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Snipping away at the causes of disease
Manufacturing Chemist, May 2003

Biotechnology will generate at least half of all new medicines by the end of the decade. So said Dr Kalevi Kurkijärvi of BioFund Management at the recent Biotech 03 event held in Helsinki, Finland, where there is a burgeoning biotech industry. ‘There are close to 1,900 biotech companies in Europe, compared to 1,500 in the US,’ he said. ‘But maybe 60% of these have business issues and could do with being consolidated.’

And indeed, there is no large biotech company in Europe along the lines of Amgen in the US. ‘There are too many companies in Europe, and not enough world-class managers for them,’ he said. ‘Governments continue to be catalysts to the emerging markets with funding, and substantial support from capital markets is still needed for success. Darwinism is currently rationalising the market, and consolidation should be a strategy, not a destiny.’

Despite the current difficulties facing the biotech sector business-wise, the rapid strides made recently in the science of biotechnology are having a dramatic effect on drug discovery. The unravelling of the human genome, in particular, is providing a massive amount of information about the causes of diseases that just a few years ago could only have been dreamed of.

Perhaps the human genome project’s biggest surprise so far is that the number of genes is much lower than had been predicted, as Professor Leena Peltonen-Palotie of the department of medical genetics and molecular medicine at the University of Helsinki explained at the conference. ‘Even one year before the announcement, the human genome was expected to contain about 100,000 genes. The actual number is only about a third of that. And the rule “one gene, one protein” is wrong – some sequenced regions could give thousands of proteins! There is also a remarkable number of large duplications.’

Until now, only single or small groups of genes or proteins have been analysed for their involvement in the disease process. It is now becoming possible, thanks to high throughput methods such as gene chips, for information on all genes to be found using one microscale test. Bioinformatics is how science is trying to elicit useful knowledge out of the mass of information provided by genome mapping. It is used to predict transcript and protein structure, and also protein function. And it is being used to identify novel metabolic pathways and protein interactions.

Several of the speakers addressed the issue of genealogical gene databases. Perhaps the most famous – and most extensive – of these is that being created by deCODE in Iceland. As the company’s president and ceo Dr Kari Stefansson explained, in Iceland there is a whole population database stretching back over 1,100 years, which has provided a powerful basis for the genetic investigation of disease.

‘Mendelian diseases are simple,’ he said. ‘They are accidents of evolution. However, common diseases are much more complex, often involving several genes. Another problem is that many common diseases occur at the interface between genetics and the environment. When we’re looking for the genetic causes of lung disease or COPD, for example, we must make sure we are not studying the genetics of nicotine addiction!’ Isolated populations have big advantages, as they have a higher degree of homogeneity, with fewer mutations in the disease genes, hence making them easier to identify.

deCODE ran incidences of cancer within Iceland through the genealogical database, and found that some did indeed run in the family. It has now created a number of disease collections, including Alzheimer’s, stroke, rheumatoid arthritis, osteoarthritis, non-insulin dependent diabetes, obesity and osteoporosis, and some more surprising ones such as anxiety. And some responsible genes have also been pinpointed, such as one implicated in schizophrenia, where 31% of patients compared to just 14% of the control group carried the at-risk halotype. Similarly, a gene implicated in stroke has also been found. Another interesting finding was that a gene involved in myocardial infarction coded for a protein that is targeted by an existing, marketed drug for a totally different indication, and this is now being evaluated as a potential treatment for myocardial infarction.

Iceland is not the only country building up a genome database. Estonia has recently begun a similar project, with the goal of collecting data on at least 1 million of the country’s 1.4 million population and creating a health and genetic database. ‘The aims are to apply DNA based diagnosis and personalised treatment methods to achieve better healthcare service at lower cost,’ explained Professor Andres Metspalu from Estonia’s University of Tartu. ‘Estonia is ideal for such a project. The population is large enough to provide sampling for common diseases. There is a developed infrastructure and nationwide primary healthcare system, which will be the main data collector, as well as an efficient IT and data communications infrastructure, a transparent legal and ethical environment, quality staff and a nascent biotechnology industry. And it’s financially possible!’

Participation in the gene research studies is voluntary, with complete confidentiality for gene donors, who also have the right to know or not know their genetic data, as well as to apply for their data to be destroyed at any time. And non-discrimination by employers and insurers is guaranteed. Estonian academic institutions will have free access to the database, and commercial companies will have to pay a fee for access. ‘43% of informed people are willing to be gene donors already,’ Metspalu said. ‘36% would like to know more before they decide. Just 6% do not support it. By 2007, we hope that three quarters of the country’s population will be included.’

Finland, too, is an excellent candidate for a genetic library. ‘Finland is a good place to do genetics research,’ said Kari Paukkeri, ceo of Jurilab, which is located in Kuopio and uses its DNA database for drug target discovery and medical DNA microchips. ‘It is 200 times cheaper to carry out genetic research in eastern Finland than in the general population.’

‘Finland has good population records that have been kept by the church, with information on families dating back to about 1600,’ said Peltonen-Palotie. ‘Many studies have been carried out in Finland, and most loci are replicated in other populations.’ One example is a study on familial combined hyperlipidaemia, which is the most common form of genetic hyperlipidaemia, estimated to cause 10–20% of all premature coronary heart disease. It has a prevalence of 1–2%, and both genetic and environmental effects are implicated. A linkage to a gene has been found in Finland, with the gene believed to be associated also with non-insulin dependent diabetes and plasma free fatty acids. By looking for genes on the web, said Peltonen-Palotie, it is possible to identify the effect SNPs have. ‘It’s a slow and expensive process,’ she said.

Another genetic condition that has been studied in Finland is lactose intolerance. It is common in Finns, and two SNPs have been found, which perhaps surprisingly are a long way from the lactase gene. ‘This means they must be very old variants,’ she said. Based on genetic data, it appears that the lactose intolerance allele represents the ancient, original, form of the human allele – those who are lactose tolerant are actually the mutants. ‘Hundreds of different allele types have been found globally, so common disease mutations have been beneficial during evolution, and it is our current lifestyle that has made them disease forming.’

John Blangero from the department of genetics at the South West Foundation for biomedical research in San Antonio, TX, US, has been studying population isolates to investigate the genetic epidemiology of infectious diseases. ‘The big advantage is that you can obtain very large pedigrees, and thus have more powerful genetic studies,’ he said. ‘Large pedigrees provide the power for genetic analysis and multiple households per pedigree, so there is potentially much less “noise”. A sibling pair study is only 5% as powerful – big families are much better for disease mapping.’

Blangero has been working with the Jirel population in Nepal since 1985. He is looking at roundworm infections, where there is no evidence for acquired immunity. Roundworm is the most common helminthic infection. The Jiri helminth project is a long term study of the genetics of susceptibility to helminthic infections, and has been looking at over 2,000 people, all of whom are connected in a single pedigree. The group are studied every year, and the prevalence of the worms is 20.3%. Subjects are treated with albendazole to clear the infection, and the exercise repeated every year. Traditional epidemiology assumes that differential exposure is responsible for the fact that only some people are infected. But the team found that the same people are reinfected with the worm, implying a genetic susceptibility to roundworm infection. As a result of genetic analysis, Blangero has pinpointed a pair of single nucleotide polymorphisms that is highly associated with worm burden levels.

Pharmacogenetics, Single Nucleotide Polymorphism, Adverse Drug Reactions, and the Cytochrome P450 Family
Genetics also has a bearing on how our bodies respond to drugs. As Prof Magnus Ingelman-Sundberg of the Institute of Environmental Medicine at the Karolinska Institutet in Sweden explained, drug absorption, excretion, metabolism and receptor interactions all have a genetic basis. ‘Only 30–60% of patients respond to the common drugs,’ he said. ‘And serious adverse drug reactions (ADRs) are responsible for 5–7% of all hospital admissions. This costs US$100bn a year in the US alone.’ This is where pharmacogenetics can help – it can take a patient’s genetic constitution into account when a drug is developed or prescribed to increase the number of responders and decrease the incidence of ADRs.

Note:  This has engendered the subdiscipline "Pharmacogenetics", the study of variability in drug action due to genetic polymorphism.  A general discussion of the implications of this may be found at   See the section Pharmacogenetics and Developments in SNP and Halotype Mapping.

Then at we find:  "6.3 Dr Sulston observed that it was the cost per test that would determine the degree of penetration of the tests at various levels, and that as well as single nucleotide polymorphism (SNP) testing, haplotyping (identifying particular patterns of SNPs on a single chromosome) would become more common, both in clinical practice and in the market place. It would become increasingly cheaper to screen the whole genome than carry out a single targeted test, and this would have repercussions for privacy and other issues."  and "6.9 Action: Members agreed that background briefing which included a description of the reality, implications and future possibilities of SNP and haplotyping testing should be prepared. "

The genotyping issue is also discussed at "DNA as Data"  where it is suggested that the universal availability of personal genetic information would reduce public sensitivity about privacy of the information.

Ingelman-Sundberg explained that 59% of products cited in ADR studies are metabolised by polymorphic Phase I enzymes, the vast majority of these being from the cytochrome P450 family. ‘Only 20% of drugs which are substrates for non-polymorphic enzymes give ADRs,’ he says. He cited several examples, such as a polymorphism that developed within the Ethiopian population which can affect the metabolism of omeprazole. Similar populations have been established for adverse reactions to warfarin and anticancer agents. ‘The lesson for drug design is to avoid a design which makes the candidate a high affinity substrate,’ he said.

Note:  For CP450 information (a fascinating study in itself) see the following links:

Note that the socalled "grapefruit" interaction with drugs is mediated by CYP450, and that it also mediates the production of DHEA and androstenedione in the body, as well as being involved in sexual development during fetal life and at puberty.  It is an exceedingly ancient gene superfamily, involved in the protection of cellular life (maybe precellular) from hostile chemicals in the environment for as many as 3.5 billion years.  Yet it evolves rapidly in response to new environmental challenges, and is a major player in plant-animal warfare, where plants develop ways to discourage animal predators with chemical defenses, and the CYP450 system responds by developing new ways to protect the organism from the defense.  Pretty cool.

Although some big strides have been made in understanding how our genes cause illness and cause interactions with medicines, we are still a very long way from a comprehensive understanding of what each gene does. As Leena Peltonen-Palotie said, ‘We have finished the genome map. We just don’t know how to fold it.’

Karyon – targeting the cure

Cell membranes in cancer cells are different from those in normal cells, so it should be possible to target them specifically. This is the principle behind the work being carried out at Karyon, which is investigating the possibility of using targeting units to direct drugs directly at tumours, and also provide a way for detecting tumours at and early stage. Its products are intended to target tumour cell surfaces, activated endothelial cells and metastases.

Karyon is working in collaboration with clinical groups at Helsinki University Central Hospital to develop and modify peptides that have been found using targeted proteomics. The peptides are carried to the tumour through the bloodstream, and can be attached to a known cytotoxic or an investigational cancer drug. Similarly, a diagnostic moiety could be linked to it, facilitating early diagnosis.

‘We have patents pending on four peptide targeting units,’ said ceo Rabbe Slätis. ‘We believe the concept is now well established, and we hope our first product will enter Phase I/IIa trials in 2005,’ he said. He expects the technology will be attractive to big pharma and generics companies, who will be able to use it to give a new lease of life to existing products, and make them more effective in targeting cancer.


BioTie Therapies – tackling addiction

BioTie Therapies, a recent merger between BioTie, Control Pharma and Carbion, is focussing its drug discovery activities on dependence disorders, inflammatory diseases and glycobiology. The product that is furthest down the development chain is nalmefene, an opioid receptor antagonist designed to treat dependence disorders, which is in Phase III for alcoholism with the trade-name Soberal, and in Phase II as Cessal for impulse control disorders such as pathological gambling.

Nalmefene targets the mesocorticolimbic reward pathway in the brain. This is one of the most important mediators of alcohol and drug reinforcements, and disturbances in its function are believed to play a central role in substance dependence. The pathway’s activity has several modulators, of which opioidergic mechanisms are one of the most important. Nalmefene prevents [beta]-endorphin, which is responsible for pleasurable sensations, from activating opioid receptors. Excessive drinkers with alcoholic heredity show a more pronounced release of [beta]-endorphin after consuming alcohol.

‘We are expecting the results of the Phase III trial in the second quarter of this year,’ says the company’s Juhani Saarinen. ‘Unlike current treatments for alcoholism, it does not involve abstinence.’ Results from the Phase II trials indicate that the number of heavy drinking days was reduced by 60% in patients with a family history of alcoholism.


Galilaeus – microbial synthesis of chemicals

Founded in 1994, Galilaeus is developing microbial production methods for microbial derived APIs. Furthest up its product pipeline are anthracyclines, particularly the daunomycin group of antibiotics that are potent anticancer drugs. They are produced by actenomycetes, mainly Streptomyces bacteria. Since its inception, Galilaeus has been working on strains that can make antibiotics like daunomycin and doxorubicin. First, the existing strain was chemically mutated to alter its production profile. High producing mutants were found, and also used to make new compounds that could have cytotoxic potential.

Galilaeus has now developed a process for pilot and industrial scale production, and a cGMP pilot plant was set up last year. Processes for making other daunomycins, notably epirubicin and idarubicin, is being developed, with the ultimate aim of offering the ingredients as generic drug substances to the pharma industry.

‘We have a full range of established, reliable producer strains, and a proven record of the development of strains.’ said ceo Dr Kristiina Ylihonko. ‘We can develop processes for both pharma companies and fine chemicals companies, making both APIs and intermediates.’ Doxorubicin and daunomycin are already available, and many other products are in development.