that the drug was having a beneficial effect on chronic pain. Meanwhile, noeffects of the drug was seen in studies onnormal rats, demonstrating that thedrug had no effect on acute pain.

‘The complete surprise was that wecould separate the effects of acute andchronic pain. This is very exciting be-cause people taking the drug for chronicpain will still be able to experience nor-mal stimuli from the environment,’ saysBrann, suggesting that different mus-carinic receptors are involved in differenttypes of pain.

Potential for the futureACADIA think that their drug candidateswill be helpful for chronic pain associ-ated with diabetic neuropathy, herpes lesions, metastatic cancer and painassociated with autoimmune diseaseslike Guillain–Barré syndrome. They couldalso have a role in arthritic and lower-back pain.

Research is now aimed at optimizingthe candidate compounds to achievegreater potency, better in vivo stabilityand a longer duration of action. ACADIAhopes to start toxicology and safetystudies in animals within a year and thecompany is aware that it is likely to be 18months or more before they can starthuman trials

‘The ability to develop small drugsthat are highly selective for M1 receptorsis itself a significant achievement,‘ commented Alan Levey, Professor ofNeurology at Emory University School ofMedicine (Atlanta, GA, USA). ‘This hasbeen a goal for many pharmaceuticalcompanies, given the potential use of anM1 receptor-specific drug for Alzheimer’sdisease, schizophrenia, pain and othercommon problems.’ He continued,‘ACADIA used a different approach tofind small molecules that activate the re-ceptor subtypes. The finding that drugscan be discovered using this strategy sets

an important precedent that could leadto the more rapid discovery of manydrug candidates that bind selectively toa variety of receptors, and that could be applied to many common medical conditions.’

References1 Bonner, T.I. et al. (1987) Identification of a

family of muscarinic acetylcholine receptorgenes. Science 237, 527–532

2 Gomeza, J. et al. (1999) Pronouncedpharmacologic deficits in M2 muscarinicacetylcholine receptor knockout mice. Proc.Natl. Acad. Sci. U. S. A. 96, 1692–1697

3 Matsui, M. et al. (2000) Multiple functionaldefects in peripheral autonomic organs inmice lacking the muscarinic acetylcholinereceptor gene for the M3 subtype. Proc. Natl.Acad. Sci. U. S. A. 97, 9579–9584

4 Kim, S.H. and Chung, J.M. (1992) Anexperimental model for peripheralneuropathy produced by segmental spinalnerve ligation in the rat. Pain 50, 355–363

5 Bennett, G.J. and Xie, Y.K. (1988) Aperipheral mononeuropathy in rat thatproduces disorders of pain sensation likethose seen in man. Pain 33, 87–107

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Plasmin, the activated form of plasmino-gen (a protein found in normal humanplasma), has been previously overlookedas a therapeutic for conditions caused byblood clots. However, a recent preclini-cal study strongly suggests that plasminis an excellent thrombolytic agent, witha striking safety profile – better than licensed agents currently in use in theclinic1.

Lead author, Victor Marder (Universityof California, Los Angeles, CA, USA), to-gether with colleagues from the BayerCorporation (Berkeley, CA, USA andRaleigh, NC, USA), used rabbit models oflocal thrombolysis and fibrinolytic haem-orrhage to compare plasmin with tissueplasminogen activator (TPA). TPA is the

current standard ‘clot-busting’ treatmentfor the management of patients with either an acute myocardial infarction,catheter or shunt, or peripheral arterialocclusion. Although effective, TPA cancause bleeding at remote sites because itspreads systemically, even after local infusion. ‘All current thrombolytics areplasminogen activators, of which TPA isthe prototype. All have the same prob-lem with bleeding, especially intracranialhaemorrhage, which can be fatal,’ comments Marder.

Giving plasmin a second chanceDuring early investigations, plasmin wasrecognized as a direct fibrinolytic enzymewith a potential clinical application.

Desire Collen (University of Leuven,Leuven, Belgium), who is actively involvedin the development of recombinant plasmin and derivatives as therapeuticagents, points out in an editorial2 that,‘Intravenous plasmin for thrombolytictherapy was investigated in several pilotstudies in humans in the 1950s and1960s (Ref. 3).’ These showed that plas-min was well tolerated, but the studieswere terminated, probably because of a lack of understanding of the kinetics of plasmin inhibition by anti-plasmin,and the unavailability at that time of local catheter delivery. Subsequently, the plasminogen activators streptokinaseand urokinase were discovered; both caninduce plasmin generation locally within

Thrombolysis without bleedingKathryn Senior, Freelance writer

a thrombus and plasmin was side-steppedin favour of these highly effective en-zymes. ‘However, in light of the prob-lems with plasminogen activators, wedecided to look again at plasmin. Thepresence of an ‘anti-plasmin’ in the bloodcould actually be a useful mechanism toprovide safety from haemorrhage, ratherthan a problem to surmount,’ saysMarder. ‘The working hypothesis is thatpurified plasmin, totally devoid of plasminogen activator, binds to and dissolves clots when delivered locally.When excess plasmin escapes into thecirculation, it is rapidly deactivated byanti-plasmin, thereby avoiding risk ofbleeding at vascular injury sites else-where in the body,’ he explains (Fig. 1).

Supporting dataThis study assessed purified plasmin forthrombolytic efficiency and for the riskof bleeding using two standard animalmodels. In a rabbit model of thrombosis,plasmin and TPA were applied as an intra-arterial infusion into an experimentallythrombosed abdominal aorta. Underconditions of unimpeded blood flow, anequivalent level of clot dissolution andflow restoration was achieved in thethrombosed vessel with 4 mg kg−1 ofplasmin and 2 mg kg−1 of TPA. When theblood flow through the aorta of the rab-bit was restricted to reduce local plas-minogen supply but to allow plasminand TPA infusion to still take place, plas-min gave better results than TPA. Anotherrabbit model, the ear-puncture rebleedmodel, was used to assess the safety ofplasmin versus TPA. In this model, a sin-gle 3.5 mm full-thickness puncture ismade at sites in the rabbit’s ear 30 or 10min before either plasmin or TPA is in-fused into the external jugular vein. Whensimilar doses of the two compounds wereused (between 2 and 4 mg kg−1), TPA in-duced rebleeding in a dose-dependentmanner from previous puncture sites innine of ten animals. By contrast, none ofthe animals given plasmin experiencedrebleeding1.

Collen agrees that ‘the data seem con-vincing and the extrapolations reason-able.’ Bayer is also encouraged by the results obtained in the collaborativestudy. ‘These preclinical animal studiesdemonstrate that plasmin represents asafe and effective thrombolytic agentthat does not induce complications suchas haemorrhage or antibody response,’says Steve Petteway, Head of Researchand Technology at Bayer BiologicalProducts (Research Triangle Park, NC,USA). However, Collen also points outthat the plasmin production and purifi-cation method employed in the studyseems too complicated for large-scaleuse and warns that it will be necessary tofind an effective expression system to enable this large and complex moleculeto be produced in large amounts by

recombinant DNA technology. ‘Intactplasminogen cannot readily be expressedin an active form in common eukaryoticexpression systems, because of the presence of intracellular plasminogen activators,’ he says. Efforts are ongoingto produce shorter-chain derivatives thatretain fibrinolytic activity but which canbe expressed more easily. ‘We have re-cently expressed a recombinant variantof human microplasminogen in Pichiapastoris (a yeast used for large-scale recombinant-protein production) andconverted it with high yield to fully activated and stabilized microplasmin (a low molecular-weight derivative of plasmin),’ he reports4. However, Bayer has every confidence in producingenough plasmin to meet all future clinical need.

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Figure 1. Schematic representation of local and systemic effects of (a) tissue plasminogenactivator (TPA) and (b) plasmin. Local delivery of both TPA and plasmin can inducethrombolysis with local delivery. TPA activates fibrin-bound plasminogen to plasmin.Plasmin has an enzymatic effect after binding to fibrin. However, in the systemiccirculation the results are different. TPA exceeds the neutralizing capacity of plasminogenactivator inhibitor-1 (PAI-1) and binds to fibrin in haemostatic plugs. Plasmin formationresults in fibrinolytic bleeding. By contrast, plasmin is neutralized by anti-plasmin anddoes not bind to fibrin in haemostatic plugs, therefore, maintaining haemostasis. Figure reproduced, with permission, from Ref. 1.

Catheter

Local catheter delivery Systemic circulation

Haemostaticplug

Thrombus

Catheter Plasmin

Local catheter delivery Systemic circulation

Haemostaticplug

Thrombus

Anti-plasmin

(a)

(b)

PAI-1TPA

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Future studies‘The follow-up studies we are engagedin at the moment are very exciting, inthat the promise of the first study is stillsupported. We have done a dose-rang-ing study in the rabbit and are currentlyfocusing on correlations of laboratoryobservations of blood-clotting param-eters as pertains to bleeding phenomena,’he says. The group also plans to studythe interaction of aspirin and heparin

with plasmin, as a prelude to eventualclinical trials. ‘Hopefully, Phase I studieswill be initiated by Bayer in 2002 foreventual use in thrombolysis by regionalinfusions by catheter for peripheral arterial or graft occlusion and catheterocclusions,’ concludes Marder.

References1 Marder, V.J. et al. (2001) Plasmin induces

local thrombolysis without causing

haemorrhage: a comparison with tissueplasminogen activator in the rabbit. Thromb.Haemost. 86, 739–745

2 Collen, D. (2001) Revival of plasmin as atherapeutic agent? Thromb. Haemost. 86,731–732

3 Clifton, E.E. (1957) The use of plasmin inhumans. Ann. New York Acad. Sci. 68,209–229

4 Collen, D. [Thromb-X, NV] (2000)Recombinant human plasminogen andplasmin: Methods for high yield production,stabilization and use for treatment.UK0031196.9

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In the 1st January issue of Drug Discovery Today…

Editorials Combinatorial chemistry: 20 years on...by Árpád Furka

Why is the global CNS pharmaceutical market so under-penetrated?by William M. Pardridge

Plus, all the regular News and Reviews

Update news and views• James Gimzewski discusses the potential of nanobiotechnology• Embryonic stem-cell research, gene patents and decision analysis approaches are the hot

topics in the Discussion Forum• Report from the IBC’s Early ADME/Tox Studies and In Silico Screening conference• Book review of Combinatorial Library Design and EvaluationUp-to-date News, News in brief and People

Reviews Assessing cognitive function in clinical trials: latest developments and future directionsby Keith Wesnes

The past, present and future of HIV vaccine development: a critical review by Alexandra Bojak, Ludwig Deml and Ralf Wagner

Monitor Molecules and drug delivery

Combinatorial Chemistry Reviews –Special

Recent developments in combinatorial organic synthesisby A. Ganesan

Work-up strategies for high-throughput solution synthesisby David Cork and Nick Hird

Virtual screening and fast automated docking methodsby Gisbert Schneider and Hans-Joachim Böhm

Enzymatically cleavable linker groups in polymer supported synthesisby Reinhard Reents, Duraiswamy A. Jeyaraj and Herbert Waldmann