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Published on 11 April 2011

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Regulation of complex molecules


Christine Clark
BSc MSc PhD FRPharmS FCPP(Hon)

Hospital Pharmacy Europe hosted roundtable meetings in London and Paris – in June and September respectively – to discuss the use of non-biological complex molecules and the implications for regulation and evaluation. The discussions involved panels of nephrologists, researchers and leading pharmacists with expertise in medicines safety, nephrology and regulation.

Clinical equivalence of complex products
The panels first considered the properties of generic and biosimilar products and the corresponding regulatory pathways. Presentations were given by Huub Schellekens (London) and Jean-Pierre Reynier (Paris).

The generic paradigm, which assumes that pharmaceutical and bio-equivalence guarantee therapeutic equivalence, cannot be applied to biologics, which are generally large and complex molecules whose biological properties cannot be predicted from laboratory tests.

Biologics are products of living cells. Examples include Factor VIII and products synthesised by bacteria. They tend to be unstable and can change during storage. It is impossible to ensure that identical copies of the originator are produced. Even products of similar composition can vary in their spatial configuration. They are also highly immunogenic.

Biologics are difficult to characterise fully because they tend to contain mixtures of related products.  Assays of biological products are difficult and ambiguous and efficacy markers are often unclear. As yet, there is no single tool that can completely characterise a biological product.

Biologics have complex modes of action, for example, interferon-alpha has more than 200 biological actions, and there is often no relationship between effects and pharmacokinetic parameters

Conventional drugs and biologics
Europe was the first to introduce a regulatory system for biologics (Human Code, art 10 para 4) and it specifies that for products that do not meet the conditions in the definition of generic medicinal products, a full dossier is required, although clinical data can be extrapolated if the mode of action is similar. So far, three groups of biosimilar have been developed in the EU – somatotropin similars, erythropoiesis stimulating agents (ESAs) and granulocyte colony stimulating factor (GCSF) similars. However, there can be marked differences between similars and the originators such as different levels of impurities or different host cells. Qualitative differences at every level have been accepted. For example, there is aberrant glycosylation in some erythropoietin similars, although the significance of this is not known and Retacrit is 10% less active than the originator product (Eprex). In addition, clinicians assume that international units are all the same but there is no reference standard – it is an in-house measure and therefore not comparable across products. In practice this means that one biosimilar cannot be substituted for another and most EU countries (except Romania) have introduced measures to discourage therapeutic interchange.

The generic and biosimilar paradigms do not address all apects of these complex molecules. For example, copaxone (glatiramer acetate) is a random polymer, defined only by its production method. Copaxone is used in the management of multiple sclerosis but the mode of action is not fully understood. It shifts the population of T cells from pro-inflammatory Th1 cells to regulatory Th2 cells that suppress the inflammatory response. It may also act as a sort of decoy, diverting an autoimmune response against myelin. Copaxone is immunogenic in all patients, inducing antibody formation within the first month of treatment. Copaxone copies – so-called glatiramoids – have been synthesised. One such product – protiramer – was found to be toxic in animal models. This clearly shows that a small difference can cause a large change in biological effect.

Low molecular weight heparins (LMWHs) are complex sulphonated, glycosylated polymers. For example, enoxaparin, which is not yet fully characterised, is described as ‘a complex set of oligosaccharides’ (in the manufacturer’s summary of product characteristics). There appear to be specific saccharide sequences that bind to clotting factors. Generic copies of enoxaparin show clear differences in anti-IIa and anti-Xa activities. However, these products have been approved through the classical generic route so that, at least, the generic product was compliant with the specifications of the reference product.

Substitution of one LMWH for another is fairly routine and as a result some hospitals have saved considerable amounts of money over the past 10 years. This has prompted administrators to ask why other biosimilars and complex molecules should not also be substituted. It is important to explain that heparins are not proteins and so the situation is different.

LMWHs can cause thrombocytopaenia and this is believed to occur as a result of an immunogenic reaction. LMWHs bind to platelet factor four to form an antigenic complex that plays a pivotal role in the development of heparin-induced thrombocytopaenia (HIT). HIT causes bleeding and can have serious consequences.  Different heparins are associated with different rates of HIT but so far no structure-activity relationship has been defined. The formation of HIT antibodies does not automatically lead to clinical HIT. It appears that a reaction between IgG HIT antibodies and polysaccharide-PF4 complexes triggers continual activation of platelets and the clinical appearance of HIT.

In practice, patients receiving heparin are closely monitored and HIT can be managed safely by changing to a different heparin or heparinoid.

One practical response adopted by some pharmacists to the potential production problems of biosimilars has been to keep two products, the originator product and a biosimilar. This enables them to benefit from the lower cost of the biosimilar whilst keeping a back-up supply of the originator product so that patient care is not compromised.

Recent discussions in the Netherlands concluded that regulations including specified biological and clinical data are required for complex molecules. This may need to be done on a case-by-case basis because it might be difficult to design an algorithm to apply to all situations.

During the discussion the following criteria for ‘complexity’ were suggested by members of the panels:

A product that cannot be characterised fully by laboratory testing
A product with a molecular weight of more than 5000 Daltons
A product with a narrow therapeutic index – although this could be a problem with complex molecules because of inherent inter-batch variability
Lack of therapeutic equivalence /interchangeability or the need for rigorous therapeutic monitoring.An example from transplant immunosuppression illustrates this: a bioavailability study of generic tacrolimus showed that the new product delivered 80–120% of the dose compare to the originator product. For patients with impaired renal function this could cause a 30% variation in dose. Experience with this product in the USA had shown that about 30% of the clinic population required major dose adjustments.

Regarding efficacy and safety, members of the panels noted that in the EU, only biosimilar products that conform to the regulation can be used (they must have used a recognised reference products), regardless of where they are made. They also commented that if every new product were to be regarded as a new entity then a complete dossier would be required and there would be a risk of stifling competition and innovation. They recommended that:

  • Any new product should have a clinical assay
Pharmacovigilance studies should be undertaken to compile an accurate toxicity profile

Iron sucrose and iron sucrose similars
The panels then considered non-biological complex drugs including iron sucrose, iron sucrose similars and other injectable iron products. Presentations were given by George Bailie (London) and Vincent Launay-Vacher (Paris).

Iron plays a major role in the treatment of anaemia in chronic kidney disease (CKD). It is estimated that CKD affects at least 20m people in the USA – about one in 10 of the adult population. Anaemia is common in CKD, affecting 76% of those with stage five.  The anaemia of CKD can be severe, intractable and associated with hospitalisation, mortality and high costs. ESAs and iron supplementation have been used extensively to treat anaemia in CKD. Intravenous administration is the recommended route in stage five disease and several products are currently available on the market.

Ferric iron, as such, does not exist in physiological conditions; it exists only as a complex with water. Iron sucrose and iron sucrose similars (ISS) all have the same general structure – a core of ferric oxyhydroxide surrounded by a shell of carbohydrate that allows slow release of iron from the core. Iron sucrose has a long clinical track record and a good safety profile. In recent years the use of iron sucrose has increased considerably whereas usage of iron gluconate and iron dextran has remained more or less static.

CKD is a pro-inflammatory condition in which there is an increased risk of cardiovascular (CV) morbidity and mortality. It is associated with traditional CV risk factors such as hypertension and diabetes along with inflammation, oxidative stress and anaemia. Uraemic patients commonly have high levels of markers of chronic inflammation such as C-reactive protein. Biomarker levels are also high in haemodialysis patients because of increased production and decreased elimination. The inflammatory process also exacerbates clinical conditions such as malnutrition and cardiovascular disease.

Iron is increasingly being recognised as a source of oxidative stress. Both the stability of the iron complex and the reduction potential of the iron influence the degree of oxidative stress. In the ideal situation iron is released slowly from the complex, avoiding the release of labile iron into the circulation. When iron sucrose is administered intravenously, the sucrose shell is released into the circulation, and the remaining complex is phagocytosed by the reticulo-endothelial system (RES). It is plausible that some free iron is released directly from the complex into the circulation. Iron is then slowly released from RES into the circulation where it binds to transferrin. At present this is the limit of knowledge. It is not known what controls the proportion of iron that diffuses out into the circulation versus the proportion that is phagocytosed and the relationship between pharmacokinetics and activity is poor.

Concerning iron sucrose similars (ISS), small differences in the structure of iron-carbohydrate complexes can affect the product stability and the severity of oxidative stress. The physico-chemical properties and pharmacological activity of iron sucrose complexes are highly dependent on the manufacturing process. Although several ISS have now been approved in a number of countries, they are not  identical to the original iron sucrose complex (Venofer®).

A number of manufacturing variables have been demonstrated to influence significantly the end products (see ‘Manufacturing process variables’ panel).

Products may differ in the structure of the iron core, the molecular weight distribution of the iron core or of the iron sucrose complex and different complexes will have different physicochemical properties. Chemical analyses of three different ISS injections showed that each one failed to meet the USP specification on one or more parameters.  When polarograms for the three ISS and Venofer® were compared, the results showed that the peaks – related to the reduction of Fe3+ to Fe2+ – were shifted to the left, that is, they were more likely to participate in oxidative reactions at physiological pH than Venofer®, the originator product. In order to ensure high reproducibility of the product with minimum batch-batch variation, the production process needs to be rigorously controlled.

Animal studies (in rats) comparing Venofer® and ISS1,2 have examined effects including iron deposition, inflammatory markers and oxidative stress parameters. The results showed that with ISS there were increased iron deposits in the liver and kidneys but ferritin stores in the liver were reduced in comparison to Venofer®.  The presence of free iron deposits in the liver and kidneys suggests that iron was released more rapidly from ISS causing over-saturation of the iron transport system and inefficient use of iron. The pro-inflammatory cytokines, interleukin six (IL-6) and tumour necrosis factor alpha (TNF-α) were raised, indicating inflammation and/or oxidative stress. Levels of protective antioxidant enzymes including glutathione peroxidase and copper-zinc-superoxide dismutase were significantly increased with ISS and not with Venofer®. In addition, serum liver enzymes (ALT, ALP and AST) were raised and blood pressure was reduced. There was also a modest reduction in creatinine clearance and a significant increase in proteinuria compared with the originator product. In short, for every parameter there were significant differences between the ISSs and the originator product. These findings suggest that clinicians would need to exercise care with the use of ISSs in patients.

The safety and tolerability of injectable iron products is an important issue in practice. A large epidemiological study recently examined the tolerability of four injectable iron products – high and low molecular weight iron dextran, ferric gluconate complex and iron sucrose.4 The results showed that life-threatening adverse drug events occurred at frequency of 11.3/million dose with iron dextran (high molecular weight) – more than 18-fold higher than with Venofer® (0.6/million dose), which presented a favourable safety profile.

Panel members discussed the findings of the published studies and made a number of comments and suggestions. They noted that the published toxicity studies in animals had identified issues that require further investigation. Although it is not possible to extrapolate these study results to patients with renal failure, they pose interesting questions and studies in humans are warranted.

It is not clear what happens after parenteral administration of iron complexes because the reported studies did not examine free iron or kinetics of iron release. It would be interesting to repeat the studies with radio-labelled iron to see how it is distributed.

The studies used traditional biomarkers. It would be interesting to look at, for example, vascular cell adhesion molecule (VCAM) and inter-cellular adhesion molecule (ICAM), which are more specific for vascular endothelial changes.

Given that parenteral iron preparations can cause transient increases in oxidative stress parameters, an important question is whether these correlate with poor outcomes for patients and this information is currently missing.

The panels were concerned to note that the ISS did not meet the USP standards – so they failed at the first hurdle.

On the basis of the evidence presented the panels recommended that both efficacy and toxicological data should be required for approval of similars and that revalidation should be required if the originator changes the manufacturing process.

Efficacy of iron sucrose similars
The ISS Fer Mylan was introduced into all the hospitals of the Assistance Publique in Paris in June 2009 because it was 21% cheaper than Venofer® and this gave Jacques Rottembourg (Professor of Nephrology, Centre Suzanne Levy, Clinique du Mont Louis, Paris, France) the opportunity to observe the impact of switching from one iron sucrose product to another.

Patients with chronic renal failure, undergoing dialysis had been treated with Venofer® and darbepoetin exclusively from 2003 until 2009. During the three-year period 2006–2008, the rate of use of the two products had remained constant and the mean haemoglobin level had been in the range 11.72–11.88 g/dl. In July 2009, a fall in the mean haemoglobin levels for the clinic population was observed.

A study was undertaken to evaluate this phenomenon more closely. Only patients who had undergone 60 or more haemodialysis sessions and had received at least one dose of intravenous iron were included. All patients received weekly IV iron administration during the mid-week haemodialysis sessions, and darbepoetin (Aranesp®) was given once every two weeks. The target haemoglobin for the clinic is 11.5–12.0 g/dl. Data were gathered for two 27-week periods before (December 2008–June 2009) and after (June 2009–January 2010) the introduction of Fer Mylan. The study group of 75 patients (mean age 63.4 (sd 15.2) years) underwent a total of 5633 dialysis sessions in the first period and 5635 in the second period. The results showed that there was a sharp fall in mean haemoglobin after the introduction of Fer Mylan. (See Figure 1)

The mean values of haemoglobin during the first and second periods were statistically significantly different at 11.78 g/dl and 11.48 g/dl, respectively.  At the beginning of the second period the mean haemoglobin value fell to 11.2 g/dl and the dose of ESA was increased to correct the deficit. It then took three months for the mean haemoglobin level to rise to the target level. The mean serum ferritin value during the first period was 533.8 μg/L (sd 327.5) compared with 457.7 μg/L (sd 290.9) in September 2009. The corresponding mean transferrin saturation values were 49.3% (sd 10.9) and 23.3% (sd 10.2). The consumption of intravenous iron was steady during the first period but increased progressively during the second period. The cumulative iron dose during the second period was 124,250 mg compared with 92,300 mg during the first period, an increase of 34.6%. Doses of Fer Mylan had to be increased in order to raise the haemoglobin. There was also a 12.6% increase in ESA usage during the second period.

The global increase in costs of anaemia medication during the second period was about 12% and the additional costs outweighed the savings to the pharmacy budget made by using the cheaper ISS. The results showed that Venofer® and FerMylan could not be considered to be therapeutically equivalent and that the differences seen in the iron indices and other biological parameters raise questions about the stability of the Fer Mylan complex and its impact on iron distribution and oxidative stress.

Fer Mylan use was discontinued after January 2010 and patients were switched to another IV iron complex. It is of interest that other hospitals in Paris have verbally reported similar experiences, but no data have been provided.

The panels discussed Professor Rottembourg’s findings and made a number of comments and suggestions.

First, aspects of methodology and interpretation were considered. It was noted that there were no other obvious changes such as changes in the quality of water  (chloramines) or new auto-analysers to account for the findings.

The rate of fall in haemoglobin was unexpectedly rapid and the possibility that this was partly due to haemolysis was raised  (as there was a corresponding rise in serum bilirubin).

It is surprising that TSAT dropped significantly even after the dose of iron was increased and explanations included the possibility that the ISS released more free iron that was taken up by the RES and so the TSAT stayed low, and that the time of measurement could also have influenced the results, because TSAT kinetics vary with the time of administration. (In this study TSAT was always measured seven days after iron administration.) The matter could be clarified if free iron were detected within hours of Fer Mylan administration but this is not routinely measured.

One panel concluded that two questions need to be answered. First, ‘Is Fer Mylan a good iron replacement?’ And second, ‘Is it toxic – does it cause oxidative stress?’ The panel recommended that a prospective, randomised study should be undertaken to demonstrate the efficacy and safety of the ISS.

The panels discussed the regulatory status of Fer Mylan and made a number of comments and recommendations concerning the need for a suitable regulatory framework.

The status of Fer Mylan is now in question. Originally it benefited from a temporary approval process for products known as  ‘essentially similars,’ which no longer exists. No clinical data on efficacy or safety were required for this. Although it has been granted a marketing authorisation, it is not a generic because it is not a simple molecule and neither does it meet the requirements to be a biosimilar. However, it is not an original drug (a ‘princeps’) since the file presented for registration did not include the minimum data on efficacy and tolerability/safety. The long-term safety of Fer Mylan is an additional concern. This cannot be extrapolated from the Venofer® data now that experience has shown that higher doses of Fer Mylan are needed. One panel member considered this situation to be unacceptable and recommended that the product’s authorisation should now be reconsidered with additional data, including efficacy and safety in human.

The panel also concluded that until such information is available, given the poorer efficacy and unknown long-term safety of Fer Mylan, only the originator drug (Venofer®) should be recommended for patients who need to receive parenteral iron supplementation on a chronic basis.

There was general agreement that more data are required before ISS is marketed and that chemical ‘similars’ do not conveniently fit either of the existing regulatory pathways for generic medicines or for biosimilars.  Ideally there should be a consensus view of the safety and efficacy data required. The USP standard, which deals with chemical quality and not with biological aspects, could be a starting point for a new regulatory framework. There is no precedent for this type of product and so perhaps Venofer® could be used as a model. (It was emphasised that ISS is very different from insulins because the physiological changes are not as rapid.)

One panel suggested that the following elements should be defined for new chemical ‘similars’:

  • Molecular size
  • Production methods
  • Relative doses in man and animals
  • Formulation issues
  • Safety information
  • Efficacy data
  • Conversion factors between products
  • Best practice with regard to efficacy

In addition, a new product should be required to show similarity with the current gold standard – in this case it should not require a change in ESA dose.

If regulators do admit unfamiliar products then it is essential to keep systematic records in case differences become apparent in use.

All of these considerations could apply to a list of high-risk products, for example, ciclosporin, amphotericin, propofol, botulinum toxin, liposomal products. Moreover, a warning about lack of therapeutic equivalence should appear in the British National Formulary.

Looking at the wider perspective, it was noted that many pharmacists (and other professionals) are unaware of the lack of equivalence data for ISS. This is a matter of concern because in the prevailing financial climate, there will be pressure to use an ISS if it appears to be cheaper and the risks will have to be weighed carefully. It was also considered important to have a clinical pharmacy input at the purchasing/contracting stage otherwise these types of problems only become apparent when the product arrives on wards or 
in clinics.

Participants in the London roundtable, 14 June: Ms Caroline Ashley, Renal Pharmacists Group and Lead Specialist Pharmacist Renal Services, Royal Free Hospital, London; Professor George Bailie, Professor of Pharmacy Practice, Albany College of Pharmacy and Health Sciences, NY; Ms Gillian Cavell, Consultant Pharmacist, Medication Safety, Kings College Hospital, London; Dr Iain MacDougall, Consultant Nephrologist, Kings College Hospital, London; Dr Donal O’Donoghue, Clinical Director of Renal Medicine, Hope Hospital, Salford and National Clinical Director for Kidney Care; Professor Jacques Rottembourg, Professor of Nephrology, Centre Suzanne Levy, Clinique du Mont Louis, Paris; 
Professor Huub Schellekens, Professor of Medical Biotechnology, Utrecht University; Professor Roger Tredree, Independent Pharmaceuticals Professional; Mr Laurence Goldberg, Independent Pharmaceutical Consultant; and Dr Christine Clark, Editor of HPE

Participants in the Paris roundtable, 27 September: Dr Vincent Launay-Vacher, Clinical Pharmacist, Nephrology, Pitié-Salpêtrière Hospital, Paris; Dr Jean-Pierre Reynier, Head of Pharmacy Department, University Hospital of Marseille; Dr Thierry Romanet, Clinical Pharmacist, Nephrology, University Hospital, Grenoble; Dr Alain Astier, Department of Pharmacy and Toxicology, Henri Mondor University Hospital, Paris; Dr Philippe Arnaud, Hospital Pharmacist, Bichat, Paris, and President of the French Syndicate of University Hopsital Pharmacists; Dr Christine Fernandez, 
Pharmacist, Pitié-Salpêtrière Hospital, Paris and Professor in Clinical Pharmacy in South Paris University; Professor Jacques Rottembourg, Professor of Nephrology, Centre Suzanne Levy, Clinique du Mont Louis, Paris, France; Mr Laurence Goldberg and Dr Christine Clark.

1.    Toblli JE, Cao G, Oliveri L, Angerosa M. Differences between the original iron sucrose 
complex Venofer® and the iron sucrose similar Generis®, and potential implications Port J Nephrol Hypert 2009; 23: 53–63
2.    Toblli JE Cao G, Oliveri Langerosa M. Differences between original intravenous iron sucrose and iron sucrose similar preparations. 
Arzneimittelforschung 2009; 59(4): 176–90
3.    Chertow GM et al. Update on adverse drug events associated with parenteral iron. Nephrol Dial 
Transplant 2006

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