treated with epoetin beta (1'7) and three Chinese patients tieated with epoetin alpha, have recently been published (12). All these data clearly suggest that the rise in the incidence of PRCA cases in recent years is mainly associated with epoctin alpha use outside the United States.

Characteristicsoj anti-erythropoietin antibodies
Casadevall et a1 (10) extensivelystudied the characteristics of anti-eiythropoietin antibodies identified in a numbei of PRCA cases These antibodies were polyclonal, homogeneous in the single patient and all able to neutralize very high concentrations of the native protein After deglycosylation, the affinity of the antibodies increased in all the patients, excepting in the

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Fig. 1

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STRUCTURAL PROPERTIES OF rHuEPO Sequence variation Changes in ylycosylation

FORMULATION Introduction o f

Length of treatment

Contaminants
impurities

of administration _-.---

i

r---

I Patient characteristics1
only one who was treated exclusively with epoetin beta. In this patient, the antibodies were probably directed against linear and conformational epitopes (lo), suggesting that mechanisms of immunization can differ among rHuEPO types Figure 1 summarizes possible mechanisms leading to PRCA development

a

7

1

1

Assav technology --I

i

ecules ( Z l ) , but it does not explain the different immunogenicity Skibeli et a1 (22) repoited a more reduced sialylation of glycans in EPO than in rHu-EPO Even if these differences might be involved in the etiology of PRCA, artefacts due to extraction procedures of human erythropoietin explaining these differences can not he completely ruled out (23) The molecule of epoetin-omega is a sialoglycoprotein with smaller amounts of O-bound sugars, it is less Molecular structure o d i f l m t eythropoietims f acidic and has difkcnt hydrophylity than the other 2 Human erythropoietin (EPO) is a hydrophobic pro- epoetins (24) At present, no cases of PRCA have been tein of 165 aminoacids stabilized by thiee N-glyrans ieported in patients treated with epoetin omega, but (18) One O-linked sugar chain is also invariably pre- only a small number of patients have received the sent (19) Deglycosylation of the N-linked oligosac- drug charides made rHuEPO remarkably susceptible to ag- Darbepoetin alfa has recently been introduced onto gregation on heat-treatment, indicating that the car- the ma1 ket Like rHuEPO, it is produced by recombibohydrate chains are essential to stability (20) The nant DNA technology in Chinese hamster ovarian carbohydiate content also plays some impoi tant roles cells and it contains five N-linked carbohydrate in the activity and biosynthesis of the molecule. Given chains(two more than rHuEPO). To introduce new this high glycosylation content (almost 40% of total carbohydrate attachment sites into the polypeptide mass) and the requirement for sialic acid for optimal barkbone, the DKA sequence of the erythropoietin in vivo activity, mammalian cells are essential to pro- gene was modified, obtaining an aminoacid sequence that differs from EPO at five positions (25) As a result, duce rHuEPO (Chinese hamstei ovary cells) At present, three different types of rHuEPO are avail- darbepoetin alpha has a greater molecular weight, able on the market: epoetin alpha, epoetin beta and sialic acid content and negative charge than rHu-EPO (9) Since the aminoacid sequence and the carboliyepoetin omega (the last one only in Eastern Europe) All these three molecules have the same aminoacid drate content of darbepoetin alpha differ from EPO, composition, which is identical to that of EPO, but dif- it is theoretically possible that this new molecule could fer in their carbohydrate content Epoetin alpha has a be immunogenic However, the location of the slightly lower sialylation than epoetin beta; this ac- aminoacid substitutions proximal to the carbohydrate counts for the small diffeiences observed in the phai- addition site probably hides them from immune surmacokinetics and pharmacodynarnics of the two mol- veillance (9) Fuithermore, the fact that these addi-

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tion sites are distal to the receptor binding sites reduces the likelihood o developing neutralizing antif bodies (9) At piesent, no PRCA cases have been ieported in patients solely treated with this agent Even if the numbei of patients who have been tieated so far with darbepoetin alpha is much lower than that of patients treated with rHuEPO, thus significantly reducing the numbei of patients at risk of developing PRCA, the majority of the patients enrolled in clinical studies have been tested for the development of antibodies against darbepoetin alpha, but these have never been detected Two other new erythropoietic agents are under evaluation A pcgylated version (i e the covalent addition of the water soluble polyethylene glycol moiety) of epoetin beta, Ro 50-3821, has recently been synthesized and is under evaluation in a phase I1 clinical trial (26) Its aminoacid sequence is identical to EPO No antibodies against this new diug have been reported, but clinical experience is still veiy limited \%en considering the i isk of developing PRCA, the total chemical synthesis of new erythropoietic agents can add further conceins compared with EPO analogues obtained with the DNA recombinant technique. The "synthetic erythiopoiesis protein" (SEP) , a polymer consisting of a 166-aniino-acid polypeptide chain and two covalently attached, branched, polymei moieties that are negatively charged has recenrly been synthesized (27) In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantlypiolonged duration of action tn vivo, but there is still no information about its possible immunogenicity

residues (glycopeptidic epitopes) (30) Conformation or proper exposure of peptidic epitopes of glycoproteins is also frequently modulated by glycosylation due to intramoleculai cai bohydrate-protein interactions In highly glycosylated proteins, peptidic epitopes are either apparently masked by glycans, or conversely glicosylation may enhance the reaction (SO). The enhancement of antibody binding to partially or totally deglycosilated antigen suggest recognition of a peptidic epitope (31). This appears to be the case foi the majority of antibodies against rHuEPO studied so far (10) Also considering that the glycate content o epof etin alpha and beta has not been modified in recent years, it seems &likely that alteied glycosylation or differences in the carbohydrate content among erythropoietin types can explain the rise in the incidence of PRCA cases observed in the last three years.

Drug formulation and down stream processing

rHu-EPO is a poorly water-soluble diug For this reason, excipients are required to enhance its dispersion or to inhibit precipitation once the solution is mixed with water Excipients differ among rHuEPO types, possibly explaining various degree o imrnunogenicity of these f molecules and in particular the recent rise in the incidence of PRCA cases occurring mainly in patients treated with Eprex@In 1998 the formulation of this drug was modified in Europe because of the fear of the "mad cow" syndrome. Following regulatory guidance. human serum albumin (HAS) was removed from the initial formulation and substituted by polysorbate 80, which is a surfactant, and glycine Since its registration in 1990, epoetin beta has conProtein giycoglation and immumgenkity tained polysorbate 20, which is another surfactant Protein glycosylation is an ubiquitous post-translational Darbepoetin alpha contains polysorbate 80, but at a modification. Oligosaccharide units of glycoproteins lower concentiation than Eprex@The HAS-flee foiseive a variety of functions (28), including modulation mulation epoetin alpha may be more susceptible to of the biologic activity and the clearance of proteins formation of aggregates under stress conditions or from the plasma Structurcs of many oligosaccharide may be more susceptible to denaturation, leading to chains linked to pioteins are identical or closely similar increased immunogenicity This formulation may also amongst species and are not or ale only weakly im- be more susceptible to formation of aggregates in the munogenic. However, inteiest in glycobiology has in- presence of silicon oil, which is used to lubricate sycreased dramatically amongst immunologists during ringes Even if no aggregate formation was seen at the last few years due to the fact that oligosacchaiides storage conditions of 28"C, higher amounts of silicon also play a cential role in adhesion and homing events oil, with higher temperature over time, increases agduring inflammatory pi ocesses, comprise powei ful gregate formation (32) Fol this reason, tighter conxenotransplantation antigens, and may provide targets trols on product to minimize exposure to higher temfor tumor immunotherapy (29) In addition, alter- peratures during manufacturing, shipping, and prior ations in glycosylation are now known to occur in a to patient use have been developed to increase awareness about the importance of appropriate handling of number of autoimmune diseases (29) Antibodies raised against glycoprotein antigens may be the drug (32) The presence of contaminants or imspecific foi their cai bohydrate units which aie recog- puiities seem not to be a main cause, since no changes product before and after the process of bulk manunized irrespective of the protein cariicr (carhohydrate epitopes), or in tlic context of the adjacent amino acid facturing were detected (32
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Route of administration
The rise in the incidence of PRCA cases has been coincident with a major shift from intravenous to subcutaneous administration of rHuEPO, given the possibility of ieducing costs with the latter This occurred especially outside the United States and could have influenced the rise in the number of PRCA cases by two mechanisms First of all, it is well known that, the subcutaneous route is mole likely to give immunogenicity than the intravenous one (33) This because skin has a highly developed immune system with different cell types contributing to antigen presentation and immunc responses It is possible that prolonged exposure of these cells to epoetin after subcutaneous administration can increase immunogenicity (1 1) Secondly, the subcutaiieous route increases the possibility of self-administration, making home storage more common and increasing the risk of inappiopriate handling or storage It is a matter of fact that the majority of the patients developing PRCA received rHuEPO subcutaneously Furthermore, different reporting rates across various countries, all utilizing identical Eprex@, could support a role of increased immunogenicity for the subcutaneous route, For instance, in Germany the number of PRCA cases is low and only about 30% of the patients receive Eprex@ subcutaneously (Johnson & Johnson pi ess release) However, it is also tiue that in Italy the majority of the patients receive Eprex@, subcutaneously, but PRCA is rare In December 2002, regulatory authorities contraindicated the subcutaneous use of Eprex", in CKD patients The impact of this measure on the incidence of new PRCA cases is still unknown
What to do?

liminaiy observations suggest that nearly 40% of the patients remain transfusion-dependent The treatment of PRCA is still an open question, due to the little expeiience accumulated so far More than half of the patients appeal to respond to immunotherapy Corticostei oids alone, steroids associated with cyclosporine or cyclophosphamide, immunoglobulin, plasmapheresis have been used Steroid plus cyclophosphamide probably give the best results (34) Cyclospoiine also seems to be effective (34) Patients who received kidney ti ansplaiitation have had the best outcome This is piobably because immunosuppressive regimens instituted after tiansplantation aie eifective on PRCA as well

at correcting the underlying anemia, restoring enei gy

In CKD patients, rHuEPO is not only highly effective

Even if PRCA secondary to rHuEPO treatment is still a very iaie condition, physicians must constantly be aware of the possibility this disease can occur and consider it in the differential diagnosis of a patient who has rapid worsening of anemia or does not respond to the drug After a complete workup for anemia has been done (including assessment of the reticulocyte count) and other known causes of anemia have been excluded, if PRCA is still suspected, bone marrow evaluation and testing for erythropoietin antibodies are indicated In the meantime, rHuEPO therapy must be discontinued, without switching treatment to another type of erythropoietin, in order to avoid misinterpretation of the causative agent and occurrence of crossreactivity with the new compound Aftei-stopping treatment, a slow decrease in the autoantibody titre is usually observed (lo), but seium erythropoietin is detectable only when autoantibodies completely disappear Pre-

levels, and incieasing patient well-being and quality of life, but it also has considerable secondary benefits in terms of morbidity and mortality reduction, pai ticuXarly in relation to improved cardiac function The abrupt rise in the incidence of PRCA cases observed in the last few years deserves particular attention from the nephrological community However, putative causative mechanisms are very complex and peihaps nobody will be able to completely clarifji them For this reason, in the near future we will piobably have to live with PRCA, balancing its severity but extieme rarity with thc high number of CKI> patients who die each year because of cardiovascular disease that could partially be reduced by anemia treatment Also we must not forget the impressive benefits on quality of life and patient wellbeing obtained by anemia correction

Address for correspondence: Prof Frnncecco Locatelli, M 11. Divisione di Nefimlogia c Diuliri A O~pcddnl;. Mamoni 17ia Dell Tirema, 11 23900 Lecco, Itub n@ologia@oorpcduk b c o . it

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PRCA secondary lo treatment with erylhrofioieliiz
RETERENCES

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Rcceaued May 20, 2003 Accepted A!lq 27, 2003
0 Socictk Itdialla di Nefrologia