Field of the invention

The present invention relates to use of paracetamol for the treatment of ischemia, particularly cardiac ischemia.

Background of the invention

Cardiovascular disease is a major cause of death and disability both developed and developing countries. Conditions such as hypertension and myocardial infarction are also on the increase as the populations in these countries age.

A low-cost drug with few side effects for the treatment of such diseases can have the potential to significantly reduced or reversed this trend.

For years, the nonsteroidal anti-inflammatory drug (NSAID) aspirin has been prescribed for the treatment of cardiac disease. However, aspirin, while being inexpensive, has the undesirable side effect of gastrointestinal damage at the effective doses prescribed, making it unattractive for use. As such, asprin is sometimes sold formulated with an antacid to counter reduce its damage to the stomach mucosa.

Other drugs such as Ramipril, an angiotensin-converting enzyme (ACE) inhibitor, have been prescribed for myocardial infarction. However, these drugs are expensive and are not affordable to low-income patients for long-term prophylaxis or treatment of heart disease.

For these reasons, an alternative low-cost drug to aspirin to reduce the effects of cardiovascular disease, but with the advantage of having few or no side effects at the dosage used, will be welcome. In a recent epidemiology paper, Rosenberg et al (2003) reported data on the use of paracetamol and aspirin which were extrapolated in the context of data collection on many drugs and other factors on studies conducted on human subjected from 1980 to 1983. Patients admitted to the hospital were requested to fill a questionnaire and to report all medicaments, whether prescribed or not, that they had taken on a regular or chronic basis for any of the indications or for other reasons. The authors indicated that because of the nature of investigation and data collections, misclassification of use must have occurred. Further, selection bias in control selection could not be ruled out. The authors also discussed the possibility of a protective effect of long-term regular paracetamol (N-acetyl-para-aminophenol, para-acetyl-amino-phenol or acetaminophen) and aspirin against myocardial infarction (Ml) in of human subjects. However, the authors concluded that the results with reference to paracetamol were compatible with no effect.

In earlier ex vivo animal studies in an ischemia-reperfusion model (Merrill et al, 2001) and in a low-flow ischemia model (Merrill, 2002), the authors studied the antioxidant properties of paracetamol (acetaminophen) against the deleterious effects produced by hydroxyl radicals in isolated, artificially-perfused guinea pig hearts. The authors indicated that paracetamol was able to attenuate the production of hydroxyl radicals invoked by reperfusion. Unlike in permanent ischemia situations, it is known that the pathology seen in reperfusion or low circulation situations is due mainly to free radical production. Interventions in reperfusion models are thus focussed at arresting the cascade of physiological events that produce free radicals or at scavenging free radicals produced to limit their deleterious effects. That such putative effects of paracetamol can be applied in non-reperfusion models of ischemia is not apparent. Further, data from ex vivo experiments may not be readily translated to in vivo models. Accordingly, there is a need for a further investigation in this field of medicine. Further, there is a need of further and/or alternative drugs which may be used in the prophylaxys and/or treatment of ischemic conditions.

Summary of the invention

The present invention addresses the problems above, and in particular provides a new alternative prophylactic and/or treatment of ischemic conditions in vivo, in particular of cardiac ischemic conditions.

Accordingly, in a first aspect, the present invention provides a method of treatment and/or prevention of ischemia in a mammal comprising administering a therapeutically effective dose of paracetamol and/or a derivative thereof. In particular, the ischemia is cardiac ischemia. Further, the mammal is a human being and the administrating of the therapeutic dose may be done before and/or after the onset of ischemia.

According to another aspect, the present invention provides a method to induce cardioprotective effects in a mammal at risk of myocardial infarction, the method comprising: administering a therapeutically effective dose of paracetamol and/or a derivative thereof before onset of the ischemia and/or administering a therapeutically effective dose of paracetamol and/or a derivative thereof after the onset of ischemia. In particular, the ischemia is cardiac ischemia and the cardioprotective effects comprise reducing infarct size, decreasing gene expression of NOS, decreasing gene expression of COX, increasing antioxidant enzyme activities, increasing capillary density and improving cardiac function.

According to another aspect, the method of the invention comprises administering a further cardioprotective compound different from paracetamol and/or a derivative thereof. The further cardioprotective compound may be administered in conjunction with the administration of paracetamol and/or a derivative thereof. For example, the further cardioprotective drug is ramipril. For another example, the further cardioprotective drug is aspirin.

According to another aspect, the present invention provides use of paracetamol and/or a derivative thereof for the preparation of a medicament for the treatment and/or prevention of ischemia in a mammal. In particular, the ischemia is cardiac ischemia. The medicament may further comprise at least one pharmaceutically acceptable diluent, excipient and/or carrier.

The treatment may comprise inducing cardioprotective effects in a mammal at risk of myocardial infarction. Further, the medicament may be administered before and/or after the onset of ischemia.

According to another aspect, the invention provides kit for treatment and/or prevention of ischemia in a mammal comprising (i) a therapeutically effective dose of paracetamol and/or a derivative thereof, or a pharmaceutically acceptable salt thereof; (ii) at least one pharmaceutically acceptable diluent, excipient and/or carrier; and (iii) instructions for use in treating and/or preventing ischemia. In particular, the kit is the treatment and/or prevention of cardiac ischemia. The mammal may be a human being or a non-human mammal.

Brief description of the figures

Figure 1 shows the changes of blood pressure after 1 week in different treated groups: saline, paracetamol, and ramipril ('*' refers to p<0.05 vs. saline treated group).

Figure 2 shows the mortality of animals after 48 hours of myocardial infarction in different treated groups: saline, paracetamol and ramipril. Figure 3 shows the infarct size ratios of rat hearts after 48 hours of myocardial infarction in different treated groups: saline, paracetamol and ramipril ('*' refers to p<0.05 vs. saline treated group).

Figures 4(A, B, C) show an animal model of myocardial infarction induced by ligation. Figure 4A shows the opening of the thorax; Figure 4B shows a myocardial infarction being induced by ligation of left descending coronary artery; Figure 4C shows a successful ligation. The arrow in Fig.4C indicates the apex cordis.

Figures 5(A,B,C,D) shows the different sizes of the infarcts. The heart is stained in TTC solution and the infarct is analyzed by Scion Imaging system. The infarct area is shown by the lighter colour (lighter shading).

Figure 6: ECG graphs of (A) pre-treatment (day 1 ), normal ECG showing P waves, QRS complex and T wave; (B) post-treatment (day 10) vehicle group, an elevated ST elevation was observed (indicated by arrows); and (C) post- treatment (day 10) paracetamol-treated group, ST elevation was improved (arrow) The ECG was monitored and recorded over a time of one minute.

Figure 7 shows the nitrite/nitrate (Nox) concentration in plasma from animals treated with vehicle and paracetamol. *<0.05 compared to vehicle.

Figure 8 shows necrosis in ischemic heart after myocardial infarction (A) and capillary vessels observed in left ventricles in vehicle (B) and paracetamol (C) .

Figure 9 shows gene expression of NOS and COX in vehicle (lane 1), and paracetamol (lane 2) treated groups after Ml. a: housekeeping gene GAPDH; b: iNOS; c: nNOS; d: COX-1; e: COX-2. *p<0.05 compared to vehicle.

Figures 10 (A, B, C, D, E) report the results related to the gene expression shown in Figure 9 for paracetamol as compared to the housekeeping gene GAPDH. Figure A: eNOS; Figure 10 B: iNOS; Figure 1OC: nNOS; Figure 1OD: COX-1 and Figure 1OE: COX-2.

Detailed description of the invention

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole contents of such bibliographic references are herein incorporated by reference.

The drug Paracetamol (N-acetyl-para-aminophenol, para-acetyl-amino-phenol or acetaminophen), while being widely used as an analgesic, has received little attention as an alternative to aspirin in the treatment of cardiovascular disease.

Paracetamol exhibits both analgesic and antipyretic activity and as such is frequently classified alongside NSAIDs such as aspirin. However, paracetamol differs from the NSAIDs in a number of important ways. For example, unlike NSAID, paracetamol exhibits little or no anti-inflammatory activity in animals (Glenn et aλ,1977; Seegers et al., 1981) and shows very limited, if any, anti¬ inflammatory activity in man (Lokken & Skjelkbred, 1980). Furthermore and most importantly, unlike NSAIDs1 paracetamol usage is associated with minimal incidence of gastrointestinal damage (Stern et al., 1984).

The limited antinociceptive, as well as the almost complete lack of anti¬ inflammatory activity of paracetamol, has frequently been ascribed to the (at best) very weak cyclooxygenase (COX) inhibitory activity of this compound (Abdel-Halim et al., 1978; Bippi & Frolich, 1990).

In the ex vivo reperfusion model used in the two studies by Merrill et al., (2001) and Merrill (2002), the authors indicated that paracetamol was able to attenuate the production of hydroxy! radicals during reperfusion. However, there was no suggestion or indication that the same mechanism would apply in vivo. Accordingly, that such putative effects of paracetamol can be applied in non- reperfusion models of ischemia is not apparent. Further, data from ex wVo experiments may not be readily translated to in vivo models.

Accordingly, up till today, no indication or suggestion has been given that paracetamol or a derivative thereof may be used in the prophylaxis and/or therapeutic treatment in permanent occlusion models of ischemia in vivo.

According to a first aspect, the present invention provides a method of treatment of ischemia in a mammal comprising administering a therapeutically effective dose of paracetamol and/or a derivative thereof. In particular, the ischemia is cardiac ischemia.

Further, the mammal may be a human being and the therapeutic dose may be administered before and/or after the onset of ischemia.

According to another aspect, the present invention provides the use of paracetamol and/or its derivative to induce cardioprotective effects in a mammal at risk of myocardial infarction. The cardioprotective effects comprise reducing infarct size, decreasing gene expression of NOS, decreasing gene expression of COX, increasing antioxidant enzyme activities, increasing capillary density and improving cardiac function.

Paracetamol is not known as an anti-inflammatory compound. As such, the finding that paracetamol exhibits a significant reduction of COX-2 expression in Ml heart than vehicle (p<0.05) was unexpected and show that these two drugs may be useful as potent inhibitors for COX-2. Based on our results reported in the examples, tables and/or figures herein included, it can be readily seen that administration of paracetamol in conjunction with a further cardioprotective compound that exerts its action via a different mechanism, can have added or synergistic benefits. Therefore, a person skilled in the art will appreciate that it is possible to administer a further cardioprotective compound such as ramipril with paracetamol. It will also be appreciated that the further cardioprotective compound may be aspirin. Aspirin may be administered at a dosage too low to cause gastrointestinal damage, but this sub-effective dosage may not have any cardioprotective effects when administered alone. However, when supplemented by paracetamol and/or a derivative thereof, a desired level of cardioprotective action may be obtained from the conjunctional use of these compounds.

The timing of the administration of the further compound is not critical and may be done at the same time as the administration of the paracetamol or it may be separated by a span of minutes or even hours.

According to another aspect, the present invention provides a method to induce cardioprotective effects in a mammal at risk of myocardial infarction, the method comprising: administering a therapeutically effective dose of paracetamol and/or a derivative thereof before onset of the ischemia and/or administering a therapeutically effective dose of paracetamol and/or a derivative thereof after the onset of ischemia. In particular, the ischemia is cardiac ischemia and the cardioprotective effects comprise reducing infarct size, decreasing gene expression of NOS, decreasing gene expression of COX, increasing antioxidant enzyme activities, increasing capillary density and improving cardiac function. The present inventors found that the administration of paracetamol resulted in reduced mortality indicating the clinical significance of the cardioprotective effect of the therapeutically effective dose of this drug. The present invention offers an alternative therapeutic approach to Ml and high- risk Ml patients. The effect of paracetamol in reducing mortality and infarct size are greater than ramipril (an angiotensin-converting enzyme [ACE] inhibitor with indication for the treatment of hypertension and Ml (Zhu et al, 1998).

Paracetamol is a very much more cost-effective drug than ACE inhibitors. Over decades of evaluation in man, it is clear that, at least at the doses effective in the rat, paracetamol has very few side effects following chronic administration over long periods in man.

In addition, as the cascade of deleterious physiological events following ischemia among various organs and organ systems (eg stroke or cerebral ischemia) are common, a person skilled in the art will appreciate that the present invention can also be applied to these other organs and organ systems to obtain the desired prophylactic and therapeutic outcomes. For prophylaxis, administration of low-dose paracetamol at the experimental dose (equivalent to a daily dose of 375mg for a 75kg human) is sufficient.

However, the present invention is not limited by this dosage. Continued administration of these compounds throughout an ischemic episode should maintain the beneficial effects of the compounds.

The administration of the compound of the present invention by intraperitoneal injection rather than oral gavage is merely for convenience and the compound can be administered by any practical route as suits the clinical requirements. Also, it can be appreciated that paracetamol is the active compound. Therefore, any other derivative based on paracetamol comes under the scope of the present invention. According to another aspect, the present invention provides use of paracetamol and/or a derivative thereof for the preparation of a medicament for the treatment of ischemia in a mammal. In particular, the ischemia is cardiac ischemia. There is also provided the use of paracetamol and/or a derivative thereof for the preparation of a medicament for inducing cardioprotective effects in a mammal at risk of myocardial infarction. Further, the medicament may be administered before and/or after the onset of ischemia. The medicament may further comprise at least one pharmaceutically acceptable diluent, excipient and/or carrier.

The medicament according to the invention may comprise any dose of therapeutically effective paracetamol and/or a derivative thereof. In particular, the medicament comprises a low-dose of paracetamol and/or a derivative thereof at the dose indicated in the examples. For example, the dose of paracetamol for both prophylaxis and treatment may be equivalent to a daily dose of 375mg respectively for a 75kg human. The effective dosage may range from 150 to 650mg for paracetamol. In particular, the effective dosage may range from 250 to 500mg for paracetamol.

Under the present invention, the paracetamol and/or derivative thereof, as active ingredients, together with any further cardioprotective compound, may be administered in any suitable route of administration into the patient.

For example, paracetamol may be administered by oral route. This route is usually convenient and acceptable to the patient as the patient remains lucid. However, after an ischemic episode, the patient may not be capable of accepting the compound by mouth. Accordingly, alternative routes or modes of administration (eg by injection) may be used. However, these alternative modes of administration may be chosen independently by the problem which may be caused by the oral administration. The paracetamol, as active ingredient or active compound, may be prepared by any suitable method. In general, such preparatory methods include the step of bringing the active ingredient paracetamol into association with a pharmaceutically acceptable carrier, excipient and/or diluent, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

For oral administration, the active compounds may be provided as a tablet, aqueous or oil suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup, elixir, spray or beverage. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutically acceptable compositions and such compositions may contain one or more of the following agents: sweeteners, flavoring agents, coloring agents and preservatives.

The sweetening and flavoring agents will increase the palatability of the preparation. Tablets containing extracts in admixture with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture are acceptable. Pharmaceutically acceptable means that the agent should be acceptable in the sense of being compatible with the other ingredients of the formulation (as well as non-injurious to the patient). Such excipients include inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; and lubricating agents such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed. Formulations for oral use may also be presented as hard gelatin capsules wherein the compound (active ingredient) is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil. In some embodiments, aqueous suspensions may contain an extract of the invention in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, dispersing or wetting agents, one or more preservatives, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspension may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by an added antioxidant such as ascorbic acid. Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide one or more extracts in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives.

Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The active compounds for parenteral administration may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1,3-butanediol. Suitable diluents include, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectable preparations.

For parenteral administration, solutions of the active compound(s) in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques will known to those skilled in the art.

Additionally, it is also possible to administer the active compounds of the present invention topically and this may be done by way of creams, jellies, gels, pastes, patches, ointments and the like, in accordance with standard pharmaceutical practice.

The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsions may also contain sweetening and flavoring agents.

The active compounds may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

The active compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide phenyl, polyhydroxyethylaspartamide- phenol, or polyethyleneoxide-polylysine substituted with palmitoylresidues. Furthermore, the active compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The amount of extract that may be combined with the carrier material to produce a single dosage form will vary depending upon the patient treated and the particular mode or route of administration.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, the person skilled in the art will understand that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modifications. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other non-human primates.

According to another aspect, the invention provides kit for treatment and/or prevention of ischemia in a mammal comprising (i) a therapeutically effective dose of paracetamol and/or a derivative thereof, or a pharmaceutically acceptable salt thereof; (ii) at least one pharmaceutically acceptable diluent, excipient and/or carrier; and (iii) instructions for use in treating and/or preventing ischemia. In particular, the kit is the treatment and/or prevention of cardiac ischemia.. The mammal may be a human being or a non-human mammal.

Having now generally described the invention, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present invention.

In the following examples, where relevant, statistical comparison between groups were analysed using one-way analysis of variance (one-way ANOVA). All data were presented as mean ± SEM. Significant differences among the groups were defined by a p value of less than 0.05.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001 ).

Example 1 - Treatment procedure

Male Wistar rats (200g-250g) were obtained from the Laboratory Animal Centre, National University of Singapore (NUS). Animals were housed under diurnal lighting conditions and fed standard rat chow and water ad libitum according to regulations of animal care and experiments by NUS. The project was approved by the institution's animal research ethics committee.

The rats were randomly assigned to two different treatment groups, namely paracetamol 5mg/kg/day (n=79), and a vehicle group 5mg/kg/day (n=80) (10% Tween 20 dissolved in 0.9% sodium chloride).

The rats were injected intra-peritoneally once daily with their respective treatment drug. The treatment started seven days before the surgery (day 8) to induce myocardial infarction (Ml) and the tissue samples were collected two days after Ml (day 10) for morphological and biochemical and molecular studies. Blood samples were collected as needed.

The mortality rates were as follows. Mortality rate for vehicle (n=80) and paracetamol (n=79) groups were 37.5% and, 21.5% (Fig. 2, Table 1).

Table 1 : Mortality rate and infarct size of vehicle, paracetamol- treated group at the end of the ten-day treatment. *p<0.05 compared to the vehicle group.

Group Mortality (%) Infarct size (%)

Vehicle 37-5 44-8±6-1 Paracetamol 21.5* 31.3±5.6*

The mortality in the paracetamol-treated group was significantly less after Ml (p<0.05) compared to the vehicle group. It was observed that rats treated with paracetamol had lower mortality rates compared to the vehicle group.

The mortality of the paracetamol-treated group showed a lower rate compared to the vehicle group. Paracetamol reduced mortality significantly compared to vehicle group (p<0.05). Paracetamol showed significant reduced infarct size after Ml (p<0.05).

Example 2 - Animal model of myocardial infarction

Ml was induced by a permanent ligation of the left descending coronary artery (Fig. 4; Stauss et al, 1994; Zhu YZ, et al, 2000) thereby permanently occluding it. Treatment was continued for another two days after surgery. At the end of the treatment period, the animals were sacrificed. Their hearts were collected and after a quick examination for gross pathology, they were immersed in liquid nitrogen and stored at -800C for further studies. Livers were similarly collected and processed for enzyme studies. A number of hearts were processed for capillary density measurements as described below

Example 3 - Hemodynamic measurement

Thirty to thirty-three rats from each treatment group were randomly selected for the measurement of BP. Blood pressure (BP) in the experimental animals was measured using the NIBP (Non-invasive Blood Pressure) System (ML125/R, ADInstruments Powerlab System, USA) and electrocardiogram (ECG) (BioAmp amplifier, (Powerlab, ADInstrument, USA) readings were measured three times per rat throughout the animal model study BP and ECG readings were recorded prior to the start of the experiment on day 1. ECG was measured for one minute using the Animal BioAmp amplifier (ML136, ADInstruments Powerlab System, USA). The rats were anaesthetized using chloral hydrate (5mg/kg Lp) before their ECG readings were taken. Subsequently, readings were taken again on day 7 prior to the operation. The last reading was measured prior to the sacrifice of the animal on day 10. Triplicate readings of BP and ECG were obtained for comparison.

Paracetamol administration in this way did not have any discernible effects on blood pressure BP (Fig. 1). On day 1 (the day when treatment was started) the values were: vehicle, 122.7±9.5 mmHg; and paracetamol, 149.2±26.3 mmHgOn day 7 (the day before surgery) the data were: vehicle, 140.4±14.4 mmHg; and paracetamol, 143.2±9.2 mmHg. On day 10 (2 days after Ml), the results were: vehicle, 122.4±31.6 mmHg; and paracetamol, 127.9±12.3 mmHg;. It can be concluded that there is no significant change in blood pressure throughout the treatment window.

Electrocardiogram (ECG) readings were measured on the first, seventh and tenth day after the start of drug treatment. Six rats from each treatment group were randomly selected. There was no difference of ECG on days 1 and 7 (before Ml) for each group (Fig. 6A). However, differences in the ECG patterns were observed between the groups at day 10 (after Ml, ECG charts are shown in Fig. 6 B, C and D). On day 10, all groups showed significant ST elevation, which is characteristic of Ml. However, in the vehicle group, the ST-elevation had the longest duration (arrows in Fig. 6B), followed by the paracetamol- treated group. .The ST elevation improved (arrows in Fig. 6C and D) in the drug-treated group as compared to the vehicle group, indicating that the drug resulted in better myocardial recovery (ST elevation reduced, Fig. 6C).

Example 4 - Pathology: measurement of infarct size and capillary density

The infarcted area was identified by 2,3,5-Triphenyltetrazolium chloride (TTC) staining according to the method described by Ji et al, 2003. In brief, the infarcted area was judged from both epicardial and endocardial sides and outlined on paper, cut and weighed. The infarct size is defined as a ratio of the left ventricular infarct area to the whole left ventricular area. The sizes of the left ventricle and the infarct area were evaluated by computer using Scion Image (Scion Inc., California, USA) which is a well-recognized software to analyze the size/intensity of unregular area from tissue or gel (http://www.scioncorp.com). A corresponding reduction in infarction size in the groups with better survival rate was noted (Fig. 3, 5). Thus, infarction size (n=6-10) for the vehicle group was 44.8% (± 6.1 %) of the left ventricle (LV) while the infarction size for paracetamol group was 31.3% (± 5.6%) of the LV. All rats in the three treatment groups developed myocardial infarctions as confirmed by the TTC staining. Infarct sizes were significantly decreased in the paracetamol-treated groups as compared to the vehicle group (p<0.05). For capillary density measurements, the hearts were isolated and three transverse sections from each heart were obtained. Each section was stored individually in histology processing cassettes that were immersed in 10% formalin. Subsequently, the cassettes containing the tissues were processed using a LEICA TP1020 Automatic Tissue Processor (Meyer Instruments Inc.). After processing, the tissues were embedded in paraffin wax using LEICA EG1120 Paraffin Dispenser. Following, three consecutive paraffin sections of 6μm thick were sliced using a microtome (Leica RM 2135). Triplicates were made. The sections were immersed in 0.5% Sta®-On solution at 37°C for approximately 5 minutes. The sections were next mounted on a glass slide which was then tapped dry and stored in a slide holder.

For staining, the paraffin sections were deparaffinised and rehydrated by soaking the slides in xylene for approximately 10 minutes. This is followed by 100% ethanol, 95% ethanol, 80% ethanol and deionised water in the stated order. Next, the slides were stained in hematoxylin followed by tap water for three minutes and five minutes respectively. Eosin counterstaining was followed by soaking the slides in eosin for 30 seconds to 2 minutes, depending on the age of the eosin. The slides were dehydrated in graded concentrations of ethanol, starting from 70%, followed by 80% and absolute ethanol until excess eosin was removed. Slides were then soaked in xylene for 10 minutes to clear the ethanol. Thereafter the slides were mounted with a coverslip in a drop of Permount mounting medium and left to dry overnight. For determination of capillary density, sections of the myocardium were observed under a light microscope under 400X magnification. The capillaries present in the left ventricle were then counted and expressed as number per square millimetre. The capillaries can be easily distinguished by the thin layer of endothelium surrounding its walls. Capillary distribution patterns within the left ventricle (ie. infarct area, area at risk, non-infarct area) were also noted. Significantly fewer capillaries were observed in the left ventricles of the vehicle group (246.1+27.5) as compared to the paracetamol-treated (336.6±17.9) - treated (302.9±13.5) group (Table 2) In the infarct area, myocardial necrosis was observed in the tissue sections obtained from the two groups (arrow, Fig. 8A). It was generally observed that there were virtually fewer vessels the vehicle group (arrows, Fig. 8B).

Table 2: Nitric oxide concentration in plasma and capillary density counted in different groups. Values are expressed as mean ± SEM (n=3). *p<0.05 compared to the vehicle group.

Group Concentration (μm) No of capillaries per mm2 Vehicle 11.4+0.3 246.1 ±27.5 Paracetamol 6.4+0.4* 336.6117.9*

Under normal physiological conditions, capillary proliferation is generally atypical in the adult mammalian heart Crisman et al (1985). However, this rare phenomenon may be observed under certain pathological conditions such as Ml (Ji et al;2003). Vascular endothelial cells proliferate during acute Ml1 resulting in angiogenesis which compensates for the limited blood flow to the ischemic myocardium. This repair mechanism corrects the imbalance between the perfusion capacity of coronary vessels and the need for oxygen and nutrients in the ischemic myocardium (Zhu et al; 1997). paracetamol was observed to enhance the angiogenetic effect. Example 5 - Measurement of nitrite/nitrate concentration in plasma

Plasma (0.5ml) was filtered using Milipore microcon (Milipore, Billerica, USA) filters and centrifuged at 10,000 rpm for 20 mins at 40C. Flavin adenine dinucleotide (FAD; 25μl 0.1 mM), 50μl 1mM NADPH and 10μl nitrate reductase (10units/ml) were added to the supernatant to convert the nitrate to nitrite and the mixture incubated at 370C for 30 mins. The reaction was terminated by addition of 5μl lactate dehydrogenase and 50μl pyruvic acid which will oxidize any unreacted NADPH. The addition of Greiss reagents allowed the formation of a purple-pink azo dye and the concentrations of nitrite and nitrate were determined spectrophotometrically at 543nm using Magellan software as described by Whiteman et al (2002).

The nitrite/nitrate (NOx) concentration is the stable end product of NO in plasma. The paracetamol (6.39±0.42 μM) -treated group showed a significant attenuation of NOx level as compared to vehicle group (11.4± 0.33 μM) (p<0.05) (Fig. 7).

These results showed that paracetamol had a significant attenuation of NOx level as compared to vehicle group. Paracetamol significantly reduced the NOx level in plasma indicating that iNOS-derived NO is also significant lowered.

Example 6 - Hepatic Antioxidant Assays

Hepatic antioxidant assays measuring catalase (CAT), superoxide dismutase (SOD), glutathione perioxidase (GPx) and glutathione S-transferase (GST) activity were performed according to Ji et al (2003). Six liver samples from each treatment group were used in the assays. The frozen liver tissue (1g) was homogenized in 1mL phosphate buffer (lOmmol/L, pH 7.5) by a Polytron homogenizer (Janke & Kunkel, Germany). The homogenate was centrifuged at 1,00Og for 10 min at 4°C; the supernatant was then divided into two parts for different enzyme assays.

For SOD and CAT activity assays, the supernatant was centrifuged at 2,30Og for a further 10 min; while the supernatant used for GPx and GST activity assays was further centrifuged for 90 min at 10O1OOOg at 4°C in an ultracentrifuge (Beckman L8-70; Beckman Instruments Inc., Fullerton, CA, USA). After centrifugation, the pellets were discarded and the supematants used for the assessment of enzyme activity.

The supernatant used for SOD and CAT assays was defined as supernatant A hereafter; while the supernatant for GPx and GST was designated as supernatant B. Both supematants A and B were diluted with phosphate buffer (10mmol/L, pH 7.5). The dilutions of the supematants for SOD, CAT, GPx and GST assays were 1 :10, 1 :20, 1 :20 and 1 :60, respectively.

SOD activity was determined based on the ability of the enzyme to inhibit auto- oxidation of pyrogallol (10mmol/L). The inhibition of pyrogallol oxidation by SOD was monitored at 420nm in a spectrophotometer and the amount of enzyme producing 50% inhibition was defined as one unit of enzyme activity.

CAT was assayed by mixing with 1 mL 1 :20 diluted supernatant A and 0.01 ml_ ethanol and then incubating in ice for 30 min. Twenty microlitres of the incubated supernatant was removed and added to a 980μL assay mixture containing 50μL of 1mol/L Tris-HCL ethylenediamine tetraacetic acid (EDTA) buffer (5mmol/L EDTA, pH 8.0), 900μL of 20mmol/L H2O2 and 30μL distilled H2O. The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbency at 240nm, since H2O2 absorbs light at this wavelength. For the assay of GPx activity, a reaction mixture was made up with 100μL 1mol/L Tris-HCL EDTA buffer (5mmol/L EDTA, pH 8.0), 20μL glutathione (lOOmmol/L), 100μL glutathione reductase (10U/mL), 100μL nicotinamide adenine dinucleotide phosphate (NADPH) (2mmol/L), 10μL diluted supernatant B (1 :20) and 660μL distilled H2O. After a 10-min reaction period at 25°C, 10μL t- butyl hydroperoxide was added to the mixture and mixed vigorously. The rate of disappearance of reduced NADPH was immediately measured spectrophotometrically at 340nm.

GST was measured in a reaction mixture made up of 200μL phosphate buffer (pH 6.5), 20μL 1-chloro-2,4-dinitrobenzene (CDNB) in ethanol (25mmol/L) and 680μL H2O. The mixture was kept at 25°C for 10 min, then 50μL glutathione (20mmol/L) was added. After thorough mixing, 50μL of diluted supernatant B (1 :60) was added to the final mixture. The activity of GST was estimated by measuring the change in optical density at 340nm due to formation of CDNB- glutathione, since CDNB-glutathione absorbs light at 340nm. All assays were performed in triplicates at 25°C.

In the CAT assay, a significant increase in enzyme activity was observed when the paracetamol- (0.48±0.02 unit/mg protein) group was compared to the vehicle group (0.32±0.04 unit/mg protein) (p<0.05). However, there was no significant change in enzyme activity between the three treatment groups in the GPx and GST assays except there was a significant decrease in enzyme activity when paracetamol group compared to the vehicle group in the GST assay.

Table 4: Measurements of antioxidant enzyme activities in the liver in vehicle and paracetamol- treated rats. Values presented are unit/mg protein. *p<0.05 compared to the vehicle group. *p<0.05 compared to the paracetamol group.

Enzyme Vehicle Paracetamol CAT 0.32±0.04 0.48±0.02* GPx 0.62±0.10 0.50±0.12 GST 7.95±0.12 2.99±0.08 SOD 8.27±2.01 8.78+0.76

Example 7 - RNA extraction and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) amplification

Total RNA was extracted according to a standard protocol (Zhu et al, 2000). Total RNA of each sample was reverse-transcribed into first-strand complementary DNA (fs cDNA) and amplified using the OneStep RT-PCR kit (Qiagen, Germany) as reported in Lue et al (2003). One microgram of total RNA from each pooled sample was used in the reverse transcriptase-polymerase chain reaction. The RT-PCR was carried out in a total volume of 20μl, containing 4μl of Qiagen OneStep RT-PCR buffer, O.δμl dNTP, 1.2μl of sense primer, 1.2μl of anti-sense primer, 0.8μl Qiagen OneStep RT-PCR Enzyme Mix. RT-PCR was carried out in a thermocycler (GeneAmp PCR System 2700). The samples were first incubated at 5O0C for 30 minutes to allow synthesis of cDNA by reverse transcription. Samples were then subjected to PCR amplification using primers specific for COX-1 , COX-2, eNOS, nNOS, iNOS and GAPDH (gyceraldehyde 3-phosphate dehydrogenase). The three PCR steps of denaturing, annealing and extension were carried out at 940C for 30 seconds, 55°C for 45 seconds, and at 72°C for 30 seconds respectively. The annealing temperature was set at 550C for all six primers. The primer sequences and their product sizes are reported in Table 4.

Table 4: Gene sequences of GAPDH, COX-1, COX-2, eNOS, iNOS and nNOS and their corresponding product sizes and sequence listings numbers Gene Sequence Product Access no

sense 5'-CATGGTCTACATGTTCCAGT-3 (SEQ ID NO: 1 ) 349bp XM_221353 antisense δ'GGCTAAGCAGTTGGTGGTGC-S' (SEQ ID NO: 2)

COX-1 sense 51-CGAGGATGTCATCMGGAG-31 (SEQ ID NO: 3) 350bp S67721 antisense δ'-TCAGTGAGGCTGTGTTAACG-S' (SEQ ID NO: 4)

COX-2 sense δ'-CTGTATCCCGCCCTGCTGGTG-S' (SEQ ID NO: 5) 282bp AF233596 antisense 5'-ACTTGCGTTGATGGTGGCCTGTCTT-3' (SEQ ID NO: 6)

eNOS sense δ'-CTGGCAAGACCGATTACACGA-S' (SEQ ID NO: 7) 423bp NM 021838 antisense δ'-CGCAATGTGAGTCCGAAAATG-S' (SEQ ID NO: 8)

iNOS sense δ'-CTACCTACCTGGGGAACACCTGGG-S' (SEQ ID NO: 9) 442bp NM 012611 antisense δ'-GGAGGAGCTGATGGAGTAGTAGCCG-S' (SEQ ID NO: 10)

nNOS sense 5'-AATGGAGACCCCCCTGAGAAC-31 (SEQ ID NO: 11 ) 381bp NM 052799 antisense δ'-TTCAGGAGGGTGTCCACCGC-S' (SEQ ID NO: 12)

These results in Fig. 1OA showed that eNOS expression was significantly lower in paracetamol group (0.86-fold) compared to vehicle group

In contrast, not all groups produced distinct inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) PCR product bands. The level of iNOS mRNA expression was lower in paracetamol (0.83-fold) treated rats compared to vehicle group (p<0.05) (Fig. 10B). Very faint nNOS mRNA expression was detected in the paracetamol group (0.23-fold) after 48 hours of Ml. A significantly lower level of nNOS production in paracetamol treated group was observed (p<0.05) compare to the vehicle group.

The paracetamol treated group exhibited a significant reduction of COX-1 (0.75- fold) and COX-2 expression (0.79-fold) (p<0.05) after Ml compare to vehicle group (Fig. 10D and E respectively). COX-1 is expressed constitutively in most tissues and is involved in maintaining physiological functions, whereas COX-2, which is almost undetectable in basal conditions, is dramatically up regulated by pro-inflammatory and mitogenic stimuli, such as cytokines, growth factors and bacterial toxins, thus playing a role in inflammation, infection and malignant cell proliferation (Bovill, 2003). Since inflammation constitutes an important feature of ischemic heart failure especially in the initial phase of Ml, therefore, inhibition of pro inflammatory prostanoids through the use of a selective COX-2 inhibitor could significantly ameliorate myocardial injury and hence improve myocardial function. References

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