WO1998006389A1

WO1998006389A1 - Intramural delivery of nitric oxide enhancer for inhibiting lesion formation after vascular injury

Info

Publication number: WO1998006389A1
Application number: PCT/US1997/013905
Authority: WO
Inventors: John P. Cooke; Sverin Schwarzacher; Tai T. Lim; Alan C. Yeung
Original assignee: The Board Of Trustees Of The Leland Stanford Junior University
Priority date: 1996-08-12
Filing date: 1997-08-07
Publication date: 1998-02-19
Also published as: EP1003500A1; JP2000516612A; DE69637676D1; EP1003500A4

Abstract

Vessels suffering vascular injury from angioplasty are treated with L-arginine intramurally. The incidents associated with restenosis are substantially reduced providing for reduced incidents of restenosis as a result of the injury. An iontophoretic catheter can be used for local delivery of the L-arginine. In the catheter, one electrode (28) is located within the catheter body (11) while the other electrode (31) is located on the body surface or within the body of the patient. The catheter balloon (26) is then inflated with the drug entering the balloon interior (27) through lumen (23). Activation of the power supply (30) causes the drug to cross the balloon wall (26) and contact the surrounding vessel wall (15) and vascular tissue.

Description

INTRAMURAL DELIVERY OF NITRIC OXIDE ENHANCER FOR INHIBITING LESION FORMATION AFTER VASCULAR INJURY

CROSS-REFERENCE TO RE1 ΛTED APPLICATIONS This application is a continuation-in-paπ of application serial no 08/556,035, filed November 9, 1995, which is a continuation-in-part of application senal no 08/336, 159, filed November 9, 1995, which is a continuation-in-part of application senal no 08/184,519, filed January 21 , 1994, which is a continuation-in-part of application serial no 076,312, filed June 1 1 , 1993, now U S Patent No 5,428,070

BACKGROL ND The long term benefit of coronary balloon angioplastv and atherectomy is limited by the considerably high occurrence of symptomatic restenosis (40-50%) 3 to 6 months following the procedure (Holmes et al Am J Cardiol ( 1984) 53 77C-81 C) Restenosis is in part due to myointima! hyperplasia, a process that narrows the vessel lumen and which is characterized by vascular smooth muscle cell migration and proliferation (Forrester et al J Am Coll Caiduol ( 1991 ) 17 758-769) Medical therapies to prevent restenosis have been uniformly unsuccessful Intravascular stents have been successfully used to achieve optimal lumen gain, and to prevent significant remodeling However, intimal thickening still plays a significant role in stent restenosis The vascular architecture is maintained or remodeled in response to the changes in the balance of paracπne factors One of the substances that participates in vascular homeostasis is endothelium derived nitric oxide (NO) NO is synthesized from the amino acid L-arginine by NO synthase NO relaxes vascular smooth muscle and inhibits its proliferation In addition, NO inhibits the interaction of circulating blood elements with the vessel wall NO activity is reduced in hypercholesterolemia and after vascular injury We have shown that administration of the NO precursor (L-arginme) has been shown to restore vascular NO activity in animals and in humans with endothelial vasodilator dysfunction due to hypercholesterolemia, atherosclerosis, or restenosis Chronic enhancement of NO activity (by oral administration of L-arginine) is associated with a significant reduction in intimal thickening due to hypercholesterolemia and/or vascular m|ury The observations associated with the oral administration are limited to svstemic action Cooke, et a! J Clin Invest 1992, 90 1 1 68-72 McNa ara el al Biochem Biophvs Res Comm l ^l>93 1 93 2^ 1 -6 Taguchi et al, Life Sciences 1993 53 PL387- 2 Tarrv and Makhoul et al, Arteπoscler Thromb 1994 , 14 938-43 , Hamon et al Circulation 1 994, 90 1357-62, Wang, et al, J Am Coll Cardiol 1994, 23 452-8 However, oral administration of L-arginme has potential systemic side-effects

These side-effects include increases in growth hormone and insulin release— this could, potentially, exacerbate hyperglycemia m patients with diabetes (which is a large segment of the patient population that requires balloon angioplasty for coronary or peripheral artery disease) In addition, there is ex idence that in high doses (30 grams daily) oral L-arginme can increase the proliferation of tumor cells in human breast cancer Accordingly, it would be beneficial to develop an approach which Λrøuld allow one to enhance NO activity selectively in the vessel wall where this effect is needed without having systemic side-effects We have developed an approach to diminish the incidence of restenosis resulting from angioplasty and atherectomy, using arginine to enhance NO activity in the vessel wall, while at the same time avoiding potential systemic side-effects

Brief Description of the Relevant Literature

Intravenous oral administration of L-arginine can enhance the release of endothelium-derived nitric oxide from vessels of animals or humans with hypercholesterolemia and/or atherosclerosis ( 1 -8) Chronic oral administration of L-arginine also inhibits the development of atherosclerosis in hypercholesterolemic animals (9-10) Oral administration of L-arginine also inhibits restenosis following balloon injury (1 1- 13) as does oral L-arginine combined with application of L-arginine to the external surface of the vessel using a pluroπic gel ( 14) However, oral or intravenous administration of L-arginine has systemic side-effects Oral or intravenous L-arginine is known to induce the release of growth hormone and insulin, this could potentially exacerbate hyperglycemia in patients with diabetes Moreover, one study has indicated that high doses of oral arginine can increase the proliferation of tumor cells in human breast cancer

We have shown that intravascular administration of a plasmid construct containing the gene encoding nitric oxide synthase can increase NO production locally in the vessel w all and w ill inhibit l esiertosis in the l at cai otid ( l ^**- ) How ev er this approach requn ed direct exposure o! the \ essel and surgical arteπotomv as well as prolonged installation of the solution containing the NOS gene ^'I his approach would be unpractical for preventing restenosis in coronary arteries Others have shown that administration of drugs consisting of nitπc oxide, or releasing nitπc oxide, can inhibit restenosis after angioplasty Chronic inhalation of nitπc oxide inhibits restenosis following balloon-induced vascular injury of the rat carotid artery ( 16) Oral administration of NO donors (drugs which release nitπc oxide) inhibits restenosis in rat and pig models of balloon angioplastv-mduced

( 1 7, 1 8) However oi al or inhaled administration of mine oxide or nitπc oxide donoi s have systemic eilects (hypotension, headache) and are susceptible to drug tolerance (lack of effect ol drug after prolonged administration There is even some evidence in animal models that NO donors may accelerate atherosclerosis, possibly bv suppressing endogenous NO activity in the vessel wall ( 19, 20)

1 Girerd X I, Hirsch AT, Cooke JP, Dzau \'J, Creager MA L-arginfine augments endothelium-dependent vasodilation in cholesterol-fed rabbits Cu e Res 1990,67 1301 - 1308

2 Cooke JP, Andon NA, Girerd XJ, Hirsch AT, Creager MA Arginine restores cholinergic relaxation of hypercholesterolemic rabbit thoracic aorta Circulation 1991 ,83 1057-62 3 Rossitch E, Jr , Alexander E , III, Black P, Cooke JP. L-arginine normalizes endothelial function in cerebral vessels from hypercholesterolemic rabbits J Clin Invest 1991 ,87 1295- 1299

4 Drexler H, Zeiher AM, Meinzer K, Just H Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolemic patients by L-arginme Lancet 1991 ,338 1 546- 1 550

5 Creager MA, Gallagher SJ, Girerd XJ, et al L-arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans J Clin Invest

1 992.90 I 24S- 3

6 Kuo L, Davis MJ, Cannon MS, Chilian WM Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation Restoration of endothelium-dependent responses by L-arginine Circ Res 1992,70(3) 465-76

7 Tsao PS, McEvoy LM, Drexler H, Butcher EC, Cooke JP Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-argiπiπe

Circulation 1904 89 1 76-82

8 Drexler H, Fischell TA. Pinto FJ, Chenzbraun A, Botas J, Cooke JP_^_ Alderman EL Effect of L-arginine on coronary endothelial function in cardiac transplant recipients Relation to vessel wall morphology Circulation 1994,89 161 5- 1623

9 Cooke JP, Singer AH, Tsao PS, Zera P, Rowan RA, Billingham ME

Anti-alherogenic effects of L-arginine the hypei cholestci olemic rabbit J Clin Invest 1992,90 1 168-72

10 Wang B, Singer A, Tsao P, Drexler H, Kosek J, Cooke JP Dietary arginine prevents atherogenesis in the coronary aπery of the hypercholesterolemic rabbit J Am Coll Cardiol 1994,23 452-58 1 1 McNamara DB, Bedi B, Aurora H, Tena L, Ignarro LJ, Kadowitz PJ, Akers DL L-arginine inhibits balloon catheter-induced intimal hyperplasia Biochem Biophys Res Comm 1993, 193( 1 ) 291 -6

12 Tarry WC, Makhoul RG L-arginine improves endothelium-dependent vasorelaxation and reduces intimal hyperplasia after balloon angioplasty Arteπoscler Thromb 1994, 14(6) 938-43

13 Hamon M, Vallet B, Bauters Ch, Wernert N, McFadden EF, Lablanche JM, Dupuis D, Bertrand ME Long-term oral administration of L-arginme reduces intimal thickening and enhances neoendot helium-dependent acet\ lchoime-induced relaxation after arterial injury Circulation 1994,90 1 357-62

14 Taguchi J, Abe J, Okazaki H, 1 akuwa Y, Kui okawa K L-arginine inhibits neomtimal formation following balloon injury 1993,53(23) PL387-92

15 von der Leyen H, Gibbons GH, Moπshita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ Gene therapy inhibiting neomtimal vascular lesion In vivo transfer of endothelial cell nitric oxide svnthase gene Proc Natl Acad Sci USA 1995,92 1 1 37-4 1

16 Lee JS, Adπe C, Jacob HJ, Roberts JD, Jr , Zapol WM, Bloch KD Chronic inhalation of nitric oxide inhibits neomtimal formation after balloon-induced arterial injury Circ Res 1996,78(2) 337-42

17 Seki J, Nishio M, Kato Y, Motoyama Y, Yoshida K FK409, a new nitric oxide donor, suppresses smooth muscle proliferation in the rat model of balloon angioplasty Arterosclerosis 1995, 1 1 7( 1 ) 97- 106

18 Groves PH, Banning AP, Penny WJ, Newby AC, Cheadle HA, Lewis MJ The effects of exogenous nitπc oxide on smooth muscle cell proliferation following porcine carotid angioplasty Cardto Res 1995 30( 1 ) 87-96 19 Munzel t, Syegh H, Freeman B A, Tarpey MM, Harrison DG Evidence for enhanced vascular superoxide anioπ production in nitrate tolerance A novel mechanism underlying tolerance and cross-tolerance J of Clinical Investigation, 1995, ( I ) 187-94

20 Bult H, Buyssens N, DeMeyer GRY, Jordaens FH, Herman AG Effects of chronic treatment with a source of exogenous nitπc oxide on the release of endothehum-derived relaxing factor by aortae from normal and hypercholesterolemic rabbits Elsevier Science Publishers B V (Bnomedicai Division) Nitπc oxide from L- arginine a bioregulatory system Moncada S Higgs, editors 1990, Chapter l 3, pp 101 - 1 Ob

SUMMARY OF fHE I NVENTION Methods and devices are provided for inhibiting the pathology associated with vascular injury, particularly during angioplasty and atherectomy An NO precursor, particularly L-arginine, is intramurally introduced into the walls of the injured vessel in proximity to the injury in an amount to inhibit the pathology, e g restenosis, associated with the vascular injury Various conventional delivery devices may be used for intramui al delivery of the NO precui sor which are loaded with the NO precursor

BRIEF DESCRI PTION OF ^'I HE DRAWINGS FIGS 1 A and I B are schematic diagrams explaining the treatment and experimental protocol of the acute study (FIG 1 A) and the chronic study (FIG IB) FIG 2A shows the endothelium-dependent vasomotion before and following the local delivery of L-arginine The Y axis shows % constriction and dilatation, the X axis the course of the expenment Befoi e the L-arginme thei e w as only a minor change in diameter in both iliac arteries Following the local delivery of L-argmine treated segments showed significant dilatation The control segments are constricted *= p<0 001 FIG 2B is a graph showing that the vessel segments distal to the delivery site did show only minor changes in vessel diameter to acetylcholine and were not affected by the L-arginine delivery FIG 3 is a representative aortogram in the hypercholesterolemic rabbit Before Ach infusion (left panel, 10 ^SM), the vessel diameter of the iliac arteries are identical Following the local drug delivery (right panel), the right iliac artery dilates to the same dose of Ach after receiving L-arginine FIG 4 is a graph showing maximum nitric oxide production 1 hr and 1 week following drug delivery Vessels treated with L-arginme (striped bars) showed significantly higher production of nitric oxide compared to vessels treated with vehicle (dark bars) (*p<0 04, * "^*p<0/01 ) Absolute values 1 week after the delivery were considerably higher compared to those obtained 1 hr after the delivery of L-arginme FIG 5 is a bar graph of Intima/Media ratios at 2 and 4 weeks following local drug deliv erv lπtima/Media witios wei e si nificanilv lo ei in the L-aiginine treated groups after 2 and 4 weeks in comparison to vehicle-treated vessels ( *p<0 04 * *p<0 01 )

FIG 6 depicts low power microphotographs of iliac arteries of hypercholesterolemic rabbits 4 weeks after balloon catheter injury and local drug delivery Intimal thickening is markedly reduced in the vessel segment treated with L- argmine (right) in a comparison to that treated with vehicle (left)

FIG 7 are histograms illustrating the percentage of intimal lesion occupied by macrophages In vessel segments H eated w ith L-arginine ( stuped bai s) macrophage accumulation did not exceed 20% of the intimal ai ea By contrast in vehicle treated segments macrophages occupied up to 70% of the intimal area in some cases

FIG 8 is a fragmentary view, partially in section of a drug delivery apparatus for use in the subject invention positioned in a blood vessel with the dilatation balloon in its inflated state and containing a solution of an NO precursor FIG 9 is a fragmentary iew, partially in section, of the NO precursor drug delivery apparatus positioned in a blood vessel and embodying iontophoresis means to transport the drug across the balloon surface

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods and devices are provided for the treatment of pathologies associated with vascular ιn|ury, particularly in relation to angioplasty and atherectomy Of particular interest is the injury referred to as restenosis, which results from the migration and proliferation of vascular smooth muscle cells into the lntima of the vessel as well as accretions associated with the atherosclerosis

The method provides introducing into the vessel walls at the site of injury an NO precursor, which results in the enhancement of NO production in the cells at the site of injury Various delivery systems may be employed which result in the NO forming agent infusing into the vessel wall, and being available to the cells for NO production Devices which may be employed include drug delivery balloons, e g porous, sonophoretic, and lontophoretic balloons, as exemplified by the devices depicted in W092/1 1895 WO95/05866 and WO96/08286, as well as such commercial devices as Dispatch® ( Scimed) See also Santoian et al , Cath Caidiov Diag ( 1993) 30 34S-354, Mullet et al I lin ( oil ( itiiliol ( ! ^r>^<)2) 20 460-46tι and Orti? et al Cn dilation ( 1994) 89 1 8- 1 522 The NO precui sor agent is introduced in a delivers' balloon for transport bv a catheter to the sue of iury The balloon may then be expanded under pressure driving the agent from the balloon into the surrounding vessel wall The amount of agent which is employed may vary depending upon the nature of the agent, the region to be treated, and the loss of the agent from the region The infusion of the agent is maintained for sufficient time to ensure that the cells and extracellular matrix in the iniury region are exposed to the agent, so as to enhance the production of NO by these cells The agent may have a single active ingredient or be a combination of active ingredients Of particular interest aie the ammo acids, L-arginine and L-lysine, individually or in combination, as a mixture or as a oligopeptide, or a biologically equivalent compound, such as low molecular weight oligopeptides having from about 2-10 usually 2-6 amino acids or acetylated am o acids and oligopeptides, etc Other agents may be employed to enhance the amount of nitric oxide, either directly by enhancing production of nitric oxide, e g by enhancing absorption, and/or to enhance the activity of NO synthase, or to protect NO fi om degradation Generally, the dose range will be about 0 2 to 0 05 the amount that would be administered orally on a daily dosage These compounds include vitamin B_(J (5-25mg), folate(0 04-1 mg), vitamin B_n (0 05-0 l ing), cysteine or N-acetyl cvsteine (20- 100mg), vitamin C (25- 200mg), coenzyme Q (2 -9mg), glutathione (5-25mg), vitamin E (20- 100 I U ), β- carotene ( 1 -2500 1 U ) or other physiologically acceptable antioxidants, such as tocopherols, phenolic compounds, thiols, and ubiquinones These additional additives will generally be present in relatively smail amount in the formulation, generally being present in less than about 10 weight percent, more usually less than about 5 weight percent, frequently less than about 1 percent, and in at least about 0 01 weight percent of the nitric oxide precursor

Λ physiologically acceptable medium will be employed, normally an aqueous medium which may be deionized water, saline, phosphate buffered saline, etc The amount of the active NO precursor agent will vary depending upon the particular agent employed the other additives present, etc Generally, as exemplified by L-arginine, at least about 50 mg will be present and not more than about 5 g, usually at least about 100 mg and not more than about 2 <j li equentlv at least about 500 mg The concentration mav be vaπed widely, generally ranging from about 20-500 more usually from about 50-250 g/l

The time for the treatment will usually be at least about 2 minutes, and not more than about 0 5 hour, generally ranging from about 5- 1 5 minutes The rate of introduction will generally range from about 0 05-5 ml/min, depending upon all of the other factors

The subject methodology is employed with hosts who have suffered vascular iniurv, as caused by angιoplast\ and atherectomies The time for the administration of the NO precursor agent mav be vaπed widely, pi ovidmg a single administration oi multiple administrations over a relatively short time period in relation to the ime oi injury Generally, treatment may be befoi e, concurrently or after the injury, usually within 2 weeks of the in-ury, if before, and not more than about 8 weeks, usually not more than about 6 weeks, preferably in the range of 0-6 weeks (where 0 intends concurrrently or shortly after the prior procedure within 6 hours)

Tor the most part, the patients will be suffering from various conditions associated with narrowed vessels, particularly hypercholesterolemia, diabetes, tobacco use and hypertension Thus, one will normally be dealing with vessels which are narrowed to vδrying degrees as a lesult of the accumulation of plaque at the vessel wall

It is found that with one treatment of the NO precursor agent at or about the time of the injury, before or shortly thereafter, one can observe enhanced vascular NO production and reduced intimal thickening, so as to substantially reduce the potential for restenosis

As indicated, various delivery devices may be employed for the delivery of the active agent Figure 8 illustrates the drug delivery apparatus with the balloon 12 in its inflated state and within an arterial vessel in which the vessel walls are indicated by the reference numeral 15 During percutaneous transluminal coronary angioplasty ("PCTA") procedures, the guide wire 10 is first inserted into the selected artery to a point past the stenotic lesion The dilatation catheter including the catheter body 1 1 and the balloon 12 is then advanced along the guide wire 10 to the desired position in the artei lal system in which the balloon portion 12 traverses or crosses the stenotic lesion The balloon 1 2 is then inflated bv inti oducing the NO pi ecursor solution through the balloon lumen 14 into the interior chamber 1 of the balloon 12 During inflation, the outer surfaces of the balloon 12 press outwardly against the inner surfaces of the vessel wall 15 to expand or dilate the vessel in the area of the stenotic lesion, thus performing the angioplasty portion of the method as well as the intramural introduction of the NO precursor into the vessel wall

The porous balloon may be made from any of the conventional materials used for this purpose These include cellulose acetate, polyvmyl chloride, polysulfone, polvacrvlonitπle, polvui ethanes natui al and synthetic elastomei s polvolefins, polvestes, fluoropolymci s, etc Usually the film thickness willbe in the range of about 10A to l μ, with a nominal poie size oi about 0 05 to I μ Alternatively, a local drug delivery system mav be employed where the agent is delivered to the vessel wall by channels that are on the exterior surface of the balloon The balloon is placed into the diseased vessel segment as described above The balloon is then inflated in the usual manner (using saline, usually containing a contrast agent), placing the channels (on the sui tace of the balloon) in contact with the vessel wall The NO precursor solution is then infused under pressure into the channels Perforations in the channels allow the solution to exit and jet into the vessel wall under pressure to enhance intramural delivery Alternatively, a local drug delivery system may be employed where the agent is delivered to the vessel wall by channels that are on the exteuor surface of the balloon 1 he balloon is placed into the diseased vessel segment as descnbed above The balloon is then inflated in the usual manner (using saline, usually containing a contrast agent), placing the channels (on the surface of the balloon) in contact with the vessel wall The NO precursor solution is then infused under pressure into the channels Perforations in the channels allow the solution to exit and jet into the vessel wall under pressure to enhance intramural delivery

Alternatively, an lontophoretic approach may be used Figure 9 illustrates a structure utilizing iontophoresis to assist in driving the active NO precursor across the balloon wall 26 and into contact with the vessel walls 1 5 One electrode 28, the catheter electrode, is located on or within the catheter body 1 1 , while the other electi ode 1 the body surface electrode, is located on the body surface or within the body ot the patient Λn elenncal cun ent loi the lontophoi etio pi ocess is pioduced between the electrodes 28 and 3 I by an external power soui ce 30 through the electrical leads 29 and 33, respectively Direct cui i enl mav be used although other wave lorms are also utilized (e g , a series of rectangular waves producing a frequency of 100 Hz or greater)

During operation of the lontophoretic device, the balloon 26 is first positioned across the stenotic lesion The balloon interior 27 is then inflated with the drug in the lumen 23 As the balloon expands, it causes the artery to dilate This is followed by activating the power supply 30 thereby creating a current between the electrode 28 and the electrode 3 1 which passes through the balloon w all 26 1 his cuπ ent drives or drags the NO precursor within the chamber 27 aci oss the wall and into contact with the surrounding vessel wall 1 5 and vascular tissue

The following examples are offered by illustration and not by way of limitation

EXPERIMENTAL

Methods

Animals

27 male New Zealand white rabbits (NZW) weighing 3 8± 1 5 kg, were entered into the study after one week period of acclimation in the housing facilities ot the Stanford Department of Comparative Medicine All animals were inspected prior to the study by a veteπnanan, and monitoi ed daily by technicians and investigators The experimental protocols wei e appi oved by the Administrative Panel on Laboratory Animal Care of Stanford University and were performed in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care

Animals were then fed a high cholesterol diet ( 1 %, Dyets, Bethlehem, PA) for five weeks Two protocols (acute and chronic study) were carried out as follows (Figures 1 A & B) Acute .Study (n^ 13: protocol see l-^'wure I A) This study was performed to determine if intramural administration of the NO precursor L-arginme could enhance local NO synthesis

Anesthesia and Sitr ical Preparation

Six davs altei iniiiaimu the high cholester ol dici the l abbit s were anesthetized using a mixture of ketamine ( 5 mg/kg) and rompun ( 35 mg/kg) The right carotid aπery was exposed, carefullv incised and a tvgon sheath ( 5 French in diameter) was inserted under fluoroscopic control into the descending aorta An angioplasty balloon (ACS, balloon diameter 3 mm) was advanced into either iliac artery and inflated distal to the deep femoral artery at 8 ATM for 6 times with 30 second increments between each inflation Subsequently, the same procedure was repeated in the contralateral iliac artery After four additional weeks of diet, the animals were anesthetized and the left carotid artery cannulated for catheteπzation - and local drug deliver-y

Local Drug Delivery

A local drug delivery balloon (3 mm, Dispatch®, Scimed) was advanced to the left or right iliac artery and placed at the same position as the previous balloon injury The proximal end of the delivery catheter was placed at the internal iliac branch under fluoroscopic conti ol foi landmai i eference The balloon was inflated to six atmospheres and L-aiginme (800 mg/5 ml), or saline w as infused for 1 minutes at a rate of 0 2 ml/minute Subsequently, this procedure was repeated in the contralateral iliac artery The iliac artery to receive arginine treatment was randomly determined An intravenous bolus injection of Kefzol® was given for prevention of infections Identification of Endo/he urn-Dependent Vaso otion and Quantitative Angiographv

After local administration of arginine or saline, a control angiogram was obtained Subsequently two infusions containing acetylcholine ( 10 ^s 10 ⁶ M) were administered at a rate of 0 8 ml/minute for 3 minutes through a Swan Ganz catheter (4 French in diameter), placed above the iliac bifurcation Immediately following each infusion an angiogram of the iliac arteries was performed All angiograms were measured blindly by two investigators with an electronic cahper system The diameter was measured at three predetermined sites along the area of drug delivery at baseline and after each dose of acetylcholine before and after the local drug delivery The vessel diameter was also measured at a reference site distal to the infusion segment to verify downstream effects of localK deln ei ed L-ai giniπe The percent vanation in diameter compared to baseline was calculated for each dose and expressed in mean±SEM

Hai vesting of 7 issue 30 to 60 minutes following the local delivery of L-argmme animals were sacrificed and the iliac arteries carefully freed from adjacent tissue Care was taken to harvest the exact portion of the artery where the local delivery was carried out by matching the anatomy with the respective fluoroscopic picture To verify the amount of cell damage induced bv the local drug deliver, balloon electron microscopv of the delivered segment was pei toi med in t i ee rabbits

Measui e men Is o/Nili ogen Oxide

The harvested iliac artery rings were placed in cold physiological solution The vessel was opened longitudinally and incubated in 2 ml of Hanks buffered saline (HBSS) medium (Irvine Scientific ) containing calcium lonophore ( 1 μmol/L, A23 1 87

Sigma St Louis MO) and L-arginine ( 1 00 μl/L Sigma, St Louis MO) at 37°C

At selected time points (0 30 60 120 minutes), samples of the medium were collected for measurements of nitrogen oxide (NOx) and replaced with 2 ml of fresh media After incubation, the segment was weighed and NOx was measured with a commercially available chemiluminescence apparatus (model 2108, Dasibi) 100 μl of the samples were in-ected into a reduction chamber containing boiling acidic vanadium

(III) In the reduction chambei NO,- and NO,- aie l educed to NO, which is then quantified by the chemiluminescence detector after reaction with ozone Signals from the detector were analyzed by a computerized integrator and recorded as areas under the curve Standard curves for NOJNO, were linear over the range of 50 pmol to 10 nmol

Chi onii Stitdv (n ^*** I-l, pi oiotol see Tig i e IB)

This study was performed to determine if a single intramural administration of L-arginine could induce a persistent augmentation of NO activity and inhibit mvointimal hyperphasia and/or macrophage accumulation One week after initiation of the diet, a balloon injury of the iliac arteries was performed under anesthesia Immediately thei eatiei L-arginine w as admmisiei ed into the w all of the πghi oi the left iliac artery by the local delivery system Saline was admmisieied using the same catheter system to the contralateral iliac ai tei y I he dose ot I -arginine and the infusion rate was identical to that used in the acute study One, two, or four weeks (n=4, 4, and 6 respectively) after balloon injury and local drug delivery, endothelium-dependent vasomotion was assessed angiographically and/or vessels were harvested for histomorphometπc measurements lmmunohistochemistry, or chemiluminescence

Moiphomeli it Ana si s (Inlinia Media Ratio) The harvested vessels wei e fixed in 10% buflered formalin and then embedded in paraffin The embedded vessels weie sectioned into thin slices and stained with hematoxyhn and eosin foi light microscopy and histomorphometry Measurements of intimal and medial cross-sectional area were made by experienced observers blinded to the treatment group Hi tologic cross-sections were scanned with one magnification and digitized, using the Image Analysist The following boidei s were highlighted with a trackball external elastic lamina internal elastic lamina lumen/intima border Cross sectional areas of the respective vessel wall layers were then calculated and an lntima/media ratio calculated The media was defined as the area between the external and the internal elastic lamina, the intima was defined as the vessel layer between the internal elastic lamina and the lntimal/luminal border Immiinohistochemistiy

Immunohistochemical analysis was performed on tissue fixed in formaldehyde and embedded in paraffin as described above Antibodies against rabbit macrophage (RAM 1 1 , Dako Corp , Carpenteπa, CA) was used to identify macrophages Sections were incubated with the primary antibody for one hour at room temperature, anti-rabbit IgG (biotin conjugate) foi 30 minutes and avidin peroxidase for 20 minutes Peroxidase was then visualized with chromogen (Zymed Laboratories, Inc , South San Francisco, CA) Three respective cross-sections were lmmunostained for each vessel segment treated with either L-arginine or saline Macrophage staining was assessed by two experienced observers using a light microscope Areas of the vessel defined as media and iniima and the pei ce t ol the \ essel ained loi maci ophage w as determined

Niti Or'en ()\ ιde Λ leasiii emails

In four rabbits, tissue was harvested one week following the local delivery of L- arginine for measurements of NOx levels Chemiluminescence measurements were made as descπbed above

Data Analysis

Data are expressed as mean±SEM The difference in vasoreactivity to acetylcholine was expi essed as pei centage variation in diameter compared to baseline The mean change of all anei ies in each ti eatment group (L-arginine or saline) was used for comparison An unpaired t-test was performed to compare values between the two-treatment groups for each dose of acetylcholine before and following either L- arg ine or saline Additionally, a two-factor analysis of variance was performed to venfy the difference within the treatment group and between the g^roups Significantly different changes were assumed at a p-value ol •- 0 05 Differences between NOx levels were also identified using Student's t-test with Bonferroni correction for multiple comparisons Results

Acute Study (Figure I A)

Vasoreactivity

Figure 2 A shows the response of vessel segments to acetylcholine before and after the local delivery of L-arginine or saline. Baseline vessel diameters were identical before and after local drug delivery in both iliac arteries There was little change in vessel diameter before local drug delivery. This probably reflects the fact that acetylcholine-induced endothelium-dependent vasodilation is attenuated in the endothelium that regenerate after vascular injury, particularly in the setting of hypercholesterolemia After local delivery of L-arginine, endothelium-dependent vasodilation was restored By contrast, after local delivery of the vehicle, no vasodilation was observed The effect of L-arginine was localized to the segment which was exposed to intramural delivery Almost no change in vessel diameter was seen at the site distal to the local delivery of L-arginine (Figure 2B) Thus, using this method only local (rather than systemic) effects of L-arginine were observed

Nitrogen Oxide Levels

NOx measurements were made in vessel segments harvested 30-60 minutes after local drug delivers' Vessel segments treated with arginine exhibited a significant increase in nitrogen oxide levels throughout the incubation periods of 30, 60 and 120 minutes

Chronic Study (Figure I B Vasoreactivity Vasomotion studies were performed two or four weeks after the local drug delivery Iliac arteries treated with vehicle tended to vasoconstπci in response to acetylcholine whereas those treated with L-arginine tended to vasodilate although the observed differences did not reach statistical signance (Ach 10 'M , 2.2± 1 3% vs - 4.2±3.8%, L-arginine vs vehicle. Ach I 0^'5; 7.2± I .0 vs -4 0±7.5). Intinia Media Ratio

Figure 5 shows the results obtained two and four weeks following local drug delivery Administration of L-arginine significantly inhibited mtimal lesion formation in comparison to vehicle control This phenomenon was even more apparent four seeks following local drug delivery I nuinohistochemisti v

Figure 7 shows the percentage of the intimal lesion surface area which stained positively for macrophages Only 0- 10% of the intimal area was infiltrated by positively stained cells in the L-arginine treated segments whereas in vessel segments treated with vehicle the mtimal area involved by macrophages was markedly higher, in some segments exceedinu ι0^u o ol the mtimal area

Niti ogen Oxide Levels

In vascular segments fiom four rabbits, NO was measured NO production ex vivo was significantly higher one week following the delivery of L-arginine compared to segments exposed to vehicle These levels were also higher compared to those achieved one hour following the delivery (Figure 4)

It is obvious from the above results, that the subject methodology and devices provides an alternative ti eatment to substantially reduce the occui rence of restenosis after vascular injury 1 he methodology is simple can be performed in conjunction with the procedure resulting in the vascular ιn^*urv, and is found to be effective Importantly, this approach avoids systemic side effects associated with oral or intravenous adminstration of L-arginme, while providing effective treatment In this way, a procedure which has been commonly used can find expanded application as a result of the reduced incidents of restenosis

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spn it oi scope of the appended claims

Claims

WHAT IS CLAIMED IS1 A method for reducing the probability of restenosis resulting from injury caused by angioplasty or atherectomy, said method comprising introducing intramurally proximal to the site of said injury a nitric oxide precursor at a time and in an amount to increase the amount of nitπc oxide formation of the cells proximal to said injury, whereby a reduced incidence of restenosis occurs2 A method according to Claim I w herein said nitric oxide precursor is at least one of L-ai ginine L-lvsme oi an oligopeptide compi ismϋ at least one of L- argmine and L-lysme3 A method according to Claim I , wherein said time is at the time of said injury and not later than four weeks thereafter4 A method according to Claim 1 , wherein said NO precursor is in an aqueous medium5 A method according to Claim 1 w hei em said introducing is by means of a local delivery catheter6 A method for reducing the probability of restenosis resulting from injury caused by angioplasty or atherectomy, said method comprising introducing intramur liv at the site of said injury L-arginine at the time of injury or not later than two weeks thei eafter as an aqueous solution at a concentration in the range of 20 to 500 g/l to provide an amount to increase the amount of nitric oxide formation of the cells at the site of said injury, whereby a reduced incidence of restenosis occurs7 A method according to Claim 6, wherein said introducing is by means of a local delivery cathetei 8 A porous balloon catheter comprising a balloon with a solution of an NO precursor in the lumen of said balloon9 A catheter according to Claim 8, wherein said catheter is lontophoretic10 A catheter according to Claim 8, wherein said catheter comprises a balloon with exterior channels or compartments for local delivery of a solution comprising the NO precursor AMENDED CLAIMS[received by the International Bureau on 5 November 1997 (05.11.97); original claims 1-10 replaced by amended claims 1-9 (2 pages)]

1. A method for reducing the probability of restenosis resulting from injury caused by angioplasty or atherectomy, said method comprising: introducing intramurally proximal to the site of said injury a nitric oxide precursor within six hours of the injury and in an amouni to increase the amount of nitric oxide formation of the cells proximal to said injury, whereby a reduced incidence of restenosis occurs

2. A method according to Claim 1 , wherein said nitric oxide precursor is at least one of L-arginine. L-lysme or an ohgopeptide comprising at least one of L-arginine and L-lysine

3. A method according to Claim 1 , wherein said NO precursor is in an aqueous medium.

4. A method according to Claim 1. wherein said introducing is by means of a local delivery catheter

5 A method for reducing the probability of restenosis resulting from injury caused by angioplasty or atherectomy . said method comprising introducing intramurally at the site of said injury L-arginine at the time of injury or not later than six hours thereafter as an aqueous solution at a concentration in the range of 20 to 500g/l to provide an amount to increase the amount of nitric oxide formation of the cells at the site of said injury , whereby a reduced incidence of restenosis occurs

6 A method according to Claim 6. wherein said introducing is by means of a local delivery catheter.

7. A porous balloon catheter comprising a balloon with a solution of an NO precursor in the lumen of said balloon.

8. A catheter according to Claim 8. wherein said catheter is iontophoretic.

9. A catheter according to Claim 8, wherein said catheter comprises a balloon with exterior channels or compartments for local delivery of a solution comprising the NO precursor.