The invention relates to treating and preventing ischemic cell damage in a reperfusion situation . The active principle is reduced forms of stable nitroxides ( >NO ^* — τ>>NOH) and some compounds which are readily convertible thereto . The compounds employed according to the invention comprise the structural element ( I ) :

- C - N - C -

. ' I

OR

In this formula (I) , R may be selected from among one unpaired electron, the compound in that case being a nitroxide; a cation, such as a physiologically acceptable metal ion; hydrogen, the compound in that case being a hydroxylamine derivative; or a protective group which is of the type as is known per se for hydroxyl groups and which is readily removable so that R then will be hydrogen or an unpaired electron. The protective group may form an ester or ether being acyl, tetrahydropyranyl, triorganosilyl (the organo groups therof being alkyl and/or aryl groups) etc. The nitroxides to which the invention is applicable are stable free radicals. It is known that in order to fulfil this condition the alpha and alpha' carbon atoms may be aromatic or aliphatic with certain substituents. An aliphatic alpha carbon atom is preferably tetrasubstituted. It can be a tertiary carbon atom. For a review of stable nitroxides see for example Spin Labelling in Pharmacology, Ed. Holteman J.L. , Academic Press 1984, especially pages 1-85. In the light of such knowledge as has been gained up to now, the most preferred compounds to be used according to the invention are cyclic compounds containing structure (I) as three members in a 5- or 6-membered ring structure. Such compounds have the general structure (II) :

OR

In formula (II) :

(i) A is a 2- or 3-membered organic bridge containing at least one or two groups CR-R, and optionally also ether, thioether, ester, amide, k tone or other structure giving a stable ring according to the formula;

(ii) ^R ₇ ~ ^R ₄ ^av k ^e hydrocarbon groups attached to the ring via the aliphatic or aromatic alpha carbon atoms. The groups may be the same or different or may pairwise form cyclic structures (be organic bridges) and optionally contain functional groups such as ester, ether, thioether, carboxyl, amine (primary, secondary, tertiary, or quaternary'') , alcohol, amide etc., but at least three R,-R, being preferably lower alkyl such as C,-C as e.g. methyl;

(iii) R _ς and R _fi are both preferably hydrogen although at ^: least one of them may be selected from among the aforesaid hydrocarbon groups or contain one of the functional groups, R^ and R _fi in CR-P.., at different positions of the ring being identical or different; and

(iv) p has the meanincr.s set forth above.

A prerequisite applying to all of the substituents R_I. to R,o, in formula (II) is that they must not adversely affect the stability of the nitroxide structure or ring structure. Compounds of formula (II) having different R substituents but identical structures in other respects are convertible to one another. It is easy, for instance, to convert compounds where R is a cation or protective group to the corresponding hydroxylamine compounds (R=H) . The hydroxylamine compounds and corresponding nitroxides are convertible to one another by means of oxidation and reduction, respectively.

The hydroxylamine compounds comprising structure (I) are known to be very reactive with free radicals to thus form a stable nitroxide (nitroxyl radical) . This phenomenon has been utilized for studying free radical reactions both in biological and in non-biological systems. In these studies, a suitable hydroxylamine compound comprising structure (I) (R=H) was added, the resultant nitroxide (R = unpaired electron) then being quantified by ESR. For this earlier domain of practical use, several types of general synthesic methods have been ^" developed which will give compounds comprising structure (II) . See for example Spin Labelling in Pharmacology, Ed. Holteman J.L., Academic Press 1984, especially p. 1-85, and Rotantsev, E.G., et al., Synthesis (1971), p. 190-202 and 401-14.

Therapeutical uses and biological effects other than that of the invention have been indicated in a great number of publications. See for instance Chemical Abstracts:

78 106130t (1973) 95: 215492J (1981) i 33187s (1974) ϋ ^: 62729k (1982) 83. 22254b (1975) 961: 97252b (1982)

69326n (1976) .'' 34470s (1983) 87 62729z (1977) 98: 65171z (1983)

87_ 95618r (1977) 103: 64744y (1985) ii 17029b (1978) 104: 28491a (1986) ii 49273J (1979) 104: 81560y (1986) ϋ 33843e (1980) 105: 183407s (1986) , 93 179363r (1980) SE-B-499,105 and EP-A-93,945

WO-A-86/01110 describes the use of hydroxylamine (H-NOH) as an absorption enhancer for a free radical scavanger in vivo. In addition to the publication above Chemical Abstracts 95: 215492J (1981) has been cited during the first year of the prosecution of the priority application. The title and summary of this latter C . reference are inconsistent with each other. The title refers to the use of a compound different from formula I in ischemia, while the text of the abstract refers to compounds of formula I but for other uses.

The exact reason as to why the compounds may exert beneficial effects in reperfusion as now set forth have not yet been fully elucidated. It is probable, however, that their radical scavengincr function and their reciprocal convertibility are important factors.

Compounds to be mentioned in the context of this invention are in particular onocyclic compounds wherein R_. -R. are individual groups other than hydrogen while A together with the nitroxide structure (I) forms a 1,3-oxazolidine, pyrro- lidine or piperidine structure. The preferred cyclic com¬ pounds are thus tetrasubstituted on the two alpha carbon atoms of the nitroxide group.

In popular terminology, the N- ( R) -1,3-oxazolidine compound in which R ₁ -R is 2-ethyl-2,4,4-trimethyl, and R is hydrogen or an unpaired electron, and which has no other substituents, is called OXANOH or OXANO ^* , respectively.

Also, the piperidine compound in which R. -R. is N- (OR) - 2,2, 6, 6-tetramethyl, and R is hydrogen or an unpaired electron, and which has no other substituents, is called in popular terminology TEMPOH or TEMPO ^* , respectively.

Damage due to insufficient blood flow (= ischemia) in an organ develops according to a complex pattern. The primary cause is the cessation of oxygen and of substrate (= nutrient) transport which will impair energy-dependent processes. The consequence will be a distrubance of cell metabolism and iόn gradients; the ultimate effects being cell death and irreversible tissue damage.

Several important components in this damaging .process have been identified. Research during recent years has focused on the so-called reperfusion damage, i.e., the damage arising when blood flow is restored after a period of ischemia. In this situation several factors co-operate to give rise to extensive formation of so-called free oxygen radicals. A feature common to these radicals is that they are highly reactive because of the presence of unpaired electrons - and thus they may give rise to cell damage.

Several of the metabolites accumulated in the cell during the ischemic period will function as substrates for enzymic processes resulting in the formation of superoxide radicals (0„.) when oxygen supply increases at the time of reperfusion. The superoxide radical may give rise to the formation of other radicals as well which are even more reactive, such as hydroxyl radicals (OH") . In the process called lipid peroxid-

3+ ation, superoxide co-operates with ferri(Fe ) ions and

nucleoside phosphates to form perferryl radicals, which initiates the peroxidation of polyunsaturated fatty- acids in the phospholipid layers of membranes. This sequential reaction is self-propagating and it may continue as long as polyunsaturated fatty acids are available. Lipid peroxid¬ ation thus involves the formation of several intermediate free radicals and leads to destruction of the membrane, which is vital for the structural integrity of the cell.

A large number of substances have been proposed as scaven¬ gers for free radicals formed in connection with reperfusion

(treatment of ischemia) and intoxication. The purpose in these cases has been to prevent damages due to free radicals

(van der Vusse, G.J. et al., Trends in Pharmacol. Sci. 6

(1985) , p. 76-9) . As examples may be mentioned substances like SOD (superoxide dismutase) , inhibitors of xanthine oxidase (e.g. allopurinol) , scavengers in the form ^' a low- molecular carbohydrates, methionine, histidine etc.

(WO-A-86/00812) . Quite recently a paper has been published which describes positive effects of a so-called spin trap in a traumatic shock model (Novelli G.P. et al., Free Rad Res Co m 1 (1985) , p. 321-7) . This spin trap is alpha-phenyl-N-t- butyl nitrone. An exhaustive explanation as to why this effect was obtained was not given. From a chemical point of view, it is well-known that nitrones act as radical scavengers in a different manner from that of nitroxides.

The invention comprises both the method of using the compounds in question for medical drug therapy and the use of these compounds for producing compositions for such therapy. The invention provides for improvements of therapies in cases where lipid peroxidation or free radical damage is part of the pathogenic process to be treated and prevented. The invention is directed to the prophylaxis and treatment of ischemic cell damage either in mammals, including humans, or

in organs that have been excised from them. The term "ischemic cell damage" here comprises not only the damage arising during ischemia but also the damage that may arise upon reperfusion.

The invention in its various aspects is intended for being utilized in acute resuscitation cases, e.g. upon cardiac arrest or other conditions involving circulatory collapse, where the brain is subject to ischemia. Further fields of practical application comprise various types of traumas in the central nervous system, cerebral hemorrhage, stroke, subarachnoid hemorrhage or in cases of intracranial vascular surgery where temporary occlusion of blood vessels is a necessary step. The drug kit may be used also in cases of ischemic conditions of other organs such as heart, kidney,- intestine, liver and skeletal muscle, in association with conditions of shock, trauma, embolisms and infarctions, and moreover also when various types of surgery are carried out such as heart surgery, vessel reconstruction, and transplan¬ tation of organs.

Another field of applicability may be the use as perfusion solution and preservation solution for organs in cases of for example cardioplegia or organ transplantations.

A therapeutically active amount of the compound comprising structure (I) may be administered in a number of different ways in accordance with the the invention. Administration thus may be parenteral, e.g. intraarterial, intravenous, subcutaneous, intramuscular etc. As a rule the compounds are administered in the form of a sterile aqueous solution buffered to a physiologically acceptable pH. The solution is prepared and injected in direct conjunction with or just before administration. The dose given should be selected in

-9 -2 the range of from 10 to 10 mol per kg of body weight but may .deviate from this general recommendation, depending on the particular compound and indication involved in each case. The administration may be repeated.

Simultaneously with a compound comprising structure (I) it may be suitable, within the concept of the invention, to administer further drugs which will have a positive effect on the particular indication involved (so-called ultifactor treatment) , for example a plasma volume expander (e.g. dextran or hydroxyethyl starch) , SOD, calcium blocking agents (such as nifedipine, nimodipine, verapamil, lido- flazine, flunarizine etc.), diuretics (especially osmotic diuretics) , antiedemics etc. It may also be advantageous to administer other types of low-molecular scavengers, for instance aliphatic or aromatic thiol or alcohol, or a low-molecular compound containing nitrogen structure such as primary amine, secondary amine or imine. See O-A-86/00812.

Within the concept of this invention, the structure (I)- containing the active compound may be packaged in the form of a drug kit containing at least one dosage unit of the compound, if desired together with at least one.of the drugs mentioned in the preceding paragraph. The dosage unit may vary, depending on such factors as the indication to be treated, the particular compound comprising the aforesaid structure (I) , and the particular type of patient (child, adult) . For a normal person (75 kg) the amount of the compound will be in the range of 7.5x10 —8 to 7.5x10—1 mol.

According to such knowledge as has been gained up to now, it is desirable that in a drug kit according to the invention the compound should be packaged in a -manner such as is common practice with this type of compounds, i.e. in an ampoule under an inert atmosphere or in vacuo, so that when to be used, the compound is to be reconstituted in a suitable, physiologically acceptable buffer.

The nitroxides that may be employed according to the inven¬ tion are stable free radicals whereas the corresponding reduced forms (R=H) are readily oxidizable to the corre¬ sponding nitroxides. The nitroxides contemplated can readily

be converted to the reduced form, e.g. by catalytic hydro- genation in aqueous solution with PtO, although then they must usually be used immediately or within one or a few hours, in view of the risk for air oxidation. Both the radical and the corresponding reduced form can be used according to the invention, though it is often practically best, for technical reasons, to use the radical.

The invention is defined more precisely in the attached claims which form an integral part of this specification. The invention will be illustrated below by means of some patent examples.

Example 1

Antiarrhythmic effect of OXANOH and OXANO" on the isolated rat heart subjected to regional ischemia and reperfusion

Male rats of the Wistar strain (220-280 g) were used. The heart was isolated and perfused via the aorta according to the Langendorff technique. Krebs-Henseleit bicarbonate buffer containing 11.1 mM glucose and 4.3 mM potassium ( )

2+ and 1.4 mM calcium (Ca ) was used as the perfusion medium

(pH 7.4, 37 °C) .

Regional ischemia was induced by ligation of the left coronary artery. After a period of 15 minutes of regional ischemia the ligation was released and the heart was reper- fused for 10 minutes. Epicardial ECG was recorded throughout the experiment by means of two silver electrodes attached to the heart. The ECG was analyzed for

1) the incidence and duration of ventricular fibrillation (VF) ;

2) the length of the reperfusion period during which the heart remained in normal sinus rhythm.

Hearts perfused with rebs-Heinseleit buffer were compared with hearts perfused with the same buffer but containing 25 ,uM OXANOH or 25 ,uM OXANO ^* .

Reduction of OXANO": In the presence of platinum oxide hydrogen gas was bubbled through a 10 mM solution of OXANO" for 45 minutes (Dulbecco's phosphate buffer) . The OXANOH thus formed was stored on ice and used within two hours.

Table 1

Ventricular fibrillation Duration of normal sinus rhyth during reperfusion period

Duration (s)

Those exhibiting

Incidence All hearts fibrillation Time (s)

Control 100 % 302 + 61 302 + 61 285 + 62 47.5 + 10.4

(n = 12) (12/12)

OXANO" (25 ,uM) 92 % 177 + 58* 193 + 61* 416 + 58* 59.4 + 0.6*

(n = 12) ' (11/12) (n =~11)

OXANOH (25 ,uM) 66 % 135 + 63* 203 + 85* 457 + 63* 76.2 + 10.5*

(n = 12) ' . (8/12) Cn = 8)

* ₌ p <0.01 vs. control.

Results: In the control group all the hearts developed ventricular fibrillation during the reperfusion period. If the perfusion medium contained 25 ,uM OXANOH the number of hearts developing arrhythmias was reduced to 8 out of 12, with a concomitant decrease of the duration of ventricular fibrillations and a concomitant increase of that part of the reperfusion period during which heart sinus rhythm was normal. These differences were statistically significant. OXANO ^* produced a similar effect, although not quite so pronounced.

Discussion and conclusion: The results suggest that a compound containing N-hydroxy-l,3-oxazolidine structure and the corresponding nitroxide may be an efficient agent for preventing and reducing negative effects of ischemia/ reperfusion on myocardial tissue. It has been proposed that radical-induced lipid peroxidation has an important role in the development of reperfusion-induced arrhythmias (Bernier M et al., Cire Res 58 (1986), p. 331-40), and it is probable, therefore, that OXANOH interacts with some radical- in this seσuence.

Example 2

Effects of OXANO" and OXANOH on lipid peroxidation

Methods: Peroxidation of phospholipid liposomes, catalyzed by Fe 3+ pyrophosphate, was carried out as described in detail by Carlin and Arfors (J. Free Rad. Biol. Med. I

(1986) , p. 437-42) . The reaction mixture consisted of phosphate buffer (Dulbecco) , Fe 3+ (30 ,uM) : pyrophosphate

(400 ,uM) and 0.4 mg/ml phospholipid (predominantly phosphatidyl serine) together with 0.2 mM hypoxanthine or human PMN leucocytes (10 eelIs/ml) . The peroxidation reaction was initiated by addition of either 1.7 mU/ml xanthine oxidase or 10 ,ug/ml phorbol myristate acetate

(which acts as an initiator of superoxide formation in PMN leucocytes) . After 15 min incubation, the reaction was terminated by addition of butylated hydroxytoluene. Thio- barbiturate reagent was then added and the samples were

__. boiled in a water bath for 15 min. After centrifugation the content of malone dialdehyde was determined by measurement of the absorbance in the samples at 532 nm.

OXANOH and OXANO" were added to the reaction mixtures in concentrations ranging from 0.01 to 1.0 mM (Tables 2A and 2B) . Formation of malone dialdehyde was expressed as % of that of the controls (without addition of OXANOH and OXANO"). By adding OXANOH and OXANO ^* to the reaction mixtures after the peroxidation reaction but before the addition of thio- barbituric acid it was possible to exclude interference of these compounds with the assay procedures.

Reduction of OXANO" : As in Example 1.

Table 2A

Effect of OXANO" and OXANOH on hypoxanthine-induced peroxida- lion of phospholipids. The results are given as absorbance units or as percentages (Mean + S.D.(n)). .

0.03 mM 88.1 + 1.3 ^* o (2) 0.01 mM 86.1 + 2.8 o. (2) 1 mM*** 92.5 + 8.2 % (4)

+ OXANOH,

1 mM -2.1 + 2.7 % (3)

0.1 mM 41.7 + 8.3 % (3)

* Absorbance value set to 100 % > ^■ ** Absorbance value set to 0 %

*** Test compound added after the peroxidation reaction

Table 2B

Effect on OXANO" and OXANOH- on PMA-stimulated PMN leucocyte- induced peroxidation of phospholipids. The results are given as absorbance units and percentages. (Mean + S.D. (n) ) .

Experiment

I II III

Buffer * 0.000 + 0.001 (4) -0.002 + 0.002 (3) -0.004 + 0.003 (4) Complete** 0.136 + 0.006 (4) 0.182 + 0.002 (3) 0.049 + 0.002 (4)

- ΓMA 1.9 + 1.0 . (4) 3.6 + 3.9 % (3) -5.7 + 3.8 % (4) ω c + OXANO" , 1 mM 11.8 + 1. (4) 9.5 + 6.6 % (3)

-. 0.3 mM 61.6 + 2. (4) 74.3 + 3.2 % (3)

H 0.1 128.9 + 8.5 % (3)

C mM H 0.03 mM 133,9 + 12.6 % (3) m 1 mM*** 97.8 + 3.9 (3) co

X 0.3 mM*** 95.4 + 0.8 (3) m q + OXANOH,

1 mM -2.6 + 1.9 % (4) -3.4 + 0.5 % (3) -10.1 + 2.7 % (4)

0.3 mM -2.4 + 1.2 % (3) -6.3 + 3.4 % (4)

0.1 mM 23.3 + 3.2 % (3). 25.3 + 4.9 % (4)

0.03 mM 81.1. + 0.9 % ^■ (3) 92,0 + 7.4 % (4)

- -

1 mM*** 90.7 + 3.7 % (3)

* Absorbance value set to 0 % ** Absorbance value set to 100 %

*** Test compound added after the peroxidation reaction

Discussion: The results presented in Tables 2A and 2B and in Figs. 1 and 2 show that compounds comprising the structure N-hydroxy-1,3-oxazolidine or the corresponding nitroxyl radicals may be of value for inhibiting lipid peroxidation. Of the compounds studied, the reduced form (OXANOH) appears to be the most potent one. Fig. 1 shows the dose-response effect of OXANO ^* and OXANOH on phospholipid peroxidation induced by xanthine oxidase. Fig. 2 shows the dose-response effect of OXANO ^* and OXANOH on phospholipid peroxidation induced by PMA-stimulated leucocytes.

At the concentrations employed, it it improbable that OXANOH will be able to efficiently compete with other, naturally occurring radical scavengers for superoxide radicals (such as SOD) . More probably, OXANOH interacts with other radicals which are involved in the lipid peroxidation process, as e.g. perferryl radicals.

Example 3

Effect on certain nitroxides, corresponding hydroxylamines and certain nitrones on lipid peroxidation

The experiment was performed as in Example 2 except that the PMN leucocytes in this case were replaced by liver microsomes from rats (0.4 mg protein/ml, final cone; produced according to Fruster L et al. , J Cell Biol 15 (1962) p. 541-56) . The

3+ Fe concentration was 1.7 ,uM, and the pyrophosphate (ADP) concentration was 1 mM.

Concentrations at which 50 % inhibition of lipid peroxidation was obtained were the following:

OXANOH (1) 63 ,uM

TEMPO ^* (2) 12.5 _/ \_M

4-OH-TEMPO"> (3) 160 ,uM

See below (4) 1 250 /

PBN (5) 4 mM

DMPO (6) >10 mM

4-POBN (7) >10 mM

In the above list, (1) , (2) , (3) and (4) are compounds comprising structure (I) . Compound (1) is N-hydroxy-2- ethyl-2,4,4-trimethyl-l ,3-oxazolidine, (2) is 2,2,6,6- tetramethylpiperidinoxyl, (3) is 4-hydroxy-2, 2, 6, 6-tetra- methylpiperidinoxyl, and (4) is 2- (2 '-ethoxycarbonylethyl) - 2,4,4-trimethyl-l, 3-oxazolidinoxyl. Compounds (5) , (6) and (7) are nitrones and do not comprise structure (I) . Compound is alpha-phenyl-N-t_-butyl-nitrone, (6) is 5 ,5-dimethyl-l- pyrroline-N-oxide and (7) is alpha- (4-pyridyl-N-oxide) -N-t- butyl-nitrone. (The nitrone group has the structure

C = N—> 0

/ the C therein being called "carbon atom"

The experiments show that the nitroxides studied and correspond¬ ing hydroxylamines, both with structure (I), are superior to nitrones with respect to inhibition of lipid peroxidation.