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Biological heparin and TSE risk
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Molecular characteristics of heparin
The consequences of BSE crisis
Quality of the raw materials sourcing
Capacity of the manufacturing process to inactivate TSE agents
Analytical control of the end product
The risks associated to the business on raw materials
Introduction
Transmission of Creutzfeldt-Jakob Disease (CJD) to children by injection of human hypophysis-extracted growth hormone has raised the non-answered question of the safety of biological tissues-extracted medicines which can transmit Transmissible Spongiform Encephalopathies (TSEs), the most notorious of them being Bovine Spongiform Encephalopathy (BSE, “mad cow” disease). The causative (non-conventional) infectious agents of these emerging diseases are highly resistant to treatments that usually inactivate bacteria and viruses. Moreover, whatever the animal species (including man), TSEs induce important disorders and symptoms which invariably lead to a fatal issue.
Among pharmaceuticals suspected to transmit such lethal diseases, heparin is particularly pinpointed. Indeed, this active ingredient was - until recently - exclusively extracted from livestock intestines (pigs, cattle or sheep). As far as ruminant intestines are concerned, scientists and Authorities have classified such materials as “tissues at risk”, i.e.: tissues with a potential infectivity in case they derive from TSE-contaminated animals. Back to the summary
Molecular characteristics of heparin
In 1916, Jay Mac Lean discovered a molecule endowed with strong anti-coagulating activities in bovine liver. In relation to its tissue origin, he named it: “heparin”. The anticoagulating and antithrombotic properties of this molecule greatly improved public health during the last sixty years. Now, it has become the active component of medicines widely used for the prevention and the treatment of venous thrombosis and cardio-vascular disorders. The worldwide consumption of heparin is higher than 500 million doses/year.
Heparin exerts its anticoagulant effect by binding to serine protease inhibitors of the blood coagulation cascade such as the antithrombin III (AT III). Moreover, heparin interacts with a wide range of plasma proteins and membrane receptors which regulate several important biological functions such as fat metabolism (Merchant et al, 1986) or smooth muscles and blood capillaries proliferation (Jackson et al, 1991). Furthermore, anti-inflammatory (Ekre et al, 1992), anticancer (Jackson et al, 1991) and antiviral properties (Spear et al, 1992; Holodniy et al, 1991) have been evidenced as well as potential activity in Alzheimer disease (Cornelli, 1996; McLaurin et al, 1999).
Heparin is produced by mast cells of most mammalian connective tissues ; it is a serglycin proteoglycan with a protein core to which about 10 polysaccharide chains (molecular weight of 100 000 Dalton) are attached. As the biosynthesis continues, heparins chains are cleaved by endo-ß-D-glucuronidase and stored in the secretory granules of mast cells under the form of glycosaminoglycan-heparin which binds to basic proteases (Matsumoto et al., 1995; Humphries et al., 1999). This stored heparin belongs to a family of heterogeneous linear polysaccharide chains made of repeating units of highly sulphated disaccharides containing an uronic acid residue (either D-glucuronic acid or L-iduronic acid) and D-glucosamine. The high content in trisulphated disaccharides makes heparin the most acidic polyanion of all the biological molecules ever known.
The identification of the minimal sequence in heparin required for binding to AT III was carried out concurrently in several laboratories (reviewed by Conrad, 1998). This work requested the isolation of short fragments formed by the partial cleavage of heparin as well as chemical synthesis of oligosaccharides. The results evidenced that the smallest oligosaccharide that bound with high affinity to antithrombin was a pentasaccharide. The sequence of this pentasaccharide represents only one-third of the chains of pig mucosa heparin. It contains a unique 3-O-sulfated GlcNSO3 at unit 4, that is a minor component of heparin, and one rigid GlcA residue at unit 3. The chemical synthesis of this pentasaccharide has been successfully completed (Sinäy et al., 1984; van Boeckel et al., 1985; Petitou et al., 1991), leading to an entirely synthetic heparin which is now developed as a medicinal anticoagulating drug. Back to the summary
The purification of heparin
Heparin can be extracted from various highly vascularized mammalian tissues (Nader et al, 1980). Its pharmaceutical form has been purified for decades from gut or lung tissues obtained from pigs, cattle and, to a lesser extent, from sheep (Coyne, 1981 ; Watt et al, 1997). The purification process generally includes the destruction of proteins by heat coagulation or enzymatic proteolysis (Scott 1960 ; Duclos, 1984). Then, heparin is extracted by ion-exchange chromatography (Volpi, 1993 ; Griffin et al, 1995), or complexation with quaternary ammonium salt (Scott, 1960; Lindahl, 1969). After decomplexation or elution from the resin by concentrated sodium chloride, heparin is selectively precipitated by ethanol or methanol, concentrated and heat dried. This "crude" heparin is then cleared of its coloured pigments and depyrogenated (elimination of endotoxins) by potassium permanganate or hydrogen peroxide (Griffin et al, 1995). These reagents are eliminated by methanol-, ethanol- or acetone-precipitation. The resulting “pure” natriated-heparin can be modified by ion-exchange of its natrium ions for potassium, lithium, calcium or magnesium ions (Duclos, 1984). At this stage, heparin is a mixture of glycosaminoglycan chains of varying lengths, with an average molecular weight (MW) 900-15000, making it polydisperse and heterogeneous (Edens et al., 1995).
Low molecular weight heparins (LMWH, mean MW 5 kDA) derive from purified heparins, either by chemical depolymerization (nitrous acid or periodic acid followed by an acidic or alkaline hydrolysis), or by enzymatic depolymerisation using Flavobacterium heparinum–derived heparinases (Kessler, 1997 ; Linhardt et al., 1992). These heparins interact in a less extent with plasmatic proteins, endothelium cells and macrophages (Kessler, 1997), leading to improved bio-availability and minor side effects (bleeding and thrombocytopenia). Back to the summary
The consequences of BSE crisis
In 1985, further to the description of the first bovine BSE case in the U.K., attention was immediately focused on the risk for man to be infected by ruminant-derived pharmaceuticals.
In 1988, the European Authorities acknowledged the transmission of BSE through meat and bone meal (MBM). Representing the first-line tissues in contact with the pathogen prion protein after an oral infection, bovine intestines were rapidly classified as Specified Risk Materials by international Health Authorities (W.H.O. 1991). This measure was all the most relevant that bovine intestines are infectious several months before the first symptoms of the disease appear and before any biological testing gives positive results.
In 1991, the European Agency for the Evaluation of Medicinal Products (EMEA) published its first “Note for guidance on minimising the risk of TSE agent via medicinal product”. This “Note for guidance” is regularly updated ; its last version is due before end 03.
In 1992, the French regulation anticipated the European decisions with a new concept, the “precautionary principle”, to offer the highest protection to patients. As a consequence, medications prepared from bovine tissues were forbidden in France in July 1992 (J.O. R.F., 11/07/92).
On March 20th 1996, the U.K. Health minister, Stephen Dorell, announced 10 human cases contaminated by BSE agent, the variant of the CJD (v-CJD).
In 1997, the EMEA ordered the total withdrawal from the European market of all medicines containing any active ingredients derived from specified risk materials, to take effect on January 1st, 1999.
During the following years, the accumulation of scientific knowledge led Health Authorities to strengthen the regulation. The importation and intra-E.U. exchange of bovine small intestine and of bovine small intestine-derived products were also forbidden (O.J.E.C. 30/06/2000 ; J.O.R.F. 11/07/2000). Currently, the European regulation on animal-derived medicinal products stipulates that ruminant tissues have to be systematically avoided and replaced by tissues from TSE-free species (EMEA, 2000).
Now, in the USA, Canada and the E.U. Members States, for each animal-derived drug product a detailed BSE risk-assessment dossier is requested to apply for a Marketing Authorisation or for clinical trials.
This set of stringent regulations regarding TSEs concerns all ruminant species, i.e.: not only cattle but also sheep and goats, all species which can be experimentally (and - potentially –naturally) easily infected by the oral route (Foster et al.,1993). As a consequence, bovine, ovine and caprine intestines have been classified by the European Union Authorities as tissues with a potential infectious risk (Scientific Steering Committe, S.S.C., 2002).
Moreover, in 2001, the European Union Council has stressed "the importance for the public to be aware of the gravity of the different sanitary risks in order to choose with full knowledge of the facts.” It encouraged State Members “to promote appropriate information methods towards the public”. It also invited the European Commission to “re-examine permanently, in the light of the available knowledge, the measures of protection against possible iatrogenic contamination risks, and particularly those related to pharmaceutical products, cosmetic products and medical devices, using the precautionary principle should the occasion arise”.
Recently, a detailed directive of the European Parliament (CE n° 1774/2002) and of the European Council (03/10/2002) settled stringent sanitary rules applicable to animal by-products not intended for human consumption. Back to the summary
The TSE risk for human health
At a meeting (june 14, 2001) organised by the World Health Organisation (WHO), the UN Food and Agriculture Organisation (FAO) and the World Animal Health Organisation (OIE), scientists warned that BSE and variant Creutzfeldt-Jakob Disease (vCJD) "has joined AIDS as a major health challenge facing the world". These diseases should be considered as an international issue as potentially infected BSE materials have been distributed throughout the world through trade of live cattle, certain cattle products and by-products.
In 1988, some time after MBM was suspected of spreading BSE, the British government banned its use in feed for ruminants such as cattle, sheep and goats in Britain. However, the government allowed the export of MBM until 1996 when it finally admitted a link between BSE and vCJD. Records show that over one million tonnes of MBM were exported from Britain to Asia between 1988 and 1996. For 11 years Britain exported the remains of BSE-infected cows to more than 80 countries, including Canada and U.S.A., where, in some countries, it was often repackaged and re-exported.
In the E.U., the risks of transmitting animal spongiform encephalopathies to humans has been reduced significantly by the ban of MBM in livestock feeding, the systematic elimination at slaughter of tissues at risk (such as brain, spinal cord and intestine) and by BSE-testing on carcasses from cattle aged over 24 months. Yet, these measures are not fully implemented all over the E.U. Moreover, the situation is still less clear in the other countries which imported British BSE-contaminated MBM. Moreover, as for any commercial activity, possibilities of fraud on animals or feedstuff cannot be excluded, as mentioned by newspapers and professional literature.
Since 1992, a sharp decrease has been recorded in the number of BSE-affected cattle in the U.K. Accordingly, from an optimistic point-of-view, the average incubation period being estimated to 13 years in humans (Knight, 2002), v-CJD cases should disappear within 10 years in the U.K. From another point-of-view, ignorance about different aspects of the disease prompts scientists to more cautiousness regarding predictions. These uncertainties explain the epidemiologists‘ previsions in human cases which range between 3 digit- and 5 digit-figures.
As an example of our ignorance, the hundred deaths recorded in the U.K. are all individuals genetically BSE-susceptible, all of them being homozygotes Met-Met on the codon 129 of the gene controlling the synthesis of the prion. Thus, it is still impossible to estimate the incubation period for the heterozygotes Met-Val, and for the most "resistant" homozygotes Val-Val as well, which represent the majority of the population. For Kuru, which is the first TSE-related disease demonstrated in human beings, the incubation period is longer in individuals who are not "susceptible" homozygotes. According to Professor John Collinge (Imperial College School of Medicine, London), the incubation period for the human form of mad cow disease could average 30 years, with some patients not showing symptoms for as long as 50 years, as observed in kuru. Therefore what we see could be only the first wave of the most susceptible people.
As further example, the quantity of infectious agents necessary to induce the infection in humans is totally unknown. For the common mouse model, results depend on the strain used. Professor Dormont’s team (CEA, Saclay, France) demonstrated that an histologically-normal brain extract, sampled from clinically healthy BSE-infected mice, remains pathogen when injected to fresh animals. In 2002, Stanley Prusiner’s team (University of California-San Francisco, U.S.A.) demonstrated that some transgenic murine strains are 10,000 times more sensitive to prion than classical strains. Thus, after the same infectious challenge, a transgenic mouse brain will contain 10,000,000 IU/g (“Infectious Units per gram”) while a classical mouse brain contains only 1,000 IU/g. These results call into question the definition of “non-infectivity level” for tissues (especially milk and muscle). Indeed, though no prions have been detected in these tissues by means of classical mouse models, one must expect that more sensitive models are able to evidence significant residual infectivity levels. Moreover, extrapolation from murine models to human pathology represents another challenge for scientists.
The incubation period of TSE is considered proportional to the infectious dose; assuming that BSE-infected bovine meat has never been demonstrated to be infectious in experimental animal models, should we conclude that the hundredth of British consumers who died from v-CJD have ingested high-risk tissues and that the other thousands - genetically more resistant or having ingested smaller quantities of infectious agent - are now in a phase of non-symptomatic (silent) incubation ?
Recently, Professor Adriano Aguzzi (Institute of Neuropathology, University Hospital, Zurich, Switzerland), made the assumption that, currently in the E.U. or elsewhere, healthy BSE-contaminated people might be responsible for a man-to-man transmission that would be more rapid and more effective in the absence of species barrier (Aguzzi, 2002).
Concerning biologically-derived heparin, its intestinal origin (potentially infective tissue) and the quantity of raw material used for the production of one daily dose (about an entire small intestine) represent the 2 major risks factors. Yet, the parenteral administration of the drug and the several-day treatment schedules associated must be considered as additional aggravating factors. Consequently, Regulation Authorities have banned the use of ruminant (bovine, ovine, caprine) tissues and have restricted the raw material sourcing to porcine intestinal mucosa, pigs being considered as TSE-free in natural conditions. Indeed, due to the implementation of stringent regulations, pigs bred in the E.U. and in the U.S.A. do not receive ruminant-derived feedstuffs any longer. Though passive contamination of pig intestines by feedstuff of ruminant origin cannot be avoided in emerging countries such as Asia or Eastern Europe, the major risk is represented by the accidental or fraudulent use of biological heparin extracted from intestine of BSE-infected ruminants. The possibilities to secure biologically-derived heparin
In order to minimize the risk of transmitting TSEs by pharmaceutical products, regulations have been regularly revised. The focus has been put on 3 pivotal points: (1) sourcing of the raw materials; (2) capability of the manufacturing process to inactivate pathogens, (3) controls on the final product.
In the U.S.A., the geographical origin of raw materials is the major criteria to warrant quality and safety of porcine heparins:
- for porcine heparins derived from U.S. sources: (1) animal husbandry (including Animal Health status) and slaughterhouse operations are regulated by the USDA; (2) pesticides and herbicides used by the farmers are regulated by the EPA; (3) antibiotics used in animals and antibiotics residues in pig carcasses are regulated by the USDA-CVM; (4) the resulting quality of the porcine heparin is regulated by the FDA-CDER;
- for foreign sources, additional information is requested by the CDER on three aspects: (1) quality of the starting material or animal organs; (2) capability of the process to inactivate pathogens; (3) testing on raw materials for antibiotics, herbicids, pesticids and pathogens.
Controlled sourcing is now generally considered as the most important criterion to achieve acceptable safety of the product because of the documented resistance of TSE agents to most inactivating procedures. Back to the summary
Quality of the raw materials sourcing
The manufacturer is legally responsible for the quality (an safety) of the raw materials he processes. For heparin, the starting raw material is intestinal mucosa; in practice, crude heparins represent the intermediate “raw materials” which are supplied worldwide by the heparin manufacturers (Europe, North and South America, Asia,…).
Currently, the strictly porcine origin of the intestinal mucosa processed by the suppliers of crude heparin relies on traceability documents. This safety criterion is acceptable only if raw materials are exclusively supplied by mono-species (pig)-processing slaughterhouses. In “polyvalent” slaughterhouses (which process porcine and ruminant carcasses as well), the risks of contamination by ruminant tissues cannot be excluded.
In many countries, the (species and geographical) origin of animal-derived raw materials cannot be properly ensured. For example, China - which provides over 50 % of the worldwide heparins - has been the object of a ban to the importation of animal products intended for human or animal consumption in the E.U.(30/01/02 E.U. Decision of 30/01/02) for unreliable sanitary system. Consequently, Chinese porcine heparin is not a sufficiently securized raw material. Indeed, while Chinese producers claim to provide both porcine and bovine heparins, batches of heparin may be made of hundreds of sub-batches obtained without any sanitary controls or traceability procedures. Moreover, China has imported tonnes of contaminated MBM from Britain and is currently the first world importer of intestines, especially ovine guts imported from Spain (a BSE-contaminated country). At last, the real status of China regarding BSE is totally unknown (as a comparison with the status regarding AIDS, experts suspect the existence of more than 1 million cases in China).
Though the raw materials produced in the USA and in Canada (officially BSE-free countries) must be regarded as quite safer, it is necessary to consider the emergence of “Chronic Wasting Disease” which is a TSE that affects ruminants such as wild and domestic elk and deer (Spraker, 2002) and which looks like Scrapie in small ruminants, though more contagious. Besides, the consumption of bovine meat from "downer cows" (syndrome with decubitus linked generally with metabolic diseases or traumatisms during calving in cows) would have induced TSE in reared minks. Curiously, the USA import large quantities of Chinese crude heparins intended to be transformed into pure heparins, while the importation of pork tissues from China is theoretically prohibited by the US government (lack of information on farm practices including feed quality, husbandry and slaughterhouse conditions).
Thus, as mere statements or audits cannot ensure the strictly porcine origin of heparins, it is necessary to control their species origin by means of analytical methods.
Only one method has been described for the control of the intestinal mucosa used for the production of crude heparin (Levieux and Levieux, 2001). This method is based on a radial immuno-diffusion technique. In industrial conditions, it detects 3 parts of ruminant (bovine, ovine or caprine) mucosa in 1,000 parts of porcine mucosa. It is also used to prove the porcine origin of the mucosa during the manufacturing process. The method is commercially available from IDBiotech (IDBiotech.com) and routinely used by some european and US heparin manufacturers. In contrast, the method is not yet applied in China.
As regard to the crude heparins imported by manufacturers, the only studies published concern the detection of residual contaminants. Due to the ion-exchange or complexation steps of the process, these contaminants are mostly acidic substances such as anionic proteins and nucleic acids.
The analysis of residual anionic proteins by immunochemical techniques has revealed the presence of a bovine-specific antigen, named “Ag 1” (Rivera et al., 2002a). This antigen, which has been fully characterized (Rivera et al., 2002b), has prompted the development of an ELISA technique able to detect traces (5 ppm) of crude bovine heparin in porcine heparin (Levieux et al., 2001). A similar approach led to the development of ELISA tests able to detect ovine or caprine contaminants and to prove the porcine origin of crude heparin batches as well (Levieux et al., 2001). All these easy-to-use techniques allow the systematic control of all crude heparin batches intended for pure heparin production. They have been validated and are commercially available from IDBiotech (IDBiotech.com).
Detection of DNA traces by PCR techniques, although technically possible, could not be easily used in industrial conditions for the following reasons: (1) heparin being a very strong PCR inhibitor, its careful clearance from samples would be problematic, costly and time consuming; (2) PCR analysis of crude heparins would require very high technicity and sophisticated equipment, which would impairs its implementation in the manufacturer’s plants; (3) the very high sensitivity of the PCR could be a major drawback in giving industrially inacceptable false positive results due to the amplification technique itself, or to the amplification of environmental contaminants such as ruminant hairs or skin scales. Such contaminants cannot be totally avoided throughout the mucosa harvest, the first steps of the process or in not-specially decontaminated and dust protected laboratories. Back to the summary
Capacity of the manufacturing process to inactivate TSE agents
Some heparin manufacturers, such as Opocrin (Italy) or Celsus Laboratories (USA), referred on their website to the capability of their heparin purification processes to reduce prion infectivity levels. In experimental conditions, the reduction rates range from 5 to 6 logarithms. These statements bring the evidence that - if fully applied - the purification processes decrease significantly prion infectivity levels. Yet, the quantification and the relevance for patients’ health of these residual infectivity levels cannot be accurately established. Back to the summary
Analytical control of the end product
Heparin molecules display only few inter-species structural differences which essentially concern sulphatation and acetylation rates of the polysaccharidic chain. H3 NMR analysis has revealed that iduronic acid residues of bovine heparins are sulphated on carbon 2 at about 97% compared to only 88 % for porcine heparins (Casu et al., 1996). This analysis can differentiate a 100 % bovine heparin from a 100 % porcine heparin, but it cannot detect species blends.
Analyses by capillary electrophoresis or by ion exchange HPLC of the disaccharides obtained by enzymatic depolymerization have also evidenced interspecies differences in sulphatation and acetylation (Dabat et al., 1993; Dabat, 1994; Mascellani et al., 1996; Bianchini et al., 1997; Toida et al., 1997; Watt et al., 1997). The sensitivity of these techniques is quite better than that obtained by NMR, although still limited to about 5-10 % of bovine heparin in porcine heparin. However, it has been demonstrated that the purification process modifies the structure of heparin and that the resulting variability is more important than the interspecies variability. Consequently, the discriminating power of analysis is significantly reduced (Bianchini et al., 1997). Besides, mere chemical treatments can modify the sulphatation rate of the molecule and, thus, transform a bovine heparin into a “porcine” heparin. The last, but not the least drawback: these physico-chemical techniques cannot detect the ovine and caprine heparins.
The direct detection of the prion has not been accepted as a safety measure. Indeed, the current sensitivity threshold of the techniques, either Western-Blot or ELISA, are still too high to provide a satisfactory method applied to heparins (though ruminant intestines are potentially highly contaminated, the quantities of the pathogen prion protein is too diluted). For bovine brains, these techniques are convenient insofar as they are applied on samples obtained from of a very precise brain area (obex) in which the prion concentrates. In the case of intestine, it is necessary to remember that this tissue is infective long before any positive testing on the brain. Back to the summary
The risks associated to the business on raw materials
The importance for human health of the anticoagulating and antithrombotic properties of the heparin explains the rapid and steady increase in the sales of this pharmaceutical ingredient. At the rate of one pig intestine for one heparin dose and nearly 500 million doses a year, the offer of raw material could not, sooner or later, answer the demand. Thus, the ever-lasting shortage in supplies of raw materials and the rough competition between operators lead to a lower quality of raw materials due to frauds and regulatory infringements.
Currently, pending law cases in France evidence that these kinds of infringements have so far been responsible for the deaths of already one hundredth of young adults who received injections of CJD-contaminated pituitary-derived growth hormone in the 1985s (“Growth Hormone” scandal). With respect to v-CJD, the New-York Times (Petersen and Winter, 2001) reported that, since 1993, the Food and drug Administration has repeatedly requested pharmaceutical companies not to use materials from cattle bred in countries where there is a risk of BSE. Yet, in 2000, regulatory authorities discovered that five pharmaceutical companies - including some of the world’s largest ones - were still using ingredients from those countries to prepare nine of the widely used vaccines. One of these companies used a blood derivative from cattle bred in the Netherlands, in spite of the banning of ruminant-derived materials originating from this country since 1997. Back to the summary
Conclusion
In spite of the recent launching of a fully synthetic heparin (pentasaccharide) devoid of any TSE-associated risks, pig-derived heparin is still extensively administered and represents an useful tool for the treatment and the prevention of cardio-vascular disorders. Nevertheless, taking into account – (1) that the manufacturing processes of biologically-derived heparins can only reduce the quantity of infectious agent without ensuring that they eliminate it, and – (2) that the species origin of these heparins cannot be mastered by means of analytical control on the (pure) final products, the control of exclusive porcine origin of the raw materials is of the utmost importance. The rigorous mastering of the supply chain implies the implementation of easy-to-use and validated analytical methods able to detect the presence of ruminant tissues in the starting raw materials (intestinal mucosa) and in the in-process raw materials (crude heparins) as well. It must be reminded that – at least in the US - manufacturers are legally responsible for the quality of the pharmaceutical materials they process and that they have to implement any measures providing safer and higher quality standards of the drug products they market. Consequently, they have to justify any corporate decision or delay in the implementation of analytical methods made available to secure medicated products.
By Didier LEVIEUX
Doctor in Veterinary Medicine, Director of Research Emeritus, National Institute for Agronomical Research (INRA), France.
Send an email to Didier Levieux
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