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Basic Requirements for Investigational New Drug and New Drug Application Approval for an Antioxidant Compound in Cardiovascular Disease Joseph L. Witztum, MD A cceptance of the use of antioxidants by the pub- lic, together with the conviction that antioxidants will prevent coronary artery disease, is widespread. Nevertheless, prospective interventional trials to test this hypothesis are lacking. How should one go about designing clinical trials to test the “antioxidant hypothesis,” and what one would use as criteria to test effectiveness of therapy? The original interest in oxidized low-density lipopro- tein (LDL) was that native LDL is not taken up by macrophages fast enough to cause foam-cell forma- tion, but oxidized LDL is. The “oxidation hypothesis of atherosclerosis” states that the oxidative modifica- tion of LDL or other lipoproteins is an important, possibly even obligatory, event in atherogenesis. The corresponding clinical hypothesis is that inhibition of LDL oxidation should retard or prevent atherosclero- sis and its clinical sequelae. 1,2 There is now much evidence that LDL oxidation does occur in vivo in humans and in various experi- mental animal models, and there are now data from several animal models that antioxidants inhibit athero- sclerosis, even when plasma cholesterol levels are unchanged. What makes clinical studies difficult is that most of us believe that LDL is not oxidized in the plasma, but only after it enters the arterial intima, where under pro-oxidant conditions, it is converted to an early stage of oxidation to become “minimally modified LDL.” Further oxidation produces more ex- tensively modified LDL. Many different factors that are difficult to measure can theoretically contribute to prooxidant activity in the artery wall. A second point is that although we were originally interested in oxidized LDL because it was taken up by macrophages, we now know that there are many bio- logic effects of both the earliest and advanced stages of oxidized LDL, including effects on endothelial cell function, vasomotor properties, monocyte chemoat- traction and differentiation, cytokine generation, and cell proliferation. 3 All these effects of oxidized LDL have been described in in vitro models, but we have no idea which of them are important in vivo for atherogenesis. A third important factor is what we might call the “threshold effect.” We know that as LDL becomes progressively oxidized, its biologic properties are likely to change. Only when the LDL has become heavily oxidized is its uptake by macrophages en- hanced. Thus, whereas an antioxidant might prevent extreme oxidization and thus retard uptake by macro- phages, the LDL could still be sufficiently oxidized to have other atherogenic properties. For example, in our laboratory, Fruebis and colleagues used probucol and a probucol analog (also an antioxidant) to treat Wa- tanabe heritable hyperlipdemic rabbits. They showed that the lag time for generation of conjugated dienes from LDL isolated from probucol-treated rabbits was increased more than 8-fold, but for the probucol analog only 4-fold. The probucol-treated animals were significantly protected against atherosclerosis, whereas the analog-treated rabbits were not. 4 This suggests that there might be a threshold level of anti- oxidant protection needed to inhibit atherogenesis in response to a given oxidative stress such as hypercho- lesterolemia. It should be noted that the increase in lag time for LDL isolated from subjects supplemented with very large doses of vitamin E is only 30 –50%. 1 Further- more, there is no assurance that the ex vivo measure- ment of lag time in isolated LDL accurately or even adequately reflects the degree of oxidation occurring in the artery wall. In fact, studies from several labo- ratories, including our own, indicate a lack of associ- ation between increased lag time and protection against progression of atherosclerosis, at least under certain conditions. Measures reflecting in vivo levels of oxidation in the artery wall are urgently needed. My fourth and final point is that oxidized LDL, and the molecules it generates, may affect cardiovascular events not only by promoting atherogenesis but also by contributing to plaque instability. 5 There is much evidence that macrophage foam-cells accumu- late in the portion of the plaque that later fissures. It may be that oxidized LDL or its products affect plaque stability by stimulating the release from arterial cells (such as macrophages) of proteolytic enzymes, which could disintegrate both the matrix and the overlying endothelium. The products might also stimulate the release of tissue factor, enhancing thrombotic tenden- cies. Which of these effects are important and relevant to humans is difficult to predict. Given all this complexity, how will we go about deciding if antioxidants work? If we look at the his- tory of cholesterol-lowering drugs and coronary artery disease we find that the first drugs tested were rela- From the Department of Medicine, University of California, San Diego, California, USA. Address for reprints: Carlos A. Dujovne, MD, Kansas Foundation for Clinical Pharmacology, 10550 Quivira Road, Suite 240, Over- land Park, Kansas 66215, USA. 50F ©1998 by Excerpta Medica, Inc. 0002-9149/98/$19.00 All rights reserved. PII S0002-9149(98)00262-8

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Page 1: Basic Requirements for Investigational New Drug and New Drug Application Approval for an Antioxidant Compound in Cardiovascular Disease

Basic Requirements for InvestigationalNew Drug and New Drug Application

Approval for an Antioxidant Compound inCardiovascular Disease

Joseph L. Witztum, MD

Acceptance of the use of antioxidants by the pub-lic, together with the conviction that antioxidants

will prevent coronary artery disease, is widespread.Nevertheless, prospective interventional trials to testthis hypothesis are lacking.

How should one go about designing clinical trialsto test the “antioxidant hypothesis,” and what onewould use as criteria to test effectiveness of therapy?The original interest in oxidized low-density lipopro-tein (LDL) was that native LDL is not taken up bymacrophages fast enough to cause foam-cell forma-tion, but oxidized LDL is. The “oxidation hypothesisof atherosclerosis” states that the oxidative modifica-tion of LDL or other lipoproteins is an important,possibly even obligatory, event in atherogenesis. Thecorresponding clinical hypothesis is that inhibition ofLDL oxidation should retard or prevent atherosclero-sis and its clinical sequelae.1,2

There is now much evidence that LDL oxidationdoes occur in vivo in humans and in various experi-mental animal models, and there are now data fromseveral animal models that antioxidants inhibit athero-sclerosis, even when plasma cholesterol levels areunchanged. What makes clinical studies difficult isthat most of us believe that LDL is not oxidized in theplasma, but only after it enters the arterial intima,where under pro-oxidant conditions, it is converted toan early stage of oxidation to become “minimallymodified LDL.” Further oxidation produces more ex-tensively modified LDL. Many different factors thatare difficult to measure can theoretically contribute toprooxidant activity in the artery wall.

A second point is that although we were originallyinterested in oxidized LDL because it was taken up bymacrophages, we now know that there are many bio-logic effects of both the earliest and advanced stagesof oxidized LDL, including effects on endothelial cellfunction, vasomotor properties, monocyte chemoat-traction and differentiation, cytokine generation, andcell proliferation.3 All these effects of oxidized LDLhave been described in in vitro models, but we haveno idea which of them are important in vivo foratherogenesis.

A third important factor is what we might call the“threshold effect.” We know that as LDL becomes

progressively oxidized, its biologic properties arelikely to change. Only when the LDL has becomeheavily oxidized is its uptake by macrophages en-hanced. Thus, whereas an antioxidant might preventextreme oxidization and thus retard uptake by macro-phages, the LDL could still be sufficiently oxidized tohave other atherogenic properties. For example, in ourlaboratory, Fruebis and colleagues used probucol anda probucol analog (also an antioxidant) to treat Wa-tanabe heritable hyperlipdemic rabbits. They showedthat the lag time for generation of conjugated dienesfrom LDL isolated from probucol-treated rabbits wasincreased more than 8-fold, but for the probucolanalog only 4-fold. The probucol-treated animalswere significantly protected against atherosclerosis,whereas the analog-treated rabbits were not.4 Thissuggests that there might be a threshold level of anti-oxidant protection needed to inhibit atherogenesis inresponse to a given oxidative stress such as hypercho-lesterolemia.

It should be noted that the increase in lag time forLDL isolated from subjects supplemented with verylarge doses of vitamin E is only 30–50%.1 Further-more, there is no assurance that the ex vivo measure-ment of lag time in isolated LDL accurately or evenadequately reflects the degree of oxidation occurringin the artery wall. In fact, studies from several labo-ratories, including our own, indicate a lack of associ-ation between increased lag time and protectionagainst progression of atherosclerosis, at least undercertain conditions. Measures reflecting in vivo levelsof oxidation in the artery wall are urgently needed.

My fourth and final point is that oxidized LDL, andthe molecules it generates, may affect cardiovascularevents not only by promoting atherogenesis butalso by contributing to plaque instability.5 There ismuch evidence that macrophage foam-cells accumu-late in the portion of the plaque that later fissures. Itmay be that oxidized LDL or its products affect plaquestability by stimulating the release from arterial cells(such as macrophages) of proteolytic enzymes, whichcould disintegrate both the matrix and the overlyingendothelium. The products might also stimulate therelease of tissue factor, enhancing thrombotic tenden-cies. Which of these effects are important and relevantto humans is difficult to predict.

Given all this complexity, how will we go aboutdeciding if antioxidants work? If we look at the his-tory of cholesterol-lowering drugs and coronary arterydisease we find that the first drugs tested were rela-

From the Department of Medicine, University of California, San Diego,California, USA.

Address for reprints: Carlos A. Dujovne, MD, Kansas Foundationfor Clinical Pharmacology, 10550 Quivira Road, Suite 240, Over-land Park, Kansas 66215, USA.

50F ©1998 by Excerpta Medica, Inc. 0002-9149/98/$19.00All rights reserved. PII S0002-9149(98)00262-8

Page 2: Basic Requirements for Investigational New Drug and New Drug Application Approval for an Antioxidant Compound in Cardiovascular Disease

tively weak in their action. Clinical endpoint trialssuch as the Lipid Research Clinics (LRC) trial sup-ported the hypothesis that cholesterol lowering couldpostpone or prevent the reaching of those endpoints,although the effect was not totally convincing. How-ever, when more effective cholesterol-lowering drugswere developed and new endpoint trials were con-ducted, the effectiveness of hypolipidemic therapywas established. Now there is a real possibility (seeother articles in this supplement) that cholesterol low-ering itself will be accepted as a surrogate endpoint inconsideration of an investigational new drug or a newdrug application.

I think a similar paradigm will have to be used forantioxidant therapy. For Phase I testing, we could usethe same criteria of safety and pharmacokinetics as areused for other pharmaceuticals. But in our presentstate of knowledge we do not know what to use assurrogate markers for Phase II studies. For Phase IIIstudies, the same clinical endpoints that have beenaccepted for the other hypolipidemic drugs will haveto be used. It would of course be of great utility tohave surrogate markers for lipid peroxidation in theartery wall, and in the body as a whole, and efforts areunderway to develop such indices. For example, someinvestigators have measured isoprostanes, which arebreakdown products of nonenzymatic oxidation ofpolyunsaturated fatty acids, in plasma or urine. Otherinvestigators have tried for years to use measurementssuch as breath pentane. We and others have suggestedthat autoantibodies to oxidation-specific epitopes ofLDL might reflect a generalized level of continuingoxidation, or that immunologically measured oxida-tion-specific epitopes on LDL may also serve such apurpose.

Although many investigators have isolated LDLfrom the plasma of subjects and looked at its inherentsusceptibility to oxidation in ex vivo assays, it remainsto be proved that this is an index of the degree ofoxidation of LDL in the artery wall. As noted earlier,there is experimental evidence in animal models of a

lack of association between this measurement andprotection against atherosclerosis, but we do not knowif this applies to humans. The only studies directlyaddressing this were (1) one conducted in nonhumanprimates by Ross and colleagues in Seattle,6 whoshowed that the ability of probucol to inhibit athero-sclerosis did correlate with LDL protection in ex vivomeasurements of oxidation; and (2) a study fromBoston that showed that the ability of probucol toprotect against abnormal vasomotor properties occur-ring in the presence of human hypercholesterolemiawas related to the degree of protection of the LDL.7

It seems likely that the only way we will knowwhether any of the currently used experimental mea-sures to assess oxidation of LDL have clinical utilitywill be to take such measurements in the context of aclinical trial and to see if a decrease in clinical eventscorrelates with decreased susceptibility of LDL tooxidation. In the future it may be possible to use$1measures of antioxidant protection for the second gen-eration of antioxidant clinical trials, but at present Ibelieve that before any approval of use of an antiox-idant compound against cardiovascular disease begiven it must be shown to be effective in preventingclinical outcomes of such disease.

1. Reaven PD, Witztum JL. Oxidized low-density lipoproteins in atherogenesis:role of dietary modification.Annu Rev Nutr1996;16:51–71.2. Steinberg D. Low-density lipoprotein oxidation and its pathobiological signif-icance.J Biol Chem1997;272(34):20963–20966.3. Berliner J, Leitirger N, Watson A, Huber J, Fogelman A, Navab M. Oxidizedlipids in atherogenesis: formation, destruction and action.Thrombos Haemost1997;78(1):195–199.4. Fruebis J, Bird DA, Pattison J, Palinski W. Extent of antioxidant protection ofplasma LDL is not a predictor of the antiatherogenic effect of antioxidants.JLipid Res1997;38(12):2455–2464.5. Lee RT, Libby P. The unstable atheroma.Arteriosclerosis, Thrombosis andVascular Biology1997;17:1859–1867.6. Sasahara M, Raines EW, Chait A, Carew TE, Steinberg D, Wahl PW, Ross R.Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman pri-mate by probucol. I. Is the extent of atherosclerosis related to resistance of LDLto oxidation.J Clin Invest1994;94(1)155–164.7. Anderson TJ, Meredith IT, Charbonneau F, Yeung AC, Frei B, Selwyn AP,Ganz P. Endothelium-dependent coronary vasomotion relates to the susceptibilityof LDL to oxidation in humans.Circulation 1996;93:1647–1650.

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