An antisense inhibitor of a protein carrierof low density lipoprotein (LDL) choles-terol has been shown to reduce choles-terol levels in two different mouse modelsof hypercholesterolaemia, according to scientists from Isis Pharmaceuticals(http://www.isip.com) who presentedtheir findings at the 11th InternationalCongress on Cardiovascular Pharmacology[1]. Using antisense oligonucleotides asdrugs to treat cardiovascular disorders isa relatively new but fast moving area andFrank Bennett, Vice President of AntisenseResearch at Isis says that, ‘If further development is successful, the new antisense drug could complement thewidely used statin-based therapies.’

Homing in on an antisense targetDeveloping antisense drugs dependsvery much on identifying an appropriatetarget, and scientists at Isis began bysearching the literature to identify com-ponents of lipoprotein metabolism produced in the liver. ‘The liver is one ofthe key organs that concentratesoligonucleotides; delivery to the site ofproduction of a target is of primary importance, and exploiting the naturalpharmacokinetics of antisense technol-ogy seemed sensible,’ explains Bennett.

Approximately 30 potential targets arebeing investigated, including the ApoB-100 protein, which is the major proteincomponent of the LDL-cholesterol trans-porting molecule (Fig. 1). This protein isexpressed in the liver, and it then travelsto cells where it acts as a carrier for cho-lesterol and other lipid. Heterozygousgenetically modified ApoB-100 knockout

mice have lower cholesterol and appearto be protected from developing diet-induced hypercholesterolemia. ‘This mu-tation is not life-threatening – quite theopposite – so blocking it using antisensetechnology is not likely to affect normalbody functions,’ says Bennett.

Antisense provides new strategyagainst ApoB-100Traditional drug development strategieshave not been used to target ApoB-100 because, unlike enzymes, carrier molecules are difficult to inhibit. The molecules also gain some protection byvirtue of their residence in lipoproteinparticles. ‘Antisense technology, by con-trast, is a successful approach becausethe oligonuceotides that match the sequence of the gene stop translation of

the mRNA in the liver; they do not needto gain access to lipoprotein particles,’says Bennett. When the mouse-specificantisense oligonucleotide, currently des-ignated ISIS147764, binds to the mRNAencoding ApoB-100 protein, it recruitsRNase H, an enzyme that degrades themRNA, preventing any translation.

More thorough toxological studies arestill to be carried out in the next phaseof development, but preliminary studiesshow that ISIS147764 is taken up intomost tissues, and, as expected, accumu-lates in the liver. ‘We have now testedother antisense products in clinical trialsin almost 3000 patients, so we are notanticipating any particular toleranceproblems with the corresponding humanantisense molecule to ApoB-100,’ notesBennett.

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Antisense inhibitor provides newtreatment approach forhypercholesterolaemiaKathryn Senior, freelance writer

Figure 1. The process of low density lipoprotein biosynthesis. ApoB-100 is the coreprotein in LDL particles. By inhibiting ApoB-100 through the application of antisensetechnology using ISIS147764, the production of LDLs is reduced or blocked.Abbreviations: ACAT, Acyl-CoA cholesterol acyltransferase; IDL, intermediate densitylipoprotein; MTP, microsomal transferase protein; TAG, triacylglycerol.

TAGstorage

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Newly synthesizedcholesterol

Cholesterolester

TAG

ApoBsynthesis

Membranesynthesis

First steplipidation

IDLVLDLsecretion

Degradation

ACATMTP

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Promising mouse model resultsEfficacy is the primary objective of cur-rent studies; experiments in mice usingthe high-fat diet model of hypercholes-terolaemia and the genetically modifiedApoE knockout model have shownpromising results. According to Bennett,in both models, mice that were obeseand that had significantly elevated cholesterol levels showed a reduction inoverall cholesterol and LDL cholesterolafter 3–4 weeks of treatment withISIS147764. ‘The response was dose-re-lated, and was accompanied by a moremodest decrease in the plasma levels oftrigycerides,’ he reports.

Statin-based drugs work more quickly,but ISIS147764 could prove suitable forsubgroups of patients who are unre-sponsive to statins, or for whom statintherapy is contra-indicated. ‘From aclinical standpoint, the initial results withISIS147764 are very exciting,’ commentsRichard Honkanen (University of SouthAlabama College of Medicine; http://southmed.usouthal.edu), whose groupis also investigating the potential of anti-sense technology in cardiovascular dis-ease [2]. He points out that ISIS147764represents the first drug designed to directly treat patients that overproduceApoB-100. ‘Statin therapy, which targetsHMG-CoA reductase and lowers LDL primarily by enhancing LDL clearance, isclearly beneficial, but the cardiovascularevent reduction is limited to ~30%,’ heexplains. Because ISIS147764 attacks the

problem from the other end – prevent-ing the formation of LDLs via inhibitingthe expression of ApoB-100 – Honakanenbelieves that antisense strategies couldbring in a new era for the medical man-agement of heart disease. Bennett agrees,adding that a recent publication sug-gesting that many more people mightbenefit from cholesterol-lowering ther-apy than previously thought [2] addsnew impetus to developing antisensedrugs that complement statin therapies.

Improving antisense productformulationThe next phase of development will testthe new drug in another geneticallymodified mouse model, and also in arabbit model of hypercholesterolaemia.‘Antisense therapies are species specific,but the time to identify a rabbit orhuman ApoB-100 antisense molecule islikely to be less than a month,’ confirmsBennett. Several other antisense prod-ucts are already in clinical trials, and thetechnology is such that refinements ofearlier products will aid the developmentof those still at the preclinical stage,speeding up this process considerably.‘First-generation products are given byintravenous injection, and are shorteracting, whereas second-generationproducts have a longer duration of ac-tion and can be given subcutaneously,making it possible for patients to givethemselves the injections,’ says Bennett.The short-term goal is for a preparation

that needs to be injected only onceevery two weeks, but in the longer-term,Isis is also working towards an oral formulation.

Looking ahead to clinical trials‘Our detailed development strategy forISIS147764 has not yet been workedout; we hope that the next phase willshow equally promising results and wewill work towards Phase I clinical trials assoon as possible,’ confirms Bennett.Although the preclinical development ofantisense drugs is much quicker thancandidates identified using other meth-ods, the timing from the start of Phase Itrials will be more comparable, takingabout four years to progress to Phase IIItrials. ‘Phase III trials for an antisenseproduct for hypercholesterolaemia wouldrequire a large number of patients, inmultiple centres, and we will probablytry and set up a partnership with anothercompany to make that possible,’ saysBennett.

Reference1 Bennett, F. (2002) Inhibition of murine APOB

B100 by antisense oligonucleotides in amurine model of hyperlipidaemia.Cardiovasc. Drugs Ther. 16 (Suppl.) (in press)

2 Golden, T. et al. (2002) Use of antisenseoligonucleotides: advantages, controls, andcardiovascular tissue. Microcirculation 9, 51–64

3 Heart Protection Study Collaborative Group(2002) MRC/BHF Heart Protection Study ofcholesterol lowering with simvastatin in20536 high-risk individuals: a randomisedplacebo-controlled trial. Lancet 360, 7–22

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The HIV virus could assist in its owndownfall if two new gene therapy strat-egies succeed. Both approaches are dueto enter Phase I safety trials in late 2002,

using new vectors and protocols thattheir proponents hope will overcomemany of the obstacles of the past 10years. Researchers led by Carl June, a

molecular immunologist at the AbramsonFamily Cancer Research Institute (http://www.abramsoninstitute.org), reportedtheir progress at the 7th European

Gene therapy progress for HIVJulie Clayton, freelance writer