Editorial

10.1517/13543784.14.2.111 © 2005 Ashley Publications Ltd ISSN 1354-3784 111

Ashley Publicationswww.ashley-pub.com

Monthly focus: Anti-infectives

Making dollars and sense of antisensePhillip WigginsPharmsouth, 604 North Pontiac Avenue Dothan, AL 36303, USA

Keywords: antisense, business collaborations, codons, drug eluting stents, dsRNA, microparticle technology, mRNA, oligonucleotides, restenosis, ribosome, ribozymes, RNase P, synthetic polymers, triple-helix oligonucleotides, viral disease

Expert Opin. Investig. Drugs (2005) 14(2):111–115

1. Introduction: the end of the beginning

The subject of antisense is too broad for a single article to cover fully. The purposeof this editorial is two fold. First, a focused analysis will be covered on two smallbiotech companies, each with different approaches or platforms for antisense tech-nology. Second, a focus on disease states will primarily cover two of greatimportance – novel antisense drugs for viral disease and, within the vast cardiovas-cular market, restenosis. One of the companies has been termed to ‘lead the indus-try’ in the development of technologies to exploit RNA. This company is bestknown for its pioneering work in the development and understanding of antisensetechnology including biology, chemistries, formulations and RNase H, RNAi,microRNA and splicing mechanisms. Most importantly, this company producedthe first approved antisense drug in 1998, formirvirsen sodium (Vitravene®), afirst-generation injectable drug for the treatment of human cytomegalovirus(HCMV). The company’s Chairman and chief executive officer (CEO), Stanley TCrooke, stated in his recent book ‘Antisense Drug Technology-Principles, Strategies,and Applications' that it is important to remember that we are less than a decadeinto the aggressive creation and evaluation of antisense technology. Also, we areattempting to create an entirely new branch of pharmacology: new chemical class,oligonucleotides; a new receptor, RNAi; a new drug receptor binding motif,hybridisation; and new post receptor binding mechanisms.

In addition, Crooke states in the book that there are still more questions thananswers. Probably and arguably we are at the end of the beginning of thistechnology. There is a great deal more to do before we understand the true valueand limits of antisense, but we are encouraged by the progress to date and lookforward to the challenges ahead [1].

Obviously, this company is ISIS Pharmaceuticals, Inc., (CA, USA).The other company’s antisense platforms to be discussed are quite different

from the ISIS approaches, plus they lack any FDA-approved antisense drug thera-pies at this time. This second company is not as well recognised, or well financed,as ISIS but their technology platforms are very intriguing and may contain thescience necessary for more efficacious, safe and perhaps a more cost effectiveapproach for our healthcare systems.

Over the past 24 years, this smaller biotech organisation has developed a gene-targeting technology, generally called antisense, which uses the genetic code asthe target for drug development. This biotech firm is the only corporation withproven, patented, third-generation antisense drugs in late stage clinicaldevelopment to treat life-threatening diseases. Chairman and CEO Denis RBurger stated in 2001 to his shareholders that his company’s NeuGene® technol-ogy uses the genomic data as a simple source code, a library of gene targets, forfuture drug development.

1. Introduction: the end of the

beginning

2. Importance of business

agreements with Big Pharma

3. Three types of oligonucleotides

4. Restenosis

5. Viral infections

6. Expert opinion and conclusion

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Making dollars and sense of antisense

112 Expert Opin. Investig. Drugs (2005) 14(2)

In the genomic arena, the race to sequence the humangenome is over, and the race to define this extensive array ofgenetic data has only just begun. Burger went on to say that,to develop drugs that influence gene function, one must firstdiscover the function for each of tens of thousands of humangenes. He said it was no surprise to them that NeuGene anti-sense agents turn out to be the best way to validate gene func-tion. “This puts us in a very advantageous position”according to Burger. “If one inhibits a disease-causing genewith our NeuGene antisense technology in order to establishfunction, then the NeuGene agent becomes the drug to treatthe disease in question”. He went on to say that other drugdevelopers must then go back and spend 3 – 5 years inresearch and development to try to come up with a drug toblock the validated gene target. His company apparentlyskips those 3 – 5 years because the validated agent is the drug!

As other biotech firms were rushing new second-generation antisense compounds into clinical trials, 17 yearsago Burger’s company recognised that these early chemistriesproved problematic and they were able to create a potentialstunning, new antisense solution. Burger’s company is AVIBiopharma (OR, USA).

As a result, it is the only biotech company in clinical trialswith third-generation antisense drugs. Using a synthetic,neutral chemical backbone, NeuGene drugs have alreadyproven to be substantially target specific, potent and far lesstoxic than their predecessors. Burger and his colleagues havewritten many important papers in recent years [2-4], as has hiscompetitor Crooke [5].

2. Importance of business agreements with Big Pharma

AVI Biopharma lost a crucial business partner, Medtronic(MN, USA), for its antisense drugs for restenosis on 9 May2004, as Abbott Laboratories (IL, USA) announced that ithad entered into a worldwide agreement with Medtronic tomarket Medtronic’s proprietary next-generation stent deliv-ery systems, as well as its current over-the-wire stent deliv-ery platform and its rapid exchange stent delivery system.Through this alliance, and the recently completed acquisi-tion of Biocompatibilities, Inc. polymer-coated coronarystent line, Abbott is now well positioned to compete fully inthe US$2 billion coronary stent market.

Under a co-exclusive agreement, Abbott will simultane-ously out-license to Medtronic its lead drug-coated stentcompound, an internally developed rapamycin analogue,and its licensed proprietary phosphorocholine (PC) coatingto develop its own drug-coated stent using its rapamycinanalogue, ABT-578, and licensed PC coating.

It is worth noting at this point that a vascular medicinefranchise may include more than just the term ‘drug-coatedstents’. It may include medical devices and therapeutics; forexample, coronary, peripheral and drug-coated stents, vesselclosure, embolic capture and occlusion catheter technologies.

3. Three types of oligonucleotides

The basis of antisense is that many diseases are associatedwith either inappropriate or inadequate production of pro-teins. Antisense technology is the process of creating syn-thetic DNA or RNA to prevent these problems by blockingthe product of these faulty proteins, which generally entailstranscription of one strand of the gene’s duplex DNA byRNA polymerase to generate the complementary mRNA.The mRNA is designated the ‘sense’ strand because it codesfor the amino acid sequence of the protein product of a gene.

There are basically three different types of oligonucleotides;however, there are others, for example, RNA interference(RNAi) refers to the introduction of homologous double-stranded RNA (dsRNA), which specifically targets a geneproduct resulting in null or hypomorphic phenotypes.

Antisense oligodeoxynucleotides (ODNs) are synthetic poly-mers and the monomers are deoxynucleotides like those in DNA.Their sequence (3´→ 5´) is antisense; that is, complementary tothe sense sequence of a molecule of mRNA (Figure 1).

The first types of oligonucleotides are the first-generationantisense compounds. These are short, gene specificsequences of nucleic acids. These classical antisense inhibitorstarget specific strands of RNA within the cell and hybridisewith them to form a duplex, thus preventing the RNA fromfunctioning normally and preventing the production of theprotein in question.

Some first-generation compounds have an ability to bedigested by an enzyme called RNase H. The RNase Hdigests the RNA that the antisense drug has matched with,leaving the original antisense drug intact. This allows the

Proteins

TranslationRibosome

Normal Notranslation

Noprotein

MessengerRNA

Withantisense

Antisenseoligonucleotide

DNA

Nucleus

Pro-messengerRNA

Figure 1. First-generation antisense technology. Reproduced with permission from Bob Crimi.

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Expert Opin. Investig. Drugs (2005) 14(2) 113

antisense to target and bind with the other RNA strand.This process can be repeated over and over, allowing oneoligonucleotide to hybridise with and destroy manystrands of faulty RNA. This is the mechanism of action ofISIS’s formivirsen sodium. An important point must bemade here. The package insert of formirvirsen sodium, aphosphorothioate oligonucleotide, states on pages 2 – 3 ofthe “Draft US PI 26 August 1998” under the Resistancesection the following: “through persistent selection pres-sure in vitro it was possible to isolate a clone of HCMVthat was 10-fold less sensitive to inhibition of replicationthan the parent strain”. The molecular basis for the resist-ance has not been elucidated. It is possible that resistantstrains may occur in clinical use.

The second types of oligonucleotides are the second-generation antisense compounds, which are somewhat dif-ferent to the first generation. The difference is that, insteadof using RNase H to eradicate the RNA strand, these oligo-nucleotides use a ribozyme (an RNA molecule that catalysesbiochemical reactions) to destroy the targeted RNA. As withthe first-generation compounds, this allows a single oligonu-cleotide to target and bind with numerous strands of RNA.

One of the newer areas of dealing with this type ofoligonucleotide is external guide sequences (EGSs) [7,8].These EGSs actually recruit a ribozyme called RNase P todigest the RNA that the antisense has combined. RNase Pis an often-encountered enzyme whose primary cellularfunction is processing the 5´ leader sequence from pre-tRNA transcripts [9]. The enzyme itself does not need to bemodified to carry out reactions in trans, as this is the onlytrue ribozyme known to bind and cleave free substrate innature [10,11]. ISIS currently has several second-generationantisense drugs in clinical trials.

The third type of oligonucleotide is triple-helix DNA.The first two types of oligonucleotides target specificstrands of RNA to prevent the production offaulty proteins. Triple-helix DNA targets the double-helixDNA strand itself. These triple-helix oligonucleotides aredesigned to bind with a specific section of the DNA,preventing its transcription into RNA. This represents thethird-generation antisense compounds made by AVIBiopharma. Triple-helix oligonucleotides truly servethe purpose as classical antisense. These oligonucleotidesbasically just use a different approach by blocking the

Figure 2. The protein production process.Modified with permission from [101].

mRNA

NeuGene

DNA

DNA unzips to transcribe amessage (mRNA)

NeuGene Antisense locks ontomRNA, blocking ribosomeassembly and preventingprotein synthesis

Ribosome componentsnormally coalesce aroundmRNA, resulting in proteinproduction

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114 Expert Opin. Investig. Drugs (2005) 14(2)

production of proteins even earlier in the proteinproduction process (Figure 2).

4. Restenosis

Restenosis has remained, until now, a major limitation tothe clinical usefulness of percutaneous transluminal angio-plasty (PTA), and poor long-term results, especially after thetreatment of longer lesions in the femoropopliteal region,have been reported [12-14]. The two primary phenomena thatcontribute to restenosis after successful angioplasty arechronic vessel constriction (remodelling) and neointimalhyperplasia (excessive formation of tissue) by means of cellproliferation [15]. AVI Biopharma stated, in their 4 Novem-ber 2004 conference call, that based on excellent clinicalresults for their Resten-NG® trials, they intend to commer-cialise NeuGene-based products for the treatment of cardio-vascular disease through a combination of internaldevelopment programmes and partnering activities usingtwo distinct approaches.

First, delivering their drug locally on a stent platform andsecond, delivering their drug systemically with their micro-particle delivery system (company secret) or Resten-MP® foruse in conjunction with other drug eluding stents, namelyCypher® and Taxus®.

For delivering Resten-NG via stents, AVI intends to initiatePhase III clinical trials with their drug-eluting stents (DES) inEurope, leading to certificate of exportability (CE) markapproval within 18 months if clinically successful.

To accomplish this, AVI has recruited employees and con-sultants with extensive experience in devices used in interven-tional cardiology with direct experience in clinical trials andthe approval process on both European and American conti-nents. In this programme, there are four components thatmust fit together like pieces of a jigsaw puzzle: the stent, theballoon delivery system, the drug and finally the coatingmaterial to bind and release the drug from the stent.

In the short period of time since the unwinding of theMedtronic agreement, they have successfully assembled thesefour components into a cohesive system that AVI believes willbe the next generation DES.

The most important component in the system, of course, isthe drug and Resten-NG has advantageous characteristicswhen compared with the drug component in Cypher orTaxus DES.

The coating that AVI is qualifying is the one that is usedby Johnson & Johnson on its approved Cypher DES. Usingthis coating, AVI has been able to exceed both the loadingand elution characteristics required for their unique drugmechanism. With the addition of the new staff members,AVI has developed their own stent that matches withResten-NG and its coating. The final component is theirballoon delivery system, which is currently being qualifiedwith the sterilisation techniques suited for Resten-NG.(For strategic and competitive reasons, AVI is not providing

further detail on the four components that make uptheir DES.)

AVI continues to make progress on the Resten-NG drugdelivered intravenously using their patented microparticledelivery technology. In preclinical studies, this was as effectiveas Resten-NG delivered by catheters or stents in preventingcardiovascular restenosis and has many advantages.

AVI has planned to initiate more extensive Phase II clinicalstudies with Resten-MP at additional sites in the US in com-bination with Taxus DES and in Europe with Bayer stents(early 2005) to compliment ongoing programmes.

5. Viral infections

Viruses are some of the most insidious creations in nature.They travel equipped with just their genetic material,packed as tightly inside a crystalline case of protein, thenlatch onto cells, insert their genes, and co-opt the cell’s cop-ying and protein machinery to make billions of copies ofthemselves. Once formed, the new viruses move gradually tothe cell surface like Royal Marines, tuck away inside tinybubbles of cell membrane and either drift, or they continuereproducing until the cell finally explodes. In any case, theygo on to attach and destroy other cells, resulting in diseasesfrom AIDS and hepatitis, to the common cold. Differentviruses cause different diseases in part because each virusenters a cell by first attaching to a specific suction-cuplikereceptor on its surface. Liver cells display one kindof receptor on its surface used by one family of viruses,whereas lung cells display another receptor used by adifferent viral family.

AVI Biopharma has an extensive infectious diseaseprogramme, as does ISIS Pharmaceuticals. Based onpreclinical studies, AVI plans to file an investigationalnew drug (IND) application to initiate human clinical trialswith their NeuGene drug for hepatitis C in 2005. Theyestimate the current and potential market for hepatitis C tobe > US$10 billion.

Burger believes that AVI’s viral disease programme andoverall approach has been misinterpreted. AVI is extensivelyinvestigating their NeuGene technology against a largenumber of RNA viruses through multiple collaborations,with the objective of commercialising a product for a largemarket opportunity. Most viral diseases have few or no treat-ment options, and very limited, if any, animal models forinvestigative work in product development.

AVI maintains that if their antisense agents can be foundefficacious against RNA viruses in the hands of independentcollaborators, they have a high likelihood of being efficaciousagainst RNA viral diseases that do not have good animalmodels like hepatitis C virus (HCV).

There are ∼ 15 families of RNA viruses that cause humandisease. With a large group of outside collaborators, AVI hasalready demonstrated efficacy against viruses in 11 ofthese 15 families.

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115 Expert Opin. Investig. Drugs (2005) 14(2)

The simplest viruses, from a molecular standpoint, are inthe family containing West Nile virus, yellow fever virus,dengue virus, tick-born encephalitis and HCV.

These viruses are nearly identical in function for themRNA of mammalian cells where NeuGene agents have sofar been effective in previous preclinical and clinical studies.Studies with severe acute respiratory syndrome (SARS) anddengue virus taught AVI the subtleties of targeting RNAviruses. Studies with West Nile taught them dosing, pharma-cokinetics, delivery to the CNS and confirmed their low tox-icity profile. Studies in Ebola taught AVI dosing, routes ofadministration and combination approaches. Studies in influ-enza, Norwalk, Calcivirus and several other virusesconfirmed these findings.

Overall, these diverse studies have advanced AVI’s HCVprogramme, so that they can now move quickly and confi-dently to efficacy studies cutting ≥ 1 year from the clinicaldevelopment cycle.

6. Expert opinion and conclusion

ISIS Pharmaceuticals has the only FDA-approved antisense drug.They have more capital to fund and expand their projects, andmore collaborations with larger companies than AVI Biopharma– the most recent of which is a second-generation antisense drugfor cancer with a major partner in Eli Lilly & Company.

AVI has a proven antisense technology in anenvironment where the very mention of antisense technologycauses concerns and headshaking due to a number of spectac-ular Phase III failures by others, albeit with quite differentsecond-generation technology. However, in today’s environ-ment, with large companies such as Merck and Pfizer losingbillions in market capitalisation with the now controversial,albeit amidst some confusing data, concerning cyclooxygen-ase 2 (COX-2) inhibitors, Big Pharma may want to take amore in-depth look at these smaller biotech companies forcollaborations that could enhance their bottom line financialsheets and also provide desperately needed cash for small tomedium biotech companies that have recently, and through-out the history of biotech, provided companies such as Lilly,Roche, Novartis, Johnson & Johnson, Abbott and manyother drug giants with big time profit makers for these com-panies that seemed to be now turning into giant marketingand distribution machines. Now is the time to take anotherlong, careful look at ‘small capital’ biotech that has proven inthe past to bring in big winners for the astute managers ofBig Pharma.

Hopefully, for the patients currently with little or no hope,both of these small biotech companies discussed in this edito-rial will produce better treatment options where onlypalliative, toxic or no effective treatments existfor the moment.

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Website101. http://www.avibio.com/home.html

AVI Biopharma website.

AffiliationPhillip WigginsPharmsouth, 604 North Pontiac Avenue, Dothan, AL 36303, USA E-mail: ww1922@graceba.net

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