Lectures

'Chasing Ehrlich's dream: the quest for magic bullets'

by Sandip K Basu,

National Institute of Immunology

In the value system of human society, the priorities are health followed by wealth followed by wisdom. This quest for good health and the urge to avoid or get over illness has driven mankind over millennia to flock towards medicine men. Modern medicine based on systematic scientific studies about the causalities of various disease processes is, however, only a little over 100 years old. Modern medical practices combining improved sanitation, immunization and the use of medicinal substances of natural or synthetic origins has drastically reduced morbidity and mortality from infectious agents. However, emergence of drug resistant pathogens and new diseases such as AIDS in recent years rudely reminds us that the war against microbes is hardly over.

Healthcare in the 21st Century crucially depends on our proficiency in the sunrise technology of molecular medicine. The edifice of molecular medicine can only be built on a firm bedrock of competent and innovative new biology research. I would like to pay homage to Dr SubbaRow by citing some examples of our humble efforts in this direction to break new grounds.

Ours has been the best of times globally because this is the first century in which mankind had some respite from the constant fear of premature death from diseases. Life expectancy has risen from about 30 years at the end of the 19th century to about 80 years in most developed countries. Even an Indian can expect to live to be about 64 today whereas it was about 33 only fifty years ago.

Ours could also turn out to be the worst of times. The deluge of new and re-emerging diseases, drug-resistant microbes and the epidemic surge of AIDS pose hazards of calamitous dimension to public health. At development costs of over $500 million and 10­-15 gestation years per new drug, even chemotherapy is fast becoming unaffordable. Public trust in modern medicine is fast eroding with the spiralling rise in healthcare costs and the resultant inequity in healthcare delivery is aggravating social discord. We are living in dubious and dangerous times indeed.

Shifting Paradigms in Health Care in Century 21. As the lifestyles in the poor nations improve, a demographic transition in the disease pattern is sure to happen in our parts of the world as well. With the increased life span and an ageing population, the major goals of medicine in the 21st century are control of cancer, cardiovascular ailments, autoimmune diseases, and behavioural disorders. As the molecular defects underlying pathological conditions are elucidated, medicine in the new century will shift to an informational paradigm that will emphasise diagnosis and prevention rather than expensive therapy. An era of "predictive medicine" will emerge that will permit assessment of the risk of an individual to contract a specific disease so that many of such risks would be countered by preventive measures, counselling, lifestyle changes, and so on.

There is robust optimism that new technologies for equitable healthcare will emerge in Century 21 from the profound molecular insights into life processes that new biology provides.

New biology is the outcome of the convergence since the 1940's of the three major streams of biology: biochernistry concerned with the isolation and chemical characterization of cell substances, cell biology exploring the subcellular components of the cell, how they relate to each other and to the intact cell, and genetics dealing with the inheritance of characters by whole animals or plants. New biology seeks molecular explanations for the many ­splendoured beauty and aberrations of life, how it perpetuates and evolves, and how it originated.

The powerful molecular approaches of new biology for studying disease processes has spawned a new biopharmaceutical industry that specializes in studying mechanisms of diseases and applies that knowledge to their diagnosis, prevention and treatment. The triumphant march of the biopharmaceutical industry is reflected in the large number of new products that are approved or are in trial.

Chimeric toxin. Some of the proteins under development have no precedent in nature: they are engineered as combinations of certain domains from several proteins. For instance, Dr J.K Batra and his colleagues at NII are using gene fusion techniques to develop a molecule in which the EGF receptor-recognizing domain of the TGF molecule is fused to a fungal toxin called restrictocin that inhibits protein synthesis in animal cells. This hybrid protein selectively destroys breast and lung cancer cells that overexpress EGF receptors. For killing other types of tumour cells, which overexpress transferrin receptors, they have engineered a molecule containing the transferrin-receptor-binding domain of an antibody and a potent fungal toxin.

CDC-NII Malaria Vaccine. Tools of cell biology and genetic engineering are being used for replacing existing vaccines, with safer and more effective versions and are improving the prospects of new vaccines against AIDS, cancer, malaria and other parasitic infections. For instance, Dr Hasnain at NII has made a composite gene by stitching together several gene fragments for proteins exprissed in various stages of malaria parasite's development, and expressed this engineered gene in baculoviruses. Studies at CDC with this artificial protein look highly promising as a new lead towards a candidate vaccine against malaria.

Towards the medicines of tomorrow: targeted drugs. Despite such welcome developments, the fact is that effective treatments against new and re-emerging diseases are proving ever harder to invent. New drugs becoming increasingly difficult to come by, we Sought alternative means so that molecules with desired curative potential that are currently unusable due to toxic side effects can be made therapeutically useful. Let me illustrate our approach towards such medicines of tomorrow.

Advantages of Site specific drug delivery. Why drugs often produce toxic reactions? Current pharmacological practice is based on the central dogma that the .effect of a drug depends on its concentration in the blood or other body fluids. Of about 1013 cells comprising the human body, only a small fraction needs intervention with drugs to cure or curb a specific disease, Having no special affinity for the diseased target cells, most drugs in current use can access normal cells as well. Since most drugs enter cells by passive diffusion, a relativelyhigh dose of the drug needs to be administered to attain therapeutically effective drug concentration inside. the cells, which aggravates the problem of toxicity. Ever since Paul Ehrlich introduced the concept of magic bullets in 1906, a common priority goal of therapeutics has been the designing of vehicles containing exclusive signals for recognizing the target cells, and delivering drugs selectively to these cells.

Targets on cell surface. Two general targets on the surface of mammalian cells are exploitable for such site-specific drug delivery: 1) antigens against which specific, non-cross-reactive antibodies can be developed, and 2)receptor molecules capable of efficient transport of macromolecular ligands. The carrier for targeting a drug to specific cells thus can either be an antibody specific for the target cell, or a ligand for the receptor molecules present only on the target cells (Basu 1990). Ehrlich himself proposed the use of a specific antibody as the carrier of the chemotherapeutic agent.

Disadvantages of Anti body-med iated targeting: The antibody-mediated targeting approachr raised great hopes after fine discovery of hybridorna technology for production of highly specific monoclonal antibodies. However, it has still not been possible to solve many of the problems of this approach.

Receptor-tirtediated transport of LDL. The receptor-mediated approach is of more recent vintage and derives from our work on the role of low-density lipoprotein in atherosclerosis. This work delineated how specific receptor molecules on the surface of rnammalian cells bind macromolecules, leading to their internalisation and processing in specific intracellular compartments. This process is now called receptor-mediated endocytosis and is universally recognised as a major transport mechanism utilised by mammalian cells for a variety of purposes.

LDL receptor work established. We had great fun working out the mysteries of this process during 1975-83 but the story is textbook matter now.

Characteristics of Receptor-mediated endocytosis important for drug delivery. Twenty years ago, thanks to late Professor B.K Bachhawat, I got a job in India. Not having much of a lab to begin with, I had all the time to ponder over how to build a scientific career in India. There was no point working on LDL as my seniors in the LDL receptor adventure, Goldstein and Brown, were already on the verge of a Nobel Prize which came in 1985. Eventually I realized the potential of the process of receptor-mediated endocytosis for the purposes of drug delivery.

Macrophages pivotal cells. I also understood that macrophages in animals mount multipronged defensive responses that protect them against a variety of invading microorganisms and developing cancers. However, in many instances these defensive responses are overwhelmed, circumvented or even misdirected so that these protective cells become the focal points in a large number of diseases - infectious, metabolic or neoplastic, which affect millions of people world wide. Therefore, a generalised targeting regimen specific for macrophages would be extremely useful.

Characteristics of Scavenger receptor system: To target macrophages we needed a receptor restricted to these cells. In the course of my earlier work on lipoprotein metabolism we discovered a receptor system present primarily on cells of marophage lineage. It appears that God created scavenger receptors for my career development in India.

Pathway of receptor-mediated drug delivery. We went about exploiting the principles of receptor-mediated endocytosis for selective drug delivery. We chemically attached the desired molecule to maleylated albumin or polyguanylic acid so that the conjugate is specifically recognized by the scavenger receptors present primarily on macrophages.

Leishmania mexicana-infected hamster footpad. The power of our approach was first demonstrated in an experiment in which L. mexicana were injected into hamster footpads which swelled to about 10 times the normal size due to multiplication of the protozoa inside the macrophages. When free methotrexate (MTX) was given no substantial cure was achieved. Administering the drug in a form targeted to macrophages as MBSA-MTX conjugate brought the footpad size back to normal. All the animals remained healthy and no antibodies against the drug conjugate could be detected in these animals (Mukhopadhyay et al. 1989; Basu et al. 1994). The fact that the first paper in this series of my first PhD student in India, Amitabha Mukhopadhyay, was published in the journal Science reflects the novelty and importance of these findings.

Improved survival of MTb infected guinea pigs with PAS-MBSA. The conjugated drug improved the survival of guinea pigs infected with Mycobacterium tuberculosis to 87% compared to only 13% with free PAS.

Effect of MBSA-DOX on C11F9 multidrug resistant tumours. The conjugated doxorubicin had an antitumour effect.

Antisense strategy. Binding of oligonucleotides complementary to a critical sequence in an mRNA can reduce or prevent production of the target protein. Several such antisense molecules are about to enter clinical trials as antiviral agents. Antisense therapy is likely to be effective against viral infections, inflammatory conditions and cancer. Selective and efficient intracellular delivery of antisense molecules to the target cells is essential for success of this approach and better methods are needed.

Antisense inhibition of VSV replication. We demonstrated that scavenger receptor-mediated intracellular delivery of antisense molecules to macrophages inhibits the replication of vesicular stomatitis virus. We are now using this principle to design new antiviral agents against macrophage-trophic viruses such as dengue, Japanese encephalitis and HIV.

Scavenger receptor-mediated manipulations of macrophage metabolism. Over the last 15 years or so, we have used scavenger receptor-mediated endocytic process for manipulating macrophage metabolism for three major purposes: combating intracellular infections such as leishmaniasis, tuberculosis and vesicular stomatitis virus, controlling macrophage cancer, and modulating immune responses.

Drs Rath/Bal and their colleagues at NII added an entire new dimension to scavenger receptor-mediated modulations of macrophage metabolism. They showed that targeting of antigens to scavenger receptors led to enhanced immunogenicity, providing a novel lead for new generation adjuvant less vaccines, generation of the Th1 type of immune response, opening a new approach for immunoprophylaxis especially against intracellular pathogens; diversion of an ongoing allergic immune response to a non­allergic route, perhaps brightening the prospects of mitigating the misery of millions; abrogation of T cell tolerance to self antigens, providing a new, tool to dissect mechanisms of immune tolerance and etiopathogenesis of autoimmunity (Abraham, et al., 1995, 1997; Singh et al., 1998)

I hope I have presented some evidence to convince you that (a) newer tools of cell biology such as monoclonal antibodies and/or receptor-mediated endocytosis appears to be a rational approach for site specific drug delivery; (b) this approach merits serious consideration in designing new chemotherapeutic agents as well as resurrecting otherwise effective but highly toxic molecules for site-specific chemotherapy; and (c) the actual availability of drugs based on the targeting principles mentioned above, however, have many hurdles to cross before drugs based on these elegant principles would find their way to the market place.

Thus, Paul Ehrlich's quest is still on, magic bullets still elude us.

The worrisome schism

Let me now draw your attention to a widely held misconception about scientific progress and societal well being that threatens the entire framework of science.

Harnessing new biology for healthcare has been the preserve so far of the developed countries. Of late, these nations invoke stringent property rights regimes with proprietary controls on knowledge bases these efforts generate. Accordingly, the commercial interests of both the developed countries and of the elite of the Third World determine the priorities of biomedical research the world over, rather than the urgency of the unmet needs of poor Third World citizens. This has created a worrisome schism between expectations and realities in the Third World.

Two factors aggravate this schism: 1) The lacunae in healthcare delivery to the poor are due far more to resource constraints and implementation failures than the lack of technologies. 2) Much of new biomedical research agenda is based on hype as seen in the recent fuss with genomics, which is unlikely to translate into real life utility (other than boosting share prices) at least for the poor any time soon.

The reality is that improvements in healthcare over the short. term do not need biomedical research as critically as they need political will and administrative skill. But restraining biomedical research with near-term expectations stands guaranteed to lose us the real and enormous long-term benefits by way of unpredictable futuristic technologies that rigorous, competent research has been historically shown to bring. Conceptual insights for breaking new grounds strike competent minds unfettered by short-term utilitarian goals.

Antiscience in public policy. You might have noted how anti-science and irrational viewpoints have exerted increasing influence on public health policy matters of late. There are instances galore, both in the First World and in the Third: The irrational edge that the heated debate on genetically modified foodstuffs takes on, even in otherwise technology-savvy societies such as Germany. Closer to home is the fanatic zeal of an erstwhile influential Union Minister in India for regulating animal experimentation with ill-informed rules and their motivated implementation, which made serious real-life biomedical research nearly impossible to pursue in India and drove our fledgling drug discovery industry to Western countries for crucial animal testing with obvious increase in costs.

No sober public debate on this critical issue has so far been possible in India, given the tendency of the media to favour emotive or celebrity reportage. The controversy thus simmers without being understood.

Why use animals for experimentation? Biomedical research aims to work out how a human or animal body functions and looks for clues for interventions to correct dysfunction for ensuring better health. However in the 20th century, society at largeset the ethical norm to treat human life with utmost dignity. Therefore no human application is permitted until an intervention is proven to be safe as extrapolated from experimental studies often involving animals. Animal experimentation thus becomes necessary out of respect for human life.

Why use animals for experimentation? Biomedical research aims to work out how a human or animal body functions and looks for clues for interventions to correct dysfunction for ensuring better health. However in the 20th century, society at largeset the ethical norm to treat human life with utmost dignity. Therefore no human application is permitted until an intervention is proven to be safe as extrapolated from experimental studies often involving animals. Animal experimentation thus becomes necessary out of respect for human life.

Is such an anthropomorphic view of the animal world justified? Is there ethical coherence to it? If their strident demands result in a virtual ban on using animals for any kind of research, what do we lose?

We do lose a lot. Most wonders of modern medicine that remarkably improved human and animal health would not have been possible without the use of animals for experimentation. There are examples galore: surgical procedures for heart and kidney transplant, vaccines against polio and whooping cough, miracle medicines from Aureomycin to Zantac. The list goes on.

Infections pose a constant and evolving threat to human life. Since 1973 about 20 brand new disease entities have been identified against which we have no effective vaccine or medicine.

Effective new ways are needed to cope with these and other diseases ranging from cancers to cardiac disorders.. For such attempts to be meaningful, the pathological processes involved need to be understood.

If animals cannot be used for mimicking such human conditions in the laboratory and finding answers to problems, human beings will have to be used for extensive experimentation with potential danger to life and health in the process. Society has already rejected this as unethical.

If neither animals nor human beings can be used, all future research towards understanding the functioning of the human body and attempts to keep it healthy would have to be stopped. Since this is hardly agreeable, we must accept that there is nothing inherently 'morally evil' about experimenting on animals, and the notion of 'animal welfare' is far more tenable than any concept of 'animal rights'.

Since animal experimentation is indispensable, is regulation of animal experimentation necessary? The answer is a definite 'yes’. All social human activities must be regulated with pragmatic rules set after democratic debate.

How do we then reconcile the conflict between the ethical perceptions of a vocal fringe with an essential requirement of biomedical research?

What society must vehemently resist however is the subversion of 'human rights' by the so-called 'animal rights' activists who try to introduce unworkable rules for animal experimentation in pursuit of a covert, non-democratic 'anti-vivisectionist' agenda.

This one of the lesser-appreciated but critical examples of the labyrinths of ethical considerations in science and technology.

Dawn or dusk? What is the image of a future for humanity that new biology heralds for us? It could be the dawn of opportunity resplendent in societal wisdom - our hopes for our children. It could also portend the dusk of the long dark night -- a tired, spent-out generation mired in indecision or foolish bequeaths - a generation that will be cursed by our children. The decision is ours.

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(Adapted from slide show notes)