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Research Strategy

Fostering Venture Research: A Case Study of the DiscoveryThat Ascorbate Enhances Adrenergic Drug Activity

Robert Root-Bernsteinn and Patrick F. DillonDepartment of Physiology, Michigan State University, East Lansing, Michigan

Strategy, Management and Health Policy

Venture Capital

Enabling

Technology

Preclinical

Research

Preclinical Development

Toxicology, Formulation

Drug Delivery,

Pharmacokinetics

Clinical Development

Phases I-III

Regulatory, Quality,

Manufacturing

Postmarketing

Phase IV

ABSTRACT Ascorbic acid (vitamin C) enhances the smooth muscle and cardiac activity ofnorepinephrine, epinephrine, and other adrenergic drugs, increasing the dose response up to threefoldand the duration of activity up to fivefold. The effect is not due to the antioxidant effects of ascorbate. Themechanism by which this enhancement occurs is as yet unknown. This discovery has broad implicationsfor utilizing all existing adrenergic drugs as well as for formulating new mixtures for the delivery of suchdrugs. Despite the clear-cut pharmaceutical potential of this discovery, it was not begun as a drugdevelopment project, and at no stage prior to its patenting was the research funded. As such, the discoveryraises some basic questions about how research projects are evaluated; why this discovery was not madebefore; whether it could have been made by means of a mechanism-driven research program; and howunexpected discoveries are to be fostered within the broad arena of biotech and pharmaceutical research.Does the current system of concensus- and market-driven research actually yield the kinds of resultsneeded to drive industry innovation and keep the drug pipeline full? Drug Dev. Res. 56:xxx–xxx, 2002.�c 2002 Wiley-Liss, Inc.

Key words: epinephrine; norepinephrine; vitamin c; serendipity; risk-taking

INTRODUCTION

Is One Method of Drug Discovery Better ThanAnother?

What is the ‘‘right’’ way to go about drugdiscovery? Does one start with rational drug design(i.e., theory or computation based on known ligands forwell-characterized receptors); phenomenology (an ob-servation that some natural product or folk remedy hasan interesting or potentially important effect); or a newmechanism (discovery of a novel gene target orreceptor-second messenger system that regulates afunction essential to a disease state)? Anyone who hasworked in the biotechnology or pharmaceutical in-dustry knows that there are those who favor each to theexclusion of the others. Few organizations do all threelet alone attempt to integrate them. What is overlookedby almost every industrial research organization is a

fourth and equally important strategy: fostering seren-dipity. Scientists know that the most importantdiscoveries occur when ‘‘chance favors the preparedmind,’’ to quote Louis Pasteur’s famous phrase. Yet fewcompanies create conditions in which serendipitousfindings can be turned into valuable discoveries. Webelieve that this oversight is a major mistake.

The issue of how best to organize research inorder to obtain the most important and valuable resultsis one that should be of high priority for every

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Journal: DDR H Disk used Pgn: Prabha.GArticle : 02-082 Pages: 17 Despatch Date: 3/9/2002

DDR

nCorrespondence to: Robert Root-Bernstein, Department ofPhysiology, Biophysical Sciences Building, Michigan State Uni-versity, East Lansing, MI 48824.E-mail: rootbern@msu.eduPublished online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ddr.10110

DRUG DEVELOPMENT RESEARCH 56:1–17 (2002)

�c 2002 Wiley-Liss, Inc.

investigator and manager of research. Even as researchand development budgets expended by pharmaceuticaland biotechnology firms have grown exponentially fromless than $4 billion in 1980 to over $30 billion last year,the number of investigational new drug applicationsand drugs approved by the Food and Drug Adminis-tration has remained constant. Some observers see thetrend toward corporate mergers actually stiflinginnovation, and some studies suggest that the largestcorporations are relying more and more heavily onstart-ups for novel drug leads as their own hugeoperations more and more frequently fail to produceviable possibilities [Weisbach and Moos, 1995]. Whileit is possible to argue that all of the major discoverieshave already been made, such an argument seemsunconvincing in light of the major breakthroughs thathave occurred over the past decade in combinatorialchemistry, genomics, proteonomics, and bioinfor-matics. New data are flowing into and from biotech-nology and pharmaceutical companies at unpreced-ented rates, but for some reason without stimulatingvery many innovations.

The fact is that data and techniques are not, inand of themselves, sufficient for making discoveries.Discoveries are, by definition, unexpected surprisesthat involve rethinking the meanings and applicationsof both existing and new data. [Root-Bernstein, 1989]Therefore, important results do not come only fromdoing experiments. Deciding what experiments to do isas important as doing the experiments themselves, andanyone who works in a laboratory knows the wide rangeof political, social, economic, business, intellectualproperty, personal, technical, and scientific issues thatcan be involved in the choice and design of experi-ments. To make these choices without studying theprocess of research and its determinants seems to us tobe a mistake. Thus, one of us (R.R.-B.) has formallystudied the discovery process in detail [Root-Bernstein,1988, 1989, 1990a,b, 1994a,b, 1995] and applied hisinsights as a consultant for major pharmaceutical(Parke-Davis) and biotech (Chiron and Mitokor)companies since 1990, helping to foster breakthroughideas and technologies such as combinatorial chemistry[DeWitt et al., 1993, acknowledgments; Root-Bern-stein, 2002a]. Unfortunately, much of that workremains confidential and cannot be described publicly,so we have resorted to addressing the issues offostering breakthrough, or what Donald Braben hascalled venture research [Braben, 1994], with regard toour own research.

While we make no claims to having made anearth-shaking discovery, we do practice what wepreach. Therefore, recapitulating the road we havetaken to the discovery that ascorbic acid (vitamin C)

enhances adrenergic drug activities provides a real-world framework for illustrating some of the strategiesand tactics that can lead to discoveries. Our basicdiscovery is that ascorbic acid (vitamin C) enhances thesmooth muscle and cardiac activity of sub-maximaldoses of norepinephrine, epinephrine, and otheradrenergic drugs, increasing the dose response up tothreefold and the duration of activity up to sevenfold.The effect is not due to the antioxidant effects ofascorbate, which can be supplied to the tissuesseparately from the adrenergic compounds. We believethat this discovery has broad implications for utilizingmany, if not all, existing adrenergic drugs; forformulating new mixtures for the delivery of suchdrugs; and for designing new and more effectiveadrenergic and ascorbyl drugs in the future. None ofthe basic research that yielded this discovery wasfunded. On the contrary, attempts to obtain fundingwere repeatedly turned down, so that we had to bootlegour research off of funds for other projects, includingthose of some of our more cooperative colleagues.

Despite support problems, the path to thediscovery of ascorbate-adrenergic drug interactionshas yielded two patents, several papers, and a varietyof unexpected insights of both theoretical and appliedinterest. Thus, we can describe at first hand thediscovery of novel problems; unusual applications ofnew technologies; modeling and hypothesizing; re-peated errors; serendipity; issues of peer review andfunding, and most importantly, how to bootleg researchwhen no funding is available. Our purpose is to suggestthat there exist extremely inexpensive, time-savingstrategies of research that can, with persistence,provide large payoffs for those who are willing togamble on them. As with any form of gambling,however, one needs to be able to calculate the odds ofsuccess and bet appropriately. Our story is, therefore,presented in order to make readers consider whether adiscovery such as ours, that is to say, one that was notpredicted and could not pass peer review during itsearly stages, could have been made at their institution.If not, then readers are asked to consider what kinds ofdiscoveries the current system of research at theirorganization limits them to making, and what theconsequences of these limitations may be in terms ofthe broadest aspects of practical drug and assaydiscovery and development.

FINDING THE RIGHT PROBLEM

All research begins with the identification ordiscovery of a problem. Good problems are general,amenable to research, and likely to yield broadlyapplicable answers. What often gets overlooked is thatfinding a problem of this sort is just as much a process

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2 R. ROOT-BERNSTEIN AND P.F. DILLION

DDR : 02� 082

of research and thinking as is the attempt to findsolutions to problems. [Root-Bernstein, 1982, 2002b].Too many scientists get trapped into ‘‘me too’’ researchnot because they are inherently uncreative, butbecause they accept at the outset other people’sconstructions of their problem. Thus, successfulscientists have suggested a variety of ways to stimulateidiosyncratic forms of problem discovery.

One is Benjamin Disraeli’s principle: behaveidiosyncratically in order to discover the unexpected.Disraeli did not mean, of course, that one should wearone’s hair in an odd way, or dress strangely, but ratherdevelop a unique style of working. Perhaps the mostgraphic example of this was Alexander Fleming, whodiscovered lysozyme and then penicillin not by‘‘accident,’’ as so many books maintain, but bydeveloping a very odd hobby. Fleming ‘‘painted’’ withcolored microbes on petri dishes and, therefore, left hiscultures uncovered on his lab table when he was donewith them just to see what would show up. This habit,of course, broke all the rules of bacteriology, but asFleming himself said, ‘‘Sometimes it is pleasant tobreak the rules just to see what will happen.’’ In hiscase, his odd ‘‘hobby’’ yielded first the bright yellowbacterium Lysodiektikus, which became the standardorganism for assaying for the presence of lysozyme, andthen, a few years later, it yielded the lovely blue-greenPenicillium mold from which penicillin would even-tually be extracted. The vast experience Fleming haddeveloped from his ‘‘hobby’’ also revealed to him theextraordinary behaviors of these microorganisms sothat he paid attention to more than just their color[Root-Bernstein, 1988, 1989]. Why did one dissolve inthe presence of mucus or tears? Why did the other killoff hardy strains of pathogenic bacteria? Theseanomalous results raised important questions.

Anomalies are a general source of valuablebiomedical problems. Thomas Kuhn, the historian ofscience best known for his coining of the term‘‘paradigm,’’ argued in his influential book TheStructure of Scientific Revolution [1959] that revolu-tionary scientists are invariably those who first payattention to, and recognize the importance of, anom-alous data. Max Delbruck, one of the founders ofmolecular biology suggested yet another fecundstrategy for discovering unexpected phenomena, whichhe called ‘‘the principle of limited sloppiness.’’ We areall trained to control as many variables in everyexperiment as possible. Delbruck suggested that oneshould be sloppy enough so that something unexpectedhappens, but not so sloppy that one cannot tell whathappened. Although this strategy played no role in ourascorbate-adrenergic experiments, it was the origin ofthe discovery of norepinephrine, one of the key

adrenergic compounds we studied. Julius Axelrod,who received the Nobel Prize for its discovery, wassaid to have a particular gift for doing ‘‘quick and dirtyexperiments’’ that paid off with unexpected results[Kanigel, 1986, p 124]. The same strategy also paid offhandsomely in another project carried out by RRB. Hediscovered that both measles vaccine and myelin basicprotein could prevent the onset of adjuvant arthritis (arat model for rheumatoid arthritis). Sequence search-ing on the SwissProt database revealed a number ofregions of homology shared by the measles and myelinproteins that he had synthesized. None of thesepeptides prevented the arthritis, however, so drawingon his experience with combinatorial chemistry, hetried inoculating some rats with mixtures of thepeptides, a sloppy experiment by anyone’s standards.Surprisingly, several of the mixtures had very highlevels of anti-arthritis activity. He was then able tosynthesize single linear polypeptides incorporatingsome of the peptides in the mixtures and these alsocontained all of the anti-arthritis activity inherent in themixtures [Root-Bernstein, 1999]. Thus, a ‘‘sloppy’’experiment yielded a breakthrough when all the‘‘clean’’ ones had failed.

Albert Szent-Gyorgyi, the discoverer of vitamin Cand of the role of myosin in muscle contractions,provided yet another problem-raising strategy that wasuseful to us. He suggested that a rich source of ideaswas to renew old knowledge [Szent-Gyorgi, 1963]. Goback and look at the oldest and most basic discoveriesin your field using the latest techniques and knowledge,he advised. Since our knowledge, techniques, andtheories have evolved since the original discovery, it isvery likely that new and important facets of thefundamental work will emerge. As we will see below,that is certainly true for us.

Albert Szent-Gyorgyi also enunciated the strategythat became the basis for the observation that raisedthe original question that eventually led to ourascorbate-adernergic work: Cultivate a sense of the‘‘sublimity of the mundane.’’ That is to say, payattention to everyday processes and observations thateveryone else ignores. In Szent-Gyorgyi’s case, hediscovered vitamin C by asking why bananas turnbrown but oranges do not [Szent-Gyorgi, 1966, pp 116–117]. In practice, this strategy often comes down toasking, ‘‘why do we do it this way and not some other?’’A perfect example, which also combines Delbruck’sprinciple of limited sloppiness, is the discovery ofRinger’s solution, which is the first of many physiolo-gical solutions used to keep tissues alive and function-ing during extended experiments. Ringer studied heartfunction. Like all of his colleagues at the time, he madeup potassium solutions using the purest potassium salt

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Q1

FOSTERING VENTURE RESEARCH: A CASE STUDY 3

DDR : 02� 082

he could find dissolved in triply-distilled water. His froghearts would beat for about 5 minutes after beingexcised from the animal. One day he went on vacationand his laboratory technician, tired of triply distillingthe water, used tap water, assuming no one would everknow the difference. He was wrong. The hearts bathedin the tap-water solution beat for hours. Ringer wasecstatic, until the solution ran out. The technician, nowthat Ringer was back in the lab, returned to using thetriply-distilled water and the hearts went back tobeating for a mere 5 minutes. It was months before thetechnician’s sloppiness eventually surfaced and Ringerrealized that there must be ions in the tap water(calcium, sodium, etc.) that were also essential to heartfunction [Beveridge, 1950, p 40]. One has to wonderwhy everyone, thoughtlessly, employed pure potassiumsalts in distilled water without every asking whyy.Why did no one explore what would happen if thestandard protocol was changed? Oddly, it is exactlythese sorts of questions that initiated our discoveries.

HOW DOES ASCORBATE PROTECTNOREPINEPHRINE IN SOLUTION?

The problem that led to the adrenergic-ascorbatestory first surfaced in 1977 as a result of the innocentquestioning of accepted, daily practice. One of us(R.R.-B.) was an undergraduate volunteer working inArthur Yuwiler’s Neurobiochemistry Laboratory at theWadsworth Veterans Administration Hospital in Brent-wood, California. Yuwiler’s lab experimented withnorepinephrine. Like many other neurobiology labs,they added vitamin C (ascorbic acid) to the solutions tokeep them fresh. When lab personnel were naivelyasked how the ascorbate protected the norepinephrinefrom oxidation, the response was that the ascorbate wasan antioxidant. Other information provided at the sametime, however, seemed inconsistent with this obvious,but overly simplistic explanation. For one thing, theascorbate solutions had to be made up from scratchevery time they were needed because they only lasted aday. Norepinephrine solutions went ‘‘bad’ within hourswithout ascorbate present. Yet the combined norepi-nephrine-ascorbate solutions could be kept for weeks.It seemed illogical that two compounds, both of whichoxidize within days, should be stable together forweeks. Moreover, reference to the Merck Indexconfirmed that norepinephrine oxidizes so much fasterthan ascorbate that the concentration of ascorbaterequired to protect norepinephrine from oxidationwould have to be hundreds of times greater. The labwas using about equimolar combinations. Since thenorepinephrine oxidized faster than the ascorbate,simple kinetic reasoning suggested that the norepi-nephrine should have been protecting the ascorbate

rather than vice versa. R.R.-B., therefore, suggestedthat perhaps some other mechanism was at work thatprotected both molecules from oxidation. As is typicalin most laboratories, the people in Yuwiler’s wereuninterested in investigating something that worked.R.R.-B., however, was left with a nagging feeling thatsomething fundamental was being overlooked. Thegerm of a problem had been planted by questioningwhat seemed obvious to everyone else.

Ten years passed during which R.R.-B. learnedtwo interesting things that broadened his interest in theascorbate-adrenergic problem. One was that the use ofascorbate to protect catecholamines and adrenergicsolutions was quite general. Therefore, the problem ofhow ascorbate protected norepinephrine might bequite general and thus of more than trivial interest.The second was that many small molecules bind to oneanother with significant affinity and selectivity [Root-Bernstein and Westall, 1984a,b, 1986; Root-Bernstein,1987]. Such small molecule interactions became hismajor area of research, and around 1983, it occurred toR.R.-B. that a possible explanation of the ascorbate-catecholamine protection conundrum might be that thetwo molecules bound to one another. CPK modelsconfirmed that such a mechanism was possible (Fig.1A,B). A complex of ascorbate with norepinephrineresults in significant pi-pi overlap bonding accompa-nied by multiple hydrogen bonds that involve everyresidue of both molecules. In short, the modelpredicted that because every site that could undergoan oxidation reaction is involved in some form ofbonding, both molecules would be protected againstoxidation by their interaction. Their long-term stabilitywas a result of their interaction preventing both fromoxidizing, not due to one oxidizing more quickly thanthe other.

At the time the first ascorbate-norepinephrinemodel was built, R.R.-B. was a post-doctoral fellow atthe Salk Institute, but was unable, once again, tointerest anyone in the problem. He ended up publish-ing the model as a theoretical prediction in 1989 simplyto see if anyone would pay attention [Root-Bernstein,1989, p 263]. No one did. The research managementlesson here seems to be that people tend to ignore whatthey consider to be too mundane or too obvious. Theycare less about how their protocols work than that theywork. Our view is rather different. Those things thatare so simple and obvious are most often the bestexamples of the most fundamental processes. Findingout how things work often gives an investigator thepower to make them work better or to apply them to anew arena where nothing yet works. The ‘‘sublimity ofthe mundane’’ is that everyday things are often themost important ones.

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4 R. ROOT-BERNSTEIN AND P.F. DILLION

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TURN IT ON ITS HEADWe fast-forward 10 years to when a technical

innovation moved the adrenergic-ascorbate project toits next stage. R.R.-B. has joined the faculty of thePhysiology Department at Michigan State University.He has performed some simple experiments showingthat norepinephrine and ascorbate do bind to oneanother. pH titration studies show that the combinationacts a buffer, as can only occur if there is bindingbetween the molecules [Root-Bernstein and Dillon,1997]. And ultraviolet spectroscopy shows that there isa concentration-dependent shift in the spectrum of thecombination as compared with the individual mole-cules [Root-Bernstein and Dillon, 1997] (Figs. 2, 3).

By this time, R.R.-B. has been collaborating forover a decade with P.F.D., a smooth muscle physiol-ogist, on various aspects of molecular complementarityand the consequences of peptide-antisense peptidebinding. [Dillon et al., 1995, 1998]. P.F.D. has alsobeen working on protein-protein binding, whichunexpectedly provides the opportunity to push theadrenergic-ascorbate problem forward. Once again,there is a management lesson here. Most scientistsreport that they solve their outstanding problems andmake most of their breakthroughs while working onother related, or even unrelated, projects, not by directattack. That was certainly the case for both of us in thisinstance.

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Fig. 1. A: CPK model of norepinephrine binding to vitamin C(ascorbate). Significant pi-pi overlap bonding occurs between the ringstructures and multiple hydrogen bonds are formed, indicated byarrows. B: CPK model of norepinephrine binding to vitamin C

(ascorbate) showing hydrogen bonds not visible in A. Note that allreactive groups are bonded making the complex very stable againstoxidation.

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P.F.D. had attended a conference on muscleenergetics in 1988. One of the speakers, DavidMaughan, gave a talk on glycolytic clusters. Theseclusters consisted of different glycolytic enzymescoupled to one another. When the product of the firsttwo sequential enzymes was produced, the amount of

free energy needed to reach the second enzyme ismuch lower if that enzyme is coupled to the firstenzyme, rather than having the product released intothe surrounding solution and finding the secondenzyme by random diffusion. P.F.D. commented onhis return that this was the most interesting talk at the

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Fig. 2. UV spectra of norepinephrine and ascorbate. The values ofthe individual spectra were added to predict the expected values of the

mixture. A significant concentration-dependent change in the spec-trum was observed in the region around 280 nm.

Fig. 3. UV spectroscopic study of the concentration-dependentchange in the spectrum of norepinephrine combinations with

ascorbate. The classic sigmoidal curve indicates that binding isprobably one-to-one and the binding constant is about 110 mM.

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meeting. The field of coupled enzymes was rapidlyexpanding at this time, formalizing the process bywhich membrane-bound aggregates of enzymes form asort of assembly line within mitochondria and othercellular structures that pass substrates from oneenzyme to another without any free diffusion. Theresult of these membrane-ordered aggregates is vastlyincreased metabolic efficiency.

His mind prepared by the conference, P.F.D.then serendipitously discovered that two solubleenzymes essential to muscle energetics, creatine kinaseand pyruvate kinase, bind to one another creatingunexpected metabolic consequences and solving someproblems of muscle energetics dealing with pyruvatekinase that have existed for more than 20 years[Newsholme and Start, 1973]. P.F.D. and a graduatestudent, Joseph Clark, were interested measuring thecreatine kinase activity in smooth muscle usingsaturation transfer NMR. One of the controls for thisexperiment involved accounting for the ATPase activityin the tissue. A double saturation experiment wasrequired. Clark had developed the technology neededto do a double saturation, and together they weretesting the technique using skeletal creatine kinase andpyruvate kinase in solution [Clark et al., 1991]. Theidea was to saturate the substrate for CK, phospho-creatine, and measure the change in the gamma-ATPresonance, then saturate the substrate for PK, phos-phoenolpyruvate, and look for the change in gamma-ATP. Finally, by saturating both PCr and PEPsimultaneously, the change in ATP should be greaterthan with either individual saturation. Instead, satura-tion of PCr produced a change not of ATP, but of PEP!In the reverse experiment, the saturation of PEPproduced a change in PCr. Saturation of ATP changedboth PEP and PCr [Dillon and Clark, 1990]. P.F.D.recognized this as equivalent to the glycolytic clustersdescribed by Maughan at the energetics meeting. Theactual result was unanticipated, but the physical andfunctional coupling of PK and CK was shown using avariety of different techniques [Dillon and Clark, 1990;Dillon et al, 1995; Sears and Dillon, 1999]. One of theimportant principles here is that an unexpected findingrequires substantial and varied confirmation.

One problem the discovery created was how tomeasure the affinity of the two enzymes for oneanother. P.F.D. and another graduate student of his,Patrick Sears, had the counter-intuitive idea of usingcapillary electrophoresis to measure the bindingconstants of the complex. While attending the FASEBconference, they attended a technical session oncapillary electrophoresis, and how it could be used toseparate different chemicals. Capillary electrophoresisis, of course, normally used to separate or purify

mixtures of molecules based on their charge-to-massratio. The technique had very few applications tobiological tissue. P.F.D. and Sears reasoned thatbetween the voltage that will completely separate apair of bound molecules into two distinct peaks andzero voltage where there will be no imposed separa-tion, there should be a voltage at which half of themolecules remain complexed and half are separated.One should, therefore, be able to determine a bindingconstant at any given voltage. By varying the voltage,one should then be able to extrapolate back to zerovoltage, which would represent the binding constant asit is usually measured in a free solution. CE had neverbeen used in this manner.

It is worth pointing out that P.F.D. and Sears hadresorted to an age-old strategy that has often stimulateduseful inventions, sometimes known as ‘‘Langmuir’sprinciple’’: turn things on their head [Jaffe, 1957, p207]. A classic example concerns the invention of theGeiger counter by Hans Geiger and Walther Mueller.While attempting to measure cosmic rays, they keptencountering spurious ‘‘noise.’’ Many attempts toeliminate the ‘‘noise’’ failed, so they eventually decidedto redesign their equipment to maximize the problem.They then discovered that this ‘‘noise’’ was actually dueto radioactivity and their apparatus became the basisfor modern Geiger counters. They eventually turnedthe problem on its head, designing the circuitry toincrease the ‘‘noise’’ maximally in order to discover itssource, only to discover that they had invented amachine for detecting radiation sources. The currentuse of ‘‘dirty’’ polymerase chain reaction procedures inorder to generate DNA diversity is another relevantexample.

CE experiments with creatine kinase and pyr-uvate kinase proved frustrating. Sears found that one ofthe enzymes bound to the glass capillary tubing,interfering with measurements. Frustrated, P.F.D.wanted to at least to perform a proof-of-conceptexperiment demonstrating that the capillary electro-phoretic determination of binding constants waspossible. He turned to R.R.-B. for advice on a possiblemodel. R.R.-B. suggested ascorbate-norepinephrine.Both were small, soluble, inexpensive molecules, andeasy to detect. Two problems, one theoretical (how doascorbate-norepinephrine protect each other) and theother technical (can capillary electrophoresis be usedto determine binding constants) had merged. Each ofus was looking to another project to advance our own.

P.F.D. spent several years developing CE for usein measurement of cellular metabolites. These effortsresulted in methods that could detect dozens ofcompounds using just nanoliters of a cellular extract[Dillon and Sears, 1998] and a patent with Dr. Douglas

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Henry for the measurement of sorbitol in diabeticretinopathy [Henry and Dillon, 2000]. These straight-forward uses of CE were based on its traditionalapplication. Still, P.F.D. wanted to measure theinfluence of voltage on the coupling of two molecules.CE should work, but the technical difficulties of thekinases were daunting. Using the small molecules,ascorbate and norepinephrine, suggested by R.R.-B.,seemed like a reasonable alternative.

The results stunned both P.F.D. and R.R.-B.. Onthe first attempt, using the conditions developed for themetabolite measurements, individual solutions ofascorbate, and NE were found to have single peaks.When both were present however, a third peakappeared! (Fig. 4) this peak could be nothing but acombination of ascorbate and NE. Further experi-ments confirmed this [Dillon et al, 2000]. The keyparameter turned out to be the spatial derivative of thevoltage, the electric field. A reduction in the electricfield reduced the separation force, and the coupledpeak grew larger. At constant NE and constant electricfield, an increase in the ascorbate concentrationresulted in an increase in the size of the coupled peakas well. At any given electric field, changing theascorbate concentration produces a dissociation curveand dissociation constant, the Ke, particular for thatelectric field. Extrapolation of the Ke’s at differentelectric fields back to zero electric field produced anovel way of measuring dissociation constants forbiological compounds, confirming the alternative wayof using CE anticipated by Sears and PFD. Themethod proved to be successful for other catechola-

mines and ascorbate, and for ascorbate-related com-pounds and NE [Dillon et al., 2000]. The bindingconstant for NE to AA was found to be about 80 mM,very close (considering differences in buffers) to theu.v. spectroscopic value of 110 mM (Fig. 3). Conditionswere also eventually found demonstrating the couplingof PK and CK [Sears and Dillon, 1999].

P.F.D. subsequently found that this dissociation ofbiological compounds in an electric field had strongsimilarities to the effect of electric fields on thedissociation of weak electrolytes by Onsager [1934]and Moore [1972] termed the second Wien effect.Despite similar results in a parallel field by theseeminent scientists, our novel method of obtainingdissociation constants was not deemed worthy ofpublication by general interest journals. Publicationin a specialty journal was not difficult, however.

WHAT PROBLEM DOES OUR DISCOVERY SOLVE?

One of the oddest, and yet not uncommon,features of our discovery was that we observed thephenomenon of ascorbate-adrenergic interaction andsynergism before we were motivated to consider whatproblems these observations might solve. In otherwords, our research developed in exactly the oppositeway in which most directed research occurs. Wesuggest that this backwards approach was probablythe only way to achieve the results we did, a point towhich we will return at the end of this article when wediscuss how some major drugs were developed by otherinvestigators.

We had speculated on the physiological effects ofascorbate-catecholamine coupling. Circulating concen-trations of catecholamines in blood plasma are aboutnanomolar, while ascorbate is present at about 50 to100 micromolar [Ganong, 1997, p 337]. Using thedissociation constant determined using CE, more than80% of the circulating norepinephrine would be boundto ascorbate at physiological levels of both. Thepotential physiological significance of our findings wasobvious. Ascorbate binding would work to protectcirculating NE, as predicted in our molecular com-plementarity papers, but would it also lower the free(active) concentration of the catecholamine? The keyappears at the cell membrane. Because of the dielectricconstant of water, there is a rapid fall in voltage as onemoves away from a biological membrane. A drop in mVover a distance of nanometers produces an electric fieldof thousands of V/cm, far more than is needed forcomplete dissociation of ascorbate and NE. Thus, asthe complex approaches a membrane, there may be afull dissociation of the compounds, leaving NEavailable to bind to its receptor [Dillon et al., 2000].

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Fig. 4. Capillary electropherograms of NE alone, AA alone, and NE þAA. The lower run has a extra peak not present in the two separateruns. *Indicates the free NE peak, and the new peak is to its left. Thispeak must be a combination of NE and AA, demonstrating the physicalbinding of NE and AA. The dissociation constant for NE and AA usingthis method was 74 mM. Courtesy of Biophysical Journal.Q8

8 R. ROOT-BERNSTEIN AND P.F. DILLION

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Having now shown that ascorbate binds toadrenergic compounds, we were naturally interestedin what the physiological effects of the binding mightbe. We retreated to the traditional techniques ofsmooth muscle mechanics for this part of the study.P.F.D. had extensive experience using these techni-ques, having done so in the discovery of the latchmechanism in the regulation of smooth musclecontraction [Dillon et al., 1981]. We were led topredict that if the ascorbate-adrenergic coupling didanything physiologically, it would produce a reductionin smooth muscle contraction, since a number ofhuman studies had recently appeared showing thatvitamin C supplementation lowers blood pressure[Bendich and Langseth, 1995; Ness et al., 1997;Nyyssonen et al., 1997; Bates et al., 1998]. Hindsightbeing, if not 20/20, at least a little better than foresight,we failed to note several phenomena. Alpha adrenergicmechanisms tend to increase blood pressure, whilebeta adrenergic ones tend to lower it, so that the sameadrenergic compound acting in different parts of thecirculatory system can have compensatory effects. Also,increasing ingestion of vitamin C increases urinaryexcretion of adrenergic compounds [Igisu et al., 1982;Kallner, 1983]. Thus, the acute and chronic effects maybe different and time becomes a critical factor inpredicting the effects of adrenergic drugs. Catechola-mines are most involved in short-term blood pressureregulation, not long-term regulation. And finally,sometimes complementary pairs of molecules producesynergistic reactions, a result we, ourselves, haddemonstrated for an antisense peptide to angiotensinII [Dillon and Root-Bernstein, 1997]. Thus, we shouldhave (but did not) realize that adding ascorbate to NEmight increase or decrease its activity on aortic smoothmuscle (alpha adrenergic effects) and that these effectsmight not predict body-wide effects (which includebeta adrenergic and excretion effects).

In the event, experiments in which ascorbate wasadded along with NE did not produce the predictedreduction in NE-induced contractile force. As ex-pected, we found no contractile effect of any dose ofascorbate alone from 5 to 500 mM. But when we addedascorbate to NE, we observed a very substantialincrease of the NE-induced contraction comparedwith NE alone (Fig. 5). The difference is so great that it

easily passes what we call ‘‘the bloody obvious test.’’‘‘Bloody obvious’’ results are those for which statisticsare unnecessary to demonstrate an effect. One usesstatistics only for the benefit of skeptics who cannottrust even their own eyes, such as editors and referees.We note that such ‘‘bloody obvious’’ experimentshighlight the history of biomedical research fromRoentgen’s demonstration that X-rays can reveal thebones in one’s hand to Fleming’s demonstration thatPenicillium mold interferes with the growth ofStaphylococcus bacteria. Nobel laureate George P.Thomson has actually stated this approach to science inthe form of the admonition, ‘‘Demonstrate, don’tmeasure’’ [Thomson, 1961, p 129–131]. The object inperforming exploratory or venture research is todiscover new phenomena, not to confirm or analyzedetails. Accuracy is far less important than thedemonstration of a striking effect. Anyone in musclephysiology will recognize the 300% increase in activitythat is portrayed in Fig. 5 as a striking effect ofascorbate for which there is no comparable report inthe literature.

Another novel aspect of the ascorbate-enhancedcatecholamine contractions also revealed itself quitequickly. Normally a catecholamine-induced contractionof smooth muscle peaks between 3 to 5 minutes afterthe compound is added, showing a significant drop-offin force by 10 minutes and returning to near baselinevalues within 15 or 20 minutes. We immediatelyobserved that the ascorbate-enhanced contractionswere being maintained and so we set up someexperiments in which we monitored the long-termcourse of the contractions. As Fig. 6 shows, ascorbate

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Fig. 5. Influence of ascorbate on low-level norepinephrine contrac-tions of rabbit aorta. Physiological concentrations of ascorbateproduce a significant increase in the force generation: 3.7 7 0.7times the force generated by 30 nM NE alone (SE, N¼6, P o 0.01).Notably, AA alone at concentrations between 5 and 500 mM producesno contraction.

Fig. 6. Long-term effect of ascorbate on force generation in rabbitaorta; 300 nM epinephrine generates maximum force. There is noinfluence of ascorbate on maximum force. Force maintenance is

substantially increased by the presence of ascorbate. Ascorbate is ableto mitigate most of the tachyphyllaxis that occurs during epinephrinecontractions.

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maintained the catecholamine contractions for hours,another phenomenon without precedent in the smoothmuscle literature.

Now, since catecholamines oxidize reasonablyrapidly at pH 7.4 and 371C in aqueous solutions, thenext obvious question, which has been repeatedlyraised by every investigator who has been shown ourresults, is whether the enhancement we were observingis simply due to the antioxidant effects of ascorbate. Weaddressed this reasonable and very important questionin two ways. First, P.F.D. used the capillary electro-phoresis equipment to measure the rate at which NEoxidizes in the buffers used in our experiments at 371C,and what effect ascorbate has on this oxidation rate. Hefound that over the time course of the experiments,only about 5% of the NE was oxidized (Fig. 7).Ascorbate prevented almost all of this oxidation. Thepresence of ascorbate in solution, however, causessmooth muscle to react as if there is three times (300%)as much NE present. Thus, the antioxidant effect ofascorbate accounts for only about 2% of the enhance-ment phenomenon that we observe.

Our second proof that ascorbate was not actingprimarily as an antioxidant was to separate the additionof the ascorbate from the catecholamine. We are to thisday unsure which of us suggested this experiment orwhat our expectations were. R.R.-B., reasoning that the

active agent was the ascorbate-catecholamine complex,seems to recall thinking that the enhancement woulddisappear if the two compounds were put in separately.P.F.D. seems to recall thinking that the enhancedactivity would remain. The important point is that welost no time debating the issue. We have developed auseful strategy of doing whatever experiments either ofus suggest, even when we do not understand the otherperson’s reasoning or agree with their expectations.Sometimes the experiments do not work, but moreoften than not, something important results. Havingbeen wrong in our individual and joint predictions somany times before, we have ceased to predictexperimental outcomes and simply do the experiments.One can’t be surprised if one does not create theconditions that will yield surprises.

In this instance, the separate addition of ascor-bate and catecholamine was clearly an experiment thatneeded to be done, so we did it. The result was that asignificant proportion of the ascorbate-induced en-hancement was preserved even when the ascorbate wasadded to the tissue and washed out several minutesprior to the addition of the catecholamine (Fig. 8).Since ascorbate obviously cannot prevent oxidation ofNE when they are never in contact, this experimenteffectively proves that the role of ascorbate is notprimarily that of an antioxidant in this system. We havealso shown that when the ascorbate is washed out, itsenhancement effect disappears after 15 to 20 minutes.These data clearly demonstrate that there are two,independent effects exerted by the ascorbate and thecatecholamine, which interact at the cellular level andwhich are active for a period of only a few minutes.Such data preclude a number of mechanisms such asprotein synthesis or receptor turnover from playingroles in the phenomenon. We began considering otherpossibilities such as the antiphosphodiesterase activityof ascorbate, second messenger activation, alteration ofadrenergic receptor affinity by ascorbate, and so forth.The fact that there is a good correlation betweenascorbate-induced enhancement and ascorbate bindingto drugs with contractile activity on smooth muscle isalso an important clue to any possible mechanism

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Fig. 7. Capillary electropherograms of NE solutions. The solutionswere kept at 371C in the same tissue baths used for rabbit aortamechanics. The NE peak is indicated. There is a steady decline in thesize of the NE peak corresponding to its oxidation. Ascorbatedecreases the rate of NE oxidation, but cannot entirely prevent it atthis temperature. The rate, however, is such that the overall loss of NEduring the 20 min during which muscle mechanics experiments arerun is only about 2%.

Fig. 8. Ascorbate increases NE contractions even when added andwashed out prior to NE addition. The ascorbate was added for 2 min,indicated by the small bar, and washed out for 8 min from rabbit aortarings. The increase with 50 mM AA is less than when NE and AA are

added together, but the effect of 150 mM AA is quite clear. Thus, atleast part of the AA enhancement of NE contractions does not requireboth to be present simultaneously. Notably, AA alone at concentra-tions between 5 and 500 mM produces no contraction.

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(Table 1). The enhancement is definitely not general toany contraction, since ascorbate has no effect at anydose on angiotensin II, acetylcholine, or potassium-induced contractions.

At this point, although we did not (and, in fact,still do not) know how ascorbate enhanced catechola-mine activity, it occurred to us that the phenomenonmight have practical uses. We, in other words,discovered the potential uses of our findings after wemade them, rather than targeting a practical problemfrom the outset. We began with a basic chemicalquestion that led to a fundamental physiological one,and that resulted in a novel phenomenologicalobservation that raised, rather than answered, mechan-istic questions. The whole project evolved in ways thatwould have been impossible in almost any industrialsetting. As with other strategic points we have made,we will see below that this scenario is far from atypicalin the history of pharmaceutical research.

In our case, we reviewed the literature on the useof adrenergic drugs and realized that the ascorbateenhancement we observed might be used to producelonger-acting, lower-dose formulations of adrenergicdrugs with less side-effects for treatment of cardiacconditions, blood pressure regulation, asthma, ophthal-mic uses, anesthetic delivery, premature delivery, andperhaps even improved efficacy neurological prepara-tions such as L-DOPA. A quick literature and patentsearch revealed no precedents and so we informed theuniversity Office of Intellectual Property that we had apotentially useful invention. Having both experiencedextraordinary difficulties working with this Office ofIntellectual Property on previous occasions, we effec-tively forced them to act quickly in this case by setting adeadline by which we intended to submit an NIHgrant. The University moved quickly to patent. Aprovisional patent application was filed with the PTO in

September of 2000, followed by the patent itself inSeptember of 2001. We applied for an NIH grant inOctober 2000. Despite the fact that we coulddemonstrate a striking and completely novel phenom-enon and provide a clear-cut strategy to go aboutelucidating the mechanism by which this phenomenonmight be manifested, the grant was triaged. We havealso had our results rejected by several major generalinterest journals such as Science, PNAS, and NatureMedicine, the most common complaint being that wedo not understand the detailed mechanism by whichadrenergic enhancement works.

We suspect that the major problem people havein evaluating our results is that they have beenachieved by using equipment in unusual ways toproduce unanticipated results. Unusual claims, ofcourse require unusual proof. We, ourselves, havehad confidence in these results because of the dictumP.F.D. constantly preaches to his students: you must beso sure of how your equipment works that when youget an unusual result, you can believe it. Thousands ofCE runs and muscle stimulations provided a back-ground against which our ascorbate finding could bejudged. In all the presentations of this work, either atmeetings, in manuscripts, or in grant applications,there has never been a single criticism of the factualdata. It is only the conclusions and the unknownmechanisms behind the findings that have beenquestioned. Of course, particularly with the mechan-isms, it is often not possible to have all the answersimmediately after one discovers a new phenomenon.One can only conjecture about the reduction in the sizeof the biomedical literature if every finding had to havethe complete mechanism explained prior to anypublication of the phenomenon.

We have also noted that people in the biotech andpharmaceutical industry, even when they are favorablyimpressed by our results, almost universally warn usthat getting our work adopted will be difficult. Thestandard approach to drug discovery is, of course, todevelop a more effective single agent. Despite the factthat triple drug regimens are now the norm in treatingAIDS, and that adjuvants are commonly used in cancerchemotherapy, there appears to be no systematicprogram of looking for useful interactions betweencompounds at any major pharmaceutical or biotechnol-ogy firm. Thus, not only has our route to our discoverybeen outside of the normal routes practiced by theindustry, our solution to the problem of betteradrenergic drugs is also non-traditional. We expectthat it will take an unusual company to adopt ourinsights.

On the other hand, we see many benefits to ourapproach. Adrenergic drugs that are currently off-

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Table 1. Correlation Between Ascorbate Binding and Ascorbate-Enhanced Smooth Muscle Activation

Contractile compound Ascorbatebindinga

Ascorbateenhancementb

Epinephrine 100mM ++Norepinephrine 110mM +++Ephedrine 1.1mM +Norephedrine (PPA) 1.5mM +Pseudoephedrine 2.5mM �Acetylcholine 0 �Angiotensin II 0 �Potassium ? �

aUV spectroscopy with ascorbate at 100mM in pH 7.0 phosphatebuffer.bContractile effect on rabbit aorta with ascorbate at 50 to 500 mM.

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patent or nearing expiration may find new life asunique, and therefore patentable formulations. Sincethe safety and efficacy of the individual adrenergicdrugs have already been demonstrated to the satisfac-tion of the FDA, and since ascorbate and many of itsvariants are FDA approved as safe food and drugadditives, the only expenses incurred by a companywishing to develop an adrenergic-ascorbate formula-tion will be optimization of the combination and itshuman testing. These expenses are certain to be far lessthan the costs of discovering and developing a noveladrenergic compound from scratch and the risk ofunexpected side effects far less.

We should note also that some human experi-mentation has already been reported that makes thedevelopment of such adrenergic-ascorbate formula-tions worthwhile. Mak and Newton [2001] havedemonstrated that intracoronary infusion of vitamin Csignificantly improves the inotropic cardiac response todobutamine in human patients, concluding that the‘‘redox environment contributes to the adrenergicregulation of ventricular contractility.’’ Grossmannet al. [2001] have demonstrated that infused ascorbicacid improves the vasodilatory effects of naturallyoccurring catecholamines in venous circulation. Thus,our in vitro experiments have found clinical validation,suggesting that a wide range of ascorbate-inducedadrenergic enhancement effects may be possible.

Finally, we would like to speculate in the mode of‘‘turning it on its head’’ that the field of druginteractions in general may present a huge treasuretrove of possibilities for drug development and deliveryin the future. At present, drug interactions are mainlyperceived to be detrimental to patients and toobtaining FDA approval. We suggest that, on thecontrary, just as asking why ascorbate can be used soeffectively to stabilize catecholamines has turned out tobe useful, so might elucidation of the mechanisms ofdrug interactions. As anyone in the pharmaceuticalindustry knows, something that is deadly in one contextcan be life-saving in another. Perhaps we can learn tomanipulate drug interactions to our advantage.

OPERATING ON A SHOE-STRING

It is worth backing up for a moment andconsidering how we managed to perform the researchthat yielded these results, which were as unexpected tous as to the scientific community. We emphasize thatneither of us foresaw the initial, let alone the laterresults that we would achieve by working on theascorbate-adrenergic project. Recall that it was under-taken not for its own intrinsic merits, but as ademonstration of principle for P.F.D.’s non-traditionaluse of the capillary electrophoresis as a method for

measuring binding constants. On the other hand, wehad explored the theoretical importance of ascorbate-catecholamine interactions attempted to obtain fund-ing. In 1997, we produced two lengthy theoreticalpapers arguing that molecular complementarity is afundamental process underlying the evolution ofmetabolic regulatory systems in all living things[Root-Bernstein and Dillon, 1997; Dillon and Root-Bernstein, 1997]. Ascorbate-adrenergic interactionswere a case study of how molecules that bind to oneanother are also co-stored and co-released in manydifferent systems in the body, including adrenals andneurons. We argued that evolution has built on the factthat such interactions protect molecules from oxidativeand other forms of degradation so that survival of thefittest operates at a chemical as well as organismic level.Molecules that interact are like symbiotic species,benefiting each other’s fitness. Each year between 1996and 1999, one or the other of us sent in an NIH grantas the primary investigator for joint projects to explorethese evolutionary and physiological implications ofmolecular complementarity,. In each case, our propo-sals were deemed to be unfundable, usually on thegrounds that they were unfocussed ‘‘fishing expedi-tions.’’ These comments continued each year, even aswe focused more and more specifically on veryparticular results and model systems, such as theascorbate-adrenergic one, providing ever-increasingexperimental results to justify our strategic choices.We never succeeded in obtaining a grant.

So how did we perform the experiments de-scribed above? We have always subscribed to theprinciple that it is possible to do research as long as oneis clever enough and has access to resources of somekind. In this case, we both had small amounts offunding from other projects, which kept us in generalsupplies. In addition, we had the basic equipmentalready on hand to perform the experiments. P.F.D.had built a muscle mechanics apparatus decadesearlier, which he had used continuously ever since.He had also obtained his capillary electrophoresisapparatus in part through a previous NIH grant and inpart through an on-campus research support grant.Once the equipment is available, one of the questionswe always ask is, ‘‘what else can we do with it?’’

Another strategy we employed to keep the projectgoing was to appeal to colleagues for non-monetaryassistance. The largest single experimental expense wemight have incurred would have been the cost ofrabbits from which to excise the aortas and tracheasupon which we experimented. The most helpfulcolleague in this regard was Steven P. Arnoczky, whowas kind enough to inform us each time his laboratorywas euthanizing rabbits for their own experiments on

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bone injuries. After his team excised the bone samplesthey needed, we would then excise our tissues. Inconsequence, our only recurring expenses were verymodest ones of the drugs used in the experimentsand the chemicals required for mixing buffers andsolutions.

Finally, it is worth noting that we personally haveperformed all of the experiments on the ascorbate-adrenergic project up until a few months ago. Thelargest single budget item in most research is thatassociated with personnel. Not only did the quitesurprising nature of the experiments make it essentialthat we, as experienced investigators, were the ones torun the initial experiments and make the initialobservations, it was also the most cost-effective strategywe could employ. Again, it is worth noting that a verylarge number of successful scientists have argued that ifyou want to make lots of discoveries, you have to be inthe lab doing the experiments yourself [Root-Bern-stein, 1989].

We should also note that we eventually did obtainfunding, but as befits a non-traditional researchproject, from a non-traditional source. As a result ofsome previously patented research on arthritis, R.R.-B.had been negotiating for several years with a noveltechnology investment firm called Acorn Technologies,Inc., based in Pacific Palisades, CA. Acorn Technolo-gies is unique in the United States in attempting to findand fund projects that have potential electronic orbiotech markets before the projects have beenpatented. Their investment strategy is to makerelatively small investments in a large number ofpotential discoveries in the hopes that some willdevelop from acorns into oaks. In return for theseinvestments, they pay for the patenting of thetechnologies and obtain an exclusive license to theinventions, which they then market to larger firms. Wehave been fortunate enough to benefit from theirlargess and are presently in the process of developingour discovery to the point at which it will hopefullyattract industry interest. We consider Acorn’s strategyto be uniquely useful for people like us who havestumbled into an area from an unexpected quarter andconsequently are taking a very non-traditional ap-proach to the problems of drug development anddelivery.

RESEARCH MANAGEMENT IN A BROADER CONTEXT

The route we took to our discovery, including itsorigins in basic research concerns, lack of peer supportand funding, and erratic course, is anything but atypicalof innovations. In 1981, Comroe and Dripps analyzedthe origins of the most important developments in‘‘open-heart surgery, blood vessel surgery, treatment of

hypertension, management of coronary artery disease,prevention of poliomyelitis, chemotherapy of tubercu-losis and acute rheumatic fever, cardiac resuscitation ancardiac pacemakers, oral dureticsyintensive careunits, and new diagnostic methods’’ [Comroe andDripps, 1981, p 120]. They found that 41% of thearticles providing key findings making possible theseclinical developments had no clinical bearing orrelevance at the time they were published and 62%were reported as ‘‘basic’’ research findings involving theinvestigation of how basic physiological functions oreffects occur or can be measured. Only 38% of thestudies that yielded clinical breakthroughs were basedon research programs directed specifically at clinicaloutcomes [Comroe and Dripps, 1981, p 121]. Theseresults strongly suggest that product-directed researchis likely to miss over half of the important clinicalinnovations that are scientifically available at any giventime. The same results also suggest that drug discoveryis just as likely to occur from attempts to understand orapply already published basic research insights as it isto result from direct attempts to discover a new drug ordiagnostic technique. Novelty does not necessarilyresult from the search for novelty.

The research of Sir James Black provides twocases in point. Black was awarded the Nobel Prize forhis discovery of beta adrenergic blockers (propranalol)and histamine blockers (cimetidine). In neither casewas he attempting to discover new pharmaceuticalagents nor was he even a chemist himself. He says thathis successes have come from taking an audaciouslydifferent approach to basic physiological questions.. ‘‘Ithink the only difference between me and some othercolleagues would be temerity. I think I ask questions ofchemists and physiologists which are really quitepreposterous. And I don’t seem to mind being thoughtstupid; I don’t seem to care what people think about myquestions. So I think in so far as what I’ve done has ledto things, they have all involved turning somethingaround the other way. I think my brain automaticallydoes this. It always is challenging the accepted view ofthings. So if someone says to me, ‘The speed of light isconstant,’ then I’ll say, ‘Well, what would happen ifwasn’t?’y You must be able to ask these questionswithout feeling ashamed of them’’ [Wolpert andRichards, 1997, p 126–127]. In the case of his discoveryof beta blockers, he began with the basic researchquestion of what causes angina. The dogma at the timewas that the pain associated with angina was a result oflack of oxygen to the heart due to constricted bloodvessels, and therefore the way to treat the disease wasto dilate the coronary arteries. Black thought, ‘‘Forgetabout that, let us see if we can decrease how muchblood the heart actually needs’’ [Wolpert and Richards,

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1997, p 124–125]. A consequence of such upside-downthinking was to redirect his thinking to finding a way toblock selectively the heart’s response to epinephrine(which is mediated by beta adrenergic receptors)without affecting its response to norepinephrine (whichacts through alpha adrenergic receptors). Would mostcompanies permit such a contrarian research project tobe undertaken today? Could Black have obtained anNIH or MRC grant? We believe from our ownexperience that both possibilities would be doubtful.

The problem of justifying research in Black’sstyle, as productive as it turned out to be, wasexacerbated by its lack of obvious application. Blackemphasizes that he never entertained pharmaceuticalgoals for his research. He, therefore, denies absolutelyhaving any inkling that beta blockers would havemedicinal applications or that he had any intention oftrying to cure ulcers when he developed cimetidine: ‘‘Ithink you should understand that at no point have Iprofessionally made claims about what I thought mywork would achieve, for the simple reason I’ve neverthought that it was going to achieve anything other thananswer a question. And the same was true with thework we did later with histamine. The challenge was tomake a molecule which would have the properties ofallowing you to answer a physiological question. Now, ifthe answer had come out a different way in each case,there would have been no drugs, but there would stillhave been a scientific contribution’’ [Wolpert andRichards, 1997, p 127]. This is clearly the case in ourown research as well. It is well to recall that in bothcases, direct attacks on the problems of how to treatangina, cure ulcers, and improve adrenergic drugactivity have been made by large groups of pharma-ceutical researchers for decades without success.‘‘There are,’’ Black therefore cautions, ‘‘certain thingsyou may not seek directly. There’s a kind of obliquenessnecessary in life’’ [Wolpert and Richards, 1997, p 123–124] Therefore, we must not be afraid to go off ontangents. Tangents, we must recall, always intersect thecurve somewhere.

If drugs and diagnostic techniques are just aslikely to occur from non-product-directed as fromproduct directed research, then basic questions ariseconcerning the best use of investigators’ time. Analmost unique study by Platt and Baker [1931] isinformative. They interviewed a large number of theirchemical colleagues and found that the majority did notsolve their problems while working directly on them,but rather when working on other projects, relaxing,sleeping, or at other non-work times. A more recentand detailed study by Root-Bernstein et al. [1995] hasconfirmed Platt and Baker’s findings. The mostproductive scientists report having multiple concurrent

projects in hand at all times, so that if one is goingpoorly, another will be going well, and so that insightsand techniques from each can cross-fertilize the othersto yield unexpected insights. We certainly had thisexperience with the ascorbate-adrenergic project.Thus, our experience in conjunction with these studiesstrongly suggests that research programs are most likelyto be successful if they investigate multiple, relatedprojects concurrently, engaging researchers in severalinvestigations so that a maximum of information andtechniques flows between the projects. Intensivelyfocused research is too likely to miss useful detours.

Detours also include learning new techniquesand skills. Certainly our willingness to explore the novelpossibilities of capillary electrophoresis as well as thetried-and-true methods of muscle mechanics and u.v.spectroscopy was a key to our success. Melvin Calvinalso believed that part of ‘‘following the light’’ is ‘‘thewillingness to undertake work to learn whatever factsand techniques might be necessary to answer thescientific questionsy’’ [Calvin, 1992, p 132]. One canonly discover what one risks discovering by goingbeyond the knowledge and skills one already possesses.

On a related note, one of the most commonstatements made by highly productive scientists is thatthey never look for the answer to a problem, but ratherinvestigate the widest possible range of answers.Robert Mulliken, a Nobel laureate in Chemistry, forexample, has written that, ‘‘I have a compulsion to lookat all possibilities, both probable and improbabley.This habityhas helped me find original ideas in thecourse of my research’’ [Mulliken, 1989, p 19].Similarly, fellow Nobelist Linus Pauling wrote thatone of his research strategies was to ask, ‘‘Whatideas’’Fnote the pluralF‘‘about this question, asgeneral and as aesthetically satisfying as possible, canwe have that are not eliminated by these results ofexperiment and observation?’’ [Pauling, 1963, p 47]. Itis fairly obvious that these men would have founduncongenial and stifling the notion that research couldbe directed at a predetermined outcome such as aspecific drug or diagnostic test. As Nobel laureateMelvin Calvin has written, you must ‘‘go where thelight leads,’’ by which he means whatever directionone’s investigation reveals novel insights [Calvin, 1992,p 132]. These men would have advocated setting ageneral goal, but recognizing at all times that the mostimportant results may be the serendipitous ones thatcause the investigator to detour from the plannedcourse.

Businesses, of course, like preplanned courses.Pre-set goals allow firm plans and investments to bemade. Serendipity requires flexibility that many admin-istrators find uncongenial. Thus, Sir James Black said of

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himself that he was ‘‘bad news for industry because Iwas constantly rebelling. I was constantly challengingthe assumptions they made. I challenged theirpractices, I was awfuly It’s a big managerial problemhow to deal with somebody like me since they’rearound in every company. It wasn’t that I set out to givethem a hard time. It was simply that being in there putme in an environment which wasn’t conducive to whatI wanted to do’’ [Wolpert and Richards, 1997, p 129].He eventually left not only industry, but academia aswell, in order to find the freedom that only self-employment can allow.

Black’s case is a lesson for industry. If people likeBlack are the driving forces of the most importantpharmaceutical and biotechnology innovations, then itspeaks very poorly of industry that its prime-moversfind industry not only uncongenial, but unworkable.Reference to any number of books and articles aboutbiomedical innovations reveals histories that are tooclose to Black’s for comfort. George Olah’s Nobel prize-winning development of so-called ‘‘magic’’ or ‘‘super-acids’’ was initiated on his own time after hours while inthe employ of Dow Chemical Company, whichbelieved the work to have no industrial applicationsand therefore declined to take out patents on it [Olah,2001, p 66–69]. The trials and tribulations involved ingetting Kary Mullis to translate his idea about thepossibility of polymerase chain reactions into reality arelegendary, as are his unusual personality and workhabits [Mullis, 1998]. Note that he, too, has found nopermanent place in the industry he helped to create.

Many other innovators have met similar, if lessdrastic, fates. The revolutionary development oforganic-based combinatorial chemistry techniques atParke Davis during the 1990s by Walter Moos, SheilaDeWitt, and Mike Pavia was met by in-house disin-terest and even disdain that caused all of the majorplayers to leave for other companies [Root-Bernstein,2002]. William C. Campbell has told a similarly‘‘complicated and unglamorous’’ story about his rolein the development of the extraordinarily effectiveantiparasitic drug ivermectin, which involved elementsof basic and targeted research, serendipity, failures,successes, cooperation, and conflict [Campbell, 1992].And the same personality and management problemshave attended the births of many biomedical diagnostictechniques as well. Roger Damadian has recorded thelong and troubled road he followed in the developmentof magnetic resonance imaging in his book A MachineCalled Indomitable [1985]. The even more difficultpath to clinical ultrasound (which, on several occasionsresulted in unemployment) has been told by John J.Wild in his reminiscences [Wild, 1992]. Wild, likeMullis, has spent large portions of his life either

unemployed or employed outside of both industry andacademia. The prevalence of such histories is disturb-ing at best.

Industry need not drive its innovators out orignore them. All of the preceding studies display fourof the most important generalizations reached byGerald Holton and his colleagues from a study of thediscovery of high temperature superconductors. First,they noted that the laboratory in which Alex Muellerand his colleagues discovered high temperature super-conductivity was unusual in its ‘‘willingness to give goodpeople the freedom to pursue projects with longgestation periods,’’ which fostered serendipitous results[Holton et al., 1996, p 375]. They note that theinventions of lasers and transistors are characterized bythe same unforced research pace and multipleserendipitous discoveries. Second, they note thatsuccess breeds success (the so-called ‘‘Mattheweffect’’). Breakthroughs often follow breakthroughswhen successful scientists are given their leads. Third,they found that the research administrators in thesuperconductivity case were unusual in encouraging‘‘flexibility that promotes borrowing with and acrossdisciplines and between basic and applied research’’(Ibid.) And finally, they found, as did we, that ‘‘thecurrent debate about the relative merits of and supportwarranted for basic and mission-oriented research isoversimplified. Historical study of the cases of success-ful modern research has repeatedly shown that theinterplay between initially unrelated basic knowledge,technology and products is so intense that, far frombeing separate and distinct, they are all portions of asingle, tightly woven fabricyIf we wish to achievenoteworthy science, even if noteworthy is defined tomean only science with an economic payoff, the nationhas no alternative but to support the seamless web ofresearch.’’ (Ibid.) Clearly, we would apply this com-ment to individual companies and research projects aswell.

CONCLUSIONS: MAKE TIME FOR SERENDIPITY

Our thesis, in short, is that pharmaceutical andbiotechnology research and development costs areincreasing without yielding proportionally increasedproducts because the economic and organizationalinfrastructure that has evolved to ‘‘improve efficiency’’is actually at odds with the nature of the discoveryprocess itself and the way innovators work within it[Root-Bernstein, 1989, 1990a,b, 1995]. The need tobegin with what is already known, to provide clear roadmaps and to move quickly past predetermined bench-marks in order to attract and keep investors is counter-productive. Science of this type becomes like a roadrally: the fastest one to figure out all of the clues gets

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the prize. When the goal is absolutely clear-cut, theroad rally model may work quite well. But the roadrally mentality leaves huge areas of the scientific mapunexplored. We remind readers of the old joke aboutthe drunk who loses his key in a dark alley but searchesfor it under the street lamp at the corner because that’swhere the light is. There may be keys to be foundunder the light of the street lamp, but there are surelyjust as many waiting to be discovered out in the darkalleys where the competition is a great deal less.Companies might do better to re-educate theirinvestors about the possibilities that lie shrouded indarkness than follow the crowd searching under thelight.

Moreover, our experience of the peer reviewsystem shows an entrenched tendency to operate incomfortable ways according to the latest fashions. Ourreading of the history of science, however, warns usthat we should be most wary of those results that bestfit our expectations and most intrigued by thosefindings that challenge our preconceptions. Innovationscan only come from innovators, and the definition ofinnovation is to operate in novel, non-obvious ways. Wecannot claim as an industry to value innovation andthen penalize innovators.

So we make the following suggestions forfostering venture research into the dark and unknownareas of biotechnology: Foster breakthrough andadventure research. This means doing whatever canbe done cheaply, quickly, and inexpensively withexisting personnel, equipment, and facilities to obtainqualitative results that can be bootstrapped into a realproject if the phenomenon merits attention. The moreproblems you recognize and the more phenomena youexplore, the greater the probability that somethingunexpected will pop up. The key is to generate manymore possibilities than you can possibly investigate,using the inherent limitations of time, money, equip-ment and personnel to determine which are the mostimportant and potentially fruitful. Those problems thatare too difficult, too expensive, too time consuming, orsimply unfruitful need to be put on a back burner orsimply forgotten as quickly as possible. When resultscry out for attention as a result of their intrinsic merit,or when investigators champion them at the expense oftheir own time and effort, then you have something tonot only base a project upon, but also attract investorinterest.

Finally, we suggest that no less than 10% of allR&D time (though significantly less money) bededicated to breakthrough or venture research. In-novation, after all, is the life-blood of any company.Without the generation of novel ideas and phenomena,everyone will be competing to achieve the same goals

in the same way. Success then becomes a mere race.Winning may mean losing if there are no ideas orresources left for the next big discovery. The only wayto have a full drug or diagnostic pipeline is to havemore projects available than can ever be prosecuted.Thus, pioneering or exploratory research offers thepossibility of opening up new territories where there isno competition (and therefore much less infrastructurerequired) for the first one there. So send out the scouts!

ACKNOWLEDGMENTS

To Walter Moos, for inviting this paper, and forhis constant patronage of our unorthodox activities.

REFERENCES

Bates CJ, Walmsley CM, Prentice A, Finch S. 1998. Does vitamin Creduce blood pressure? Results of a large study of people aged 65or older. J Hypertens 16:925–932.

Bendich A, Langseth LJ. 1995. The health effects of vitamin Csupplementation: a review. J Am Coll Nutr 14:124–136.

Beveridge WIB. 1950. The art of scientific investigation. New York:Norton.

Braben D. 1994. To be a scientist. Oxford: Oxford University Press.

Calvin M. 1992. Following the trail of light. a scientific odyssey.Washington, DC: American Chemical Society.

Campbell WC. 1992. The genesis of the antiparasitic drugivermectin. In: Weber RJ, Perkins DN, editors. Inventive minds:creativity in technology. New York: Oxford University Press,p194–216.

Clark JF, Harris GI, Dillon PF. 1991. Multisite saturation usingDANTE and continuous wave. J Mag Res Med 17:274–278.

Comroe JH Jr, Dripps RD. 1981. Scientific basis for the support ofbiomedical science. In: Roberts EB, Levy RI, Finkelstein SN,Moskowitz J, Sondik EJ, editors. Biomedical innovation. Cam-bridge, MA: MIT Press, p 101–122.

Damadian R. 1985. A machine called indomitable. New York: NewYork Times Books.

DeWitt SH, Kiely JS, Stankovic CJ, Schroeder MC, Cody DMR,Pavia MR. 1993. Diversomers: An approach to nonpeptide, non-oligomeric chemical diversity. Proc Nat Acad Sci USA 90:6906–6913.

Dillon PF, Clark JF. 1990. The theory of diazymes and functionalcoupling of pyruvate kinase and creatine kinase. J Theor Biol143:275–284.

Dillon PF, Root-Bernstein RS. 1997. Molecular complementarity, II:Energetic and vectorial basis of biological homeostasis andthermodynamics. J Theor Biol 188:481–493.

Dillon PF, Sears PR. 1998. Capillary electrophoretic measurementsof tissue metabolites. Am J Physiol 274: C840–C845.

Dillon PF, Aksoy MO, Driska S, Murphy RA. 1981. Myosinphosphorylation and the crossbridge cycle in arterial smoothmuscle. Science 211:495–497.

Dillon PF, Weberling MK, Le Tarte SM, Clark JF, Sears PR, Root-Bernstein RS. 1995. Creatine kinase increases the solubility andenzymatic activity of pyruvate kinase by means of diazymaticcoupling. J Biol Phys 21:11–23.

1

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5

7

9

11

13

15

17

19

21

23

25

27

29

31

33

35

37

39

41

43

45

47

49

51

53

55

57

59

61

63

65

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69

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75

77

79

81

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85

87

89

91

93

95

97

99

101

103

105

107

16 R. ROOT-BERNSTEIN AND P.F. DILLION

DDR : 02� 082

Dillon PF, Root-Bernstein RS, Holsworth DD. 1998. Augmentationof angiotensin II activity on rabbit aorta by an antisense peptide.Hypertension 31:854–860.

Dillon PF, Root-Bernstein RS, Sears PR, Olson LK. 2000. Naturalelectrophoresis of norepinephrine-ascorbic acid complex.J Biophys 79:370–376.

Ganong, WF. Review of medical physiology. Stamford: Appleton andLange, 1997.

Grossmann M, Dobrev D, Himmel HM, Ravens U, Kirch W. 2001.Ascorbic acid-induced modulation of venous tone in humans.Hypertension 37:949–954.

Henry DO, Dillon PF. 2000. Method for measuring cellularchemical profiles. U.S. Patent no. 6,113,763.

Igisu H, Fujino T, Dohnao T. 1982. Effect of large dose of ascorbicacid on urinary excretion of catecholamines in man. Int J VitamNutr Res 52:464–469.

Jaffe B. 1957. The story of chemistry. New York: Premier Books.

Kallner A. 1983. Influence of vitamin C status on the urinaryexcretion of catecholamines in stress. Hum Nutr Clin Nutr37:405–411.

Kanigel R. 1986. Apprentice to genius. New York: Macmillan.

Kuhn TS. 1959. The structure of scientific revolution. Chicago:University of Chicago Press.

Mak S, Newton GE. 2001. Vitamin C augments the inotropicresponse to dobutamine in humans with normal left ventricularfunction. Circulation 103:826–830.

Moore WJ. 1972. Physical chemistry, 4th ed. Englewood Cliffs, NJ:Prentice-Hall; p 462.

Mulliken RS. 1989. Life of a scientist. Berlin: Springer-Verlag.

Mullis K. 1998. Dancing naked in the mind field. New York:Pantheon.

Ness AR, Chee D and Elliot P. 1997. Vitamin C and blood pressure:an overview. Human Hypertension 11:343–350.

Newsholme EA, Start C. 1973. Regulation in metabolism.Chichester: John Wiley & Sons; p130.

Nyyssonen K, Parviainen M., Salonen R, Tuomilehtom J, SalonenJT. 1997. Vitamin C deficiency and risk of myocardial infarction:prospective population study of men from eastern Finland. BMJ314:634–638.

Olah G. 2001. A life of magic chemistry. New York: WileyInterscience.

Onsager L. 1934. Deviations from Ohm’s law in weak electrolytes.J Chem Phys 2:599–610.

Pauling L. 1963. The genesis of ideas. Proceedings of the ThirdWorld Congress of Psychiatry, 1961, Vol. 1. Toronto: University ofToronto Press and McGill University Press, p 44–47.

Platt W, Baker RA.1931. The relationship of the scientific ‘hunch’ toresearch. J Chem Ed 8:1969.

Root-Bernstein RS. 1982. The problem of problems. J Theor Biol99:193–201.

Root-Bernstein RS. 1987. Catecholamines bind to enkephalins,morphiceptin, and morphine. Brain Res Bull 18:509–532.

Root-Bernstein RS. 1988. Setting the stage for discovery. SciencesMay/June:26–35.

Root-Bernstein RS. 1989. Discovering, inventing and solvingproblems at the frontiers of science. Cambridge, MA: HarvardUniversity Press.

Root-Bernstein RS. 1990a. Who discovers and invents. Res-TechnolManage 32:43–48.

Root-Bernstein RS. 1990b. Strategies of research. Res-TechnolManage 32:36–42.

Root-Bernstein RS. 1994a. Fostering exploratory research. R&DInnovator 3:1–3.

Root-Bernstein RS. 1994b. The discovery process. ChemtechMay:15–20.

Root-Bernstein RS. 1995. Fostering innovative decision-makingthrough Leadership. R4.

Root-Bernstein RS. 1999a. New agents for treating arthritis. U.S.Patent No. 5,942,491.

Root-Bernstein RS. 1999b. Discovery. In: Encyclopedia of creativity,vol 1. New York: Academic Press; p 559–571.

Root-Bernstein RS. 2002a. The role of enculturation in scientificinnovation: A case study of combinatorial chemistry at Parke-Davis. In: John Hurley, editor. Organizing research. Dublin:Dublin University Press.

Root-Bernstein RS. 2002b. Nepistemology: problem generation andevaluation. In: Shavanina L, editor. The international handbookon innovation, in press.

Root-Bernstein RS, Dillon PF. 1997. Molecular complementarity, I:The molecular complementarity theory of the origin and evolutionof life. J Theor Biol 188: 447–479.

Root-Bernstein RS, Westall FC. 1984a. Fenfluramine binds 5-hydroxytryptophan. Brain Res Bull 12:1722–1727.

Root-Bernstein RS, Westall FC. 1984b. Serotonin binding sites. I.Structures of sites on myelin basic protein, LHRH, MSH, andACTH. Brain Res Bull 12:425–436.

Root-Bernstein RS, Westall FC. 1986. Bovine pineal antireproduc-tive tripeptide binds to luteinizing hormone releasing hormone: amodel for peptide modulation by sequence specific peptideinteraction? Brain Res Bull 17:519–528.

Root-Bernstein RS, Bernstein M, Garnier H. 1995. Correlationsbetween avocations, scientific style, and professional impact ofthirty-eight scientists of the Eiduson study. Creativity Res J 8:115–137.

Sears PR, Dillon PF. 1999. Differential coupling of smooth andskeletal muscle pyruvate kinase to creatine kinase. Biochemistry38:14881–14886.

Szent-Gyorgyi A. 1963. On scientific creativity. Proceedings of theThird World Congress of Psychiatry, 1961, vol. 1. Toronto:University of Toronto Press, McGill University Press; p 47–50.

Szent-Gyorgyi A. 1966. In search of simplicity and generalizations(50 years of poaching in science). In: Kaplan NO, Kennedy EP,editors. Current aspects of biochemical energetics. New York:Academic Press, p 63–76.

Thomson GP. 1961. The inspiration of science. Oxford: OxfordUniversity Press.

Weisbach JA, Moos WH. 1995. Diagnosing the decline of majorpharmaceutical research laboratories: a prescription for drugcompanies. Drug Dev Res 34:243–259.

Wild JJ. 1992. The origin of soft-tissue ultrasonic echoing and itsearly instrumental application to clinical medicine. In: Weber RJ,Perkins DN, editors. Inventive minds: creativity in technology.New York: Oxford University Press; p 115–141.

Wolpert L, Richards A. 1997. Passionate minds. The inner world ofscientists. Oxford: Oxford University Press.

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Q1 1999a or b?See references.1

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