WO2004056376A1

WO2004056376A1 - Pharmaceutical formulations containing nitrate

Info

Publication number: WO2004056376A1
Application number: PCT/SE2003/002052
Authority: WO
Inventors: Jon Lundberg; Eddie Weitzberg
Original assignee: Nitricare Hb
Priority date: 2002-12-20
Filing date: 2003-12-19
Publication date: 2004-07-08
Also published as: SE0203817D0; AU2003291600A1

Abstract

The use of nitrate (NO3-) in pharmaceutical formulations, e.g. in combination with a pharmaceutically active substance has been found to prevent or alleviate known side effects, as well as enhance the effect of many pharmaceutically active substances without the need for modification of the active substance itself. The invention also presents methods of treatment, based on the administration of nitrate.

Description

Pharmaceutical formulations containing nitrate Field of the invention

The present invention relates to a formulation comprising nitrate (NO3 ) in combination with a pharmaceutically active substance, for medical use. The invention also presents methods of treatment, based on the administration of nitrate, as well as the use of nitrate for the manufacture of medicaments for specific indications.

Background of the invention

Nitric oxide (NO) and chemically related compounds are involved in a variety of physiological events including vascular regulation, neurotransmission, platelet function and host-defence. An adequate NO production is important for the integrity of many major organ systems including the cardiovascular system, the airways and the gastrointestinal tract.

NO-liberating drugs (e.g. nitroglycerine) have been used for medical purposes for more that a century in treatment of cardiovascular disorders, e.g. angina pectoris. More recently novel compounds have been developed that supposedly act through release of NO or by increasing its duration of effect. Examples of such compounds include sildenafil (Viagra®) used for erectile dysfunction and nitro derivatives of already existing drugs e.g. acetylsalicylic acid (NCX 4016, NicOx S.A., France) intended to be used to enhance the effect of the parent compound while having less troublesome side effects (e.g. gastrointestinal damage). Since NO is central in so many physiological and pathophysiological events, the therapeutic potential for drugs that affect this system is almost unlimited.

Nitrate (NO3 ) is ubiquitous in nature, partly as it is a very thermodynamically stable molecule. However, both plants and certain bacteria use it as a source of nitrogen for incorporation into protein as amine groups (RNH2). To accomplish this conversion, plants have developed a range of enzymes, which accomplish the eight-electron reduction required, using energy derived from photosynthesis. Green, leafy plants, such as lettuce often contain large amounts of nitrate, especially if they are grown under low light conditions [1]. There has been great concern about the amount of nitrate in our diet, as it was found in the mid 1970s that this anion was handled in a peculiar way in the human body [3,4,5]. When swallowed it is rapidly absorbed and at least 25% is concentrated in the salivary glands by an as yet uncharacterised mechanism, so that the nitrate concentration of saliva is at least 10 times that of plasma. The nitrate is then rapidly reduced to nitrite (NO2") in the mouth by mechanisms, which will be discussed below.

Saliva containing large amounts of nitrite will be acidified in the normal stomach to produce nitrous acid, which could potentially nitrosate amines to form N-nitrosamines [6,], which experimentally are powerful carcinogens [7]. From this theoretical understanding of nitrate metabolism a number of studies have been performed which looked at the relationship between nitrate intake and cancer (particularly gastric cancer) in humans. In general it was found that there was either no relationship or an inverse relationship, so that those individuals who had a high nitrate intake had a lower rate of cancer [8,9,10]. Similarly, in animal studies, it has been generally impossible to demonstrate an increased risk of cancer (or any other adverse effect) when nitrate intake is increased [11], Despite this fact the authorities in the western world are spending copious amounts of money in restricting dietary intake of nitrate.

The reduction of nitrate (NO3 ), e.g. by bacteria, results in the formation of nitrite (NO₂ ^"). Under certain circumstances this nitrite can be further reduced to nitric oxide (NO). It has been shown that large amounts of NO are produced from acidified nitrite in saliva when swallowed [12, 13]. These were the first two studies to suggest a non- enzymatic production of NO occurring in humans. Acidification of nitrite can give rise to a variety of nitrogen oxides and related compounds (e.g. NO, NO⁺, N₂O₃, NO₂, s- nitrosothiols, HNO₂). These compounds can have significant biological effects including killing of bacteria virus and fungi, increase in blood flow, regulation of protein function, regulation of secretions (e.g. mucus production, ion transport) and more [12, 13, 16].

Many useful drugs have limitations in their clinical use due to troublesome side effects. A classical example is gastrointestinal (Gl) damage (bleeding, erosions) seen during treatment with acetylsalicylic acid (ASA) or other non-steroidal-anti- inflammatory-drugs (NSAID) compounds (17). NSAID compounds act by inhibiting certain enzymes e.g. Cyclooxygeneses (COX-1 and COX-2) as well as tromboxane production. These drugs are used to treat pain, inflammation and in lower doses they can be used to prevent certain cardiovascular diseases (e.g. myocardial infarction by inhibiting intravascular blood clotting). In many cases (e.g. arthritis, cardiovascular protection) the drug is taken over a long period of time. Despite the low dose, when often used these drugs have side effects on Gl tract. This is rather common, and constitutes a factor limiting their use.

This major problem has forced the pharmaceutical industry to try to develop novel NSAID compounds with less gastrointestinal toxicity. Such compounds are now available and are named selective COX-2 inhibitors. These drugs cause only half as many serious Gl complications as conventional NSAIDs and have been an enormous commercial success. However COX-2 inhibitors seem to have the same degree of other side effects (e.g. renal toxicity) and the risk of thrombosis is probably increased (15). Another approach that has been taken to reduce the Gl and renal toxicity of NSAIDs is the coupling to these drugs of a nitric oxide (NO) releasing moiety (15). NO has been shown to exert protecting effects on the stomach and to mediate many components of Gl mucosal defence. The exact mechanism of action of these novel compounds act is currently not known.

Prior art

US 2002155174 discloses an anti-fungal, anti-viral and/or anti-bacterial composition comprising an alkali metal nitrate, and an acidifying agent, e.g. salicylic acid or ascorbic acid. The anti-fungal, anti-viral and/or anti-bacterial effect is attributed to NO released in the mouth and Gl tract. Duncan, C. et al. (Comp Biochem Physiol, Vol. 118A, No 4, pp. 913-948, 1997) discuss the importance of dietary nitrate intake, oral nitrate reduction and enterosalivary nitrate circulation as mechanisms of protection against oral and gastrointestinal diseases.

Lanas, A. et al. (J Internat Med Research, 1998; 26: 120-128) have studied the effects of organic nitrate drugs on gastrointestinal bleeding in a retrospective study, finding indications that nitrate use was associated with a reduced risk of upper gastrointestinal bleeding and that the combination of low-dose aspirin and nitrate use was not associated with an increased risk of bleeding, as was the case for chronic low-dose aspiring use alone.

Summary of the invention The present invention as set forth in the attached claims, incorporated herein by reference, relates to a pharmaceutical composition that includes nitrate (NO₃ ^") given either alone or in combination with another pharmaceutically active agent for medical purposes. The invention also makes available a method of treatment, as defined in the claims, incorporated herein by reference. The invention also includes the use of nitrate for the manufacture of pharmaceutical compositions as specified in the description and claims.

Short description of the drawings

The invention will be described in closer detail in the following description, examples, and attached drawings, in which

Figure 1 schematically shows an example of a formulation where granules of an active substance (1 , 2) are enclosed in a capsule (3) together with a source of nitrate (4). Part of the granules of active substance (2) are shown with thicker side walls, to illustrate that they can be delayed release coated. Figure 2A schematically shows an example of a formulation where a core of an active substance (5) is surrounded by the source of nitrate (6) in the shape of a tablet or the like.

Figure 2B shows an embodiment of the above, where the core of the active substance (5) has a coating (7) delaying the release of said active substance. Figure 3 schematically shows an example of a formulation where granules of an active substance (8, 10) are embedded in a tablet (9) or the like, substantially consisting of nitrate and suitable excipients and/or tabletting aids. Part of the granules of active substance (8, 10) are shown with thicker lines (10) indicating that they are slow release coated. In any of the embodiments above, an outer coating masking the taste and/or making it easier to swallow the capsule or tablet may be provided, as is common in the art.

Description It remains to find alternative pharmaceutical compositions preventing or alleviating the known side effects of NSAID and COX-2 compounds, preferably without redesigning the active compounds themselves.

Another aim of the present invention is to enhance the effect of known pharmaceuticals, again without modification of the active compounds. Further aims of the invention, or problems associated with the prior art, and the solutions offered by the invention, and their advantages, will become apparent to a skilled person after reading the description and examples.

Surprisingly, and quite contrary to the general opinion, the present inventors found that nitrate when given orally results in significantly increased levels of nitrite in the blood, stimulates release of NO and can be beneficial to health as such, when given in the formulation described here. This is mostly due to the in vivo generation of nitric oxide, s-nitrosothiols and other biologically active nitrogen oxides from nitrate. It is especially surprising that nitrate can generate NO in humans since nitrate is considered to be the stable final breakdown product of endogenous NO synthesis. By the addition of nitrate to a drug composition many extremely troublesome side effects can be eliminated or markedly reduced. In addition, nitrate may enhance the therapeutic activity of other drugs or act alone. The formulation of nitrate described in this application results in optimal control of uptake of nitrate and any other combined drug to receive maximal therapeutic effect with a minimum of unwanted side effects. In the following nitrate means any formulation that contains inorganic nitrate (NO₃ ^" ) including inorganic nitrate salts (e.g. sodium nitrate, NaNO₃, potassium nitrate KNO₃, zinc nitrate Zn(NO₃)₂ xH₂O, lithium nitrate, calcium nitrate, magnesium nitrate, or other metal or alkali metal nitrates) or natural sources of nitrate such as plant extracts from vegetables (e.g. spinach, lettuce, beet root or other plant rich in nitrate). In this context, "nitrate" must be distinguished from "organic nitrate" such as NO- donors e.g. glyceryl trinitrate, isosorbide dinitrate and isosorbide mononitrate, often referred to simply as "nitrate".

According to the present invention nitrate is incorporated into a pharmaceutical preparation either alone or in combination with another pharmaceutically active compound for medical use.

In general nitrate can be given alone e.g. as a separate tablet or packed together with another compound. This will allow for maximal freedom in dosage and timing of intake in relation to any additional drug taken. In a preferred embodiment the composition of a tablet is with nitrate placed outside a core of another compound. The composition should be prepared so that nitrate is released first followed by delayed release and uptake of the inner compound. For example the additional compound is covered (protected) by nitrate during gastric passage and slowly released in the small intestine. An example of composition is a tablet with ASA or another NSAID in the core at a clinically recommended dose. This is covered by nitrate packed so that the ASA compound is not released until after gastric passage. Optionally, the whole tablet is covered by a thin surface layer (dragee) to enhance swallowing and to achieve a neutral taste according to well known practice in the art of galenical formulations. In another example nitrate is given either alone or in combination with another compound in order to treat or prevent gastrointestinal infection. The large amounts of NO and other nitrogen oxides produced in the stomach after nitrate intake will prevent the growth of various pathogens including bacteria, virus fungi and parasites.

The dose of nitrate to be included in e.g. a tablet should be optional depending e.g. on age, gender, body size, patient condition and therapy indication but should preferably be in the interval of 1 - 1000 mg (of NO₃ ^").

Below is a list of indications and examples of drugs that could be used in combination with nitrate. See also Table 1.

General indications for the use of nitrate in the present invention include the prevention, alleviation and/or treatment of drug-induced side effects (e.g. gastrointestinal damage caused by ASA/NSAID, damage by chemotherapy); increasing the effectiveness of other drugs (e.g. in combination with ASA to use in prevention of cardiovascular disorders such as myocardial infarction); prevention, alleviation and/or treatment of any disease.

Table 1. Examples of disorders and types of drugs for use according to the present invention

I. Cardiovascular disorders hypertension hyperlipidemia atherosclerosis - congestive heart disease ischemic heart disease myocardial infarction diabetes Drugs: - Beta-adreno receptor antagonists (e.g. atenolol, metoprolol)

ACE inhibitors (e.g. captopril, enalapril)

Angiotensin II rec antagonists (e.g. losartan)

Statins (e.g. pravastatin, simvastatin)

Digoxin Insulin - Calcium channel bloc_.kers (e.g. nifedipine, felodipine)

Loopdiuretics (e.g. furosemide)

Thiazides (e.g. hydrochlortiazide)

Amiloride

Spironolactone II. Gastrointestinal disorders gastric and duodenal ulcers drug-induced gastrointestinal damage (e.g. caused by ASA/NSAID) Drugs:

ASA - NSAID^'s (e.g. ibuprofen, diclofenak, naproxen)

Cox-2 inhibitors (e.g. celoxicib) glucocorticosteroids (e.g. budesonide, beclometazone)

5-ASA sulfasalazine - proton pump inhibitors (e.g. omeprazole or its enantiomer, pantoprazole)

H2-receptor antagonists (e.g. ranitidin, cimetidine, famotidine) 111. Respiratory disorders asthma rhinitis - sinusitis nasal polyposis chronic obstructive pulmonary disorder (COPD) drugs:

- Beta-2 agonists (e.g. salbutamol, salmeterol, terbutaline) - glμkocorticosteroids (e.g. budesonide, beclometazone)

- leukotriene receptor antagonists

5-lipoxygenase inhibitors cromoglicates ipratroprium bromide IV. Infectious disorders

Any kind of viral, bacterial, fungal or parasitic disorder Drugs: antibiotics antiviral drugs antifungal drugs antiparasitic drugs V. Urologogical disorders

Erectile dysfunction (e.g. sildenafil) overactive bladder urinary tract infection

VI. Inflammatory disorders and pain - rheumatoid inflammatory disorders (e.g. rheumatoid arthritis) pain (headache, muscolusceletal pain, osteoarthitis, tumour pain etc) dermatitis Drugs: paracetamol - ASA

NSAID^'s (e.g. ibuprofen, diclofenak, naproxen)

VII. Neoplastic diseases any neoplasm, e.g. colon cancer Drugs: - ASA

Antineoplastic drugs

VIII. Degenerative disorders drugs used to treat or prevent Alzheimer's disease osteoporosis

There are several known compounds that can be used to facilitate NO release and NO related effects in the body. These include nitrovasodilators (e.g. nitroglycerine and nitroprusside), nitro derivatives of various compounds (known as e.g. NO-ASA, NO-ibuprofen, NO-flurbiprofen, NO-naproxen), fosfodiesterase inhibitors (e.g. sildanafil that increases NO effects in some tissues). Potential limitations with the compounds listed above include marked acute vasodilating effects of short duration (nitrovasodilators) and sometimes complicated synthesis procedures (nitro derivatives). Moreover the release pattern of NO from these drugs is difficult to predict. There are several advantages with the use of nitrate in this context. Nitrate has no acute effects on systemic blood pressure because the generation of NO from nitrate is slow and prolonged and takes place mainly outside the blood stream. If nitrate is placed outside a core of another compound e.g. in a slow release formulation this will result in an optimal timely delivery of both compounds. If the inner compound is e.g. ASA the result will be 1) Less direct exposure of the gastric mucosa to ASA during gastric passage and 2) A timely early increase in gastric levels of NO and related compounds. The increase in NO results in a rapid increase in mucosal blood flow and mucus secretion, thereby protecting the mucosa against the ASA slowly entering the gastric mucosa via the blood stream. 3) In addition, administration of NSAIDs /ASA has been shown to reduce mucosal blood flow and thereby reduce the pH in the sites overlying epithelial damage, ultimately leading to inhibition of restitution and the development of hemorrhagic lesions (17). When combined with nitrate, the pH lowering effects of ASA/NSAIDs will automatically generate more NO and s-nitrosothiols near the epithelial cells because the production of these compounds from nitrite in the stomach are greater at a lower pH.

NO is clearly one very important biological effector produced in the stomach following ingestion of nitrate. Because NO is a gas it will distribute evenly throughout the gastric mucosa and thereby protect all areas exposed to an irritating agent (e.g. ASA). A locally acting solid or liquid drug aimed to protect the gastric mucosa (e.g. sucralfate) will probably not cover the entire mucosa if given orally.

Most drugs undergo metabolism, which may be dependent on liver and kidney function. In addition, some drugs, (e.g. paracetamol) can be metabolized into toxic compounds that may damage major organ systems. The advantage with nitrate is that it is not metabolized to a large extent by mammalian cells. Nitrate is a stable compound that is excreted in urine and saliva. For bioactivation of nitrate in the stomach, bacteria in the oral cavity are required. In this sense nitrate can be regarded as a "pro-drug".

Nitrovasodilators (e.g. nitroglycerine) are also metabolized to nitrate in plasma and therefore these drugs should also give rise to e.g. NO production in the stomach. However there is a profound difference in the pharmacokinetics of nitrovasodilators and nitrate. In the former drugs NO is quickly released at low doses giving profound vasodilatation. Therefore these drugs cannot be given in sufficient doses to yield the amounts of nitrate needed without major effects on blood pressure. Also, by separating nitrate from another bioactive compound as described here it is possible to achieve optimal timing so that the beneficial effects of nitrate (e.g. NO production) begins before biological effects of the other drug.

In another example nitrate could be combined in a drug used for cardiovascular disorders (see Table 1) to enhance the effects of these drugs. One example is to combine nitrate with a statin or an ACE inhibitor to add the cardiovascular protective effects of nitrate reaction products (e.g. s-nitrosothiols, NO).

In another example nitrate is added to any acid inhibiting drugs (e.g. omeprazole or cimetidine see table 1). In such example, an additional supply of a weak acid (e.g. ascorbic acid or citric acid) would help to enhance conversion of nitrite to NO and other nitrogen oxides in the stomach. This conversion is decreased by acid inhibiting drugs since it is pH dependent. When gastric pH increases the reduction of nitrite is slower and thereby less NO and s-nitrosothiols are produced. This procedure will lead to 1) better antimicrobial activity of gastric contents, and 2) less risk of nitrite accumulation in the stomach with potential harmful effects including formation of carcinogens such as nitrosamines. Alternatively, a weak acid could be given alone to patients receiving acid-suppressing drugs in general for the same reasons discussed above.

Alternatively, nitrate can be given in the form of a lozenge, tablet to chew, a water- soluble powder, an i.v. formulation, or as a standard pharmaceutical composition. In other words, the invention makes available a pharmaceutical composition containing nitrate (NO₃ ^") in combination with a pharmaceutically active agent, characterized in that the amount of nitrate is sufficient to prevent, alleviate, or treat side effects associated with said active agent, and/or to enhance the effect of said active agent. Said nitrate is preferably present in the form of a salt chosen among NaNO₃, KNO₃ and other water-soluble nitrate salts, and more preferably in the form of zinc nitrate (Zn(NO₃)₂- xH₂O).

According to one embodiment, said nitrate is present in the form of plant extract.

According to a preferred embodiment, said nitrate is combined with said pharmaceutically active agent in a galenical formulation such that the gastrointestinal tract is exposed first to the nitrate portion of said composition, and later to the pharmaceutically active agent. According to another embodiment, said nitrate is combined with said pharmaceutically active agent in a galenical formulation such that nitrate is released during gastric passage and the release of said active agent is delayed until after gastric passage. Further, one or both of the above embodiments may comprise an outer layer comprising nitrate surrounding said active agent, and/or an additional coating of the nitrate layer.

Further, according to one embodiment, said active agent is present in granulated form, the granules being embedded in nitrate. According to another embodiment, said active agent and said nitrate are present in granulated form, the granules of the active substance being coated so that the release of the active substance is delayed.

In this context, see Figures 1 , 2A, 2B and the short summary of the figures.

Further, a composition according to the invention may comprise or contain suitable pharmaceutically acceptable diluents, excipients, inert carriers, disintegrating agents, colouring agents, sweeteners and the like, as is obvious to a skilled person in the relevant field.

Coated in relation to the active substance means coated in a fashion and to a degree that the active substance is released after gastric passage. Preferably the nitrate, e.g. zinc nitrate, is released before the active substance. It is however also conceived that a portion of the nitrate is enteric coated so that nitrate is released also further in the gastrointestinal tract, after gastric passage. This way the concentration of nitrate will be further increased in the gut and in the blood. More nitrate will thus be available for the enterosalivary cycle of nitrate. According to one embodiment, said pharmaceutically active agent is an NSAID compound, preferably a NSAID compound chosen among acetyl salicylic acid, indometacin, diclofenac, piroxicam, tenoxicam, ibuprofen, naproxen, ketoprofen, nabumetone, ketorolac, azapropazone, mefenamic acid, tolfenamic acid, sulindac, diflunisal, tiaprofenic acid, podophyllotoxin derivatives, acemetacin, aceclofenac, droxicam, oxaprozin, floctafenine, phenylbutazone, proglumetacin, flurbiprofen, tolmetin, and fenbufen, in the form of salts or enantiomers thereof, or mixtures thereof. According to one specific embodiment, said pharmaceutically active agent is acetylsalicylic acid.

According to another embodiment, said pharmaceutically active agent is a COX 1 or COX 2 inhibitor, preferably one chosen among rofecoxib, celecoxib, etodolac, etoricoxib, loxoprofen, lumiracoxib, nimesulide, meloxicam, valdecoxib, tilmacoxib, parecoxib sodium, flosulide, darbufelone mesylate, 2-benzyl-4-isopropoxy-5-(4- methanesulfonylphenyi)pyridazin-3-one and 1 ,2-diarylcyclopentones, in the form of salts or enantiomers thereof, or mixtures thereof.

According to another embodiment, said NSAID is an NO-donating NSAID. Examples of NO-donating NSAIDs are compounds as described in WO 96/32946, cycloalkyls as described in WO 98/25918, such as 2,2-dimethyl-cyclopropane-1-methano, or compounds as described in CN 1144092, or in WO 97/31643, or WO 95/30641 or WO 95/09831 , or WO 94/12463, or WO 94/04484, or WO 00/51988, or US 3,641,127, or WO 99/44595, or WO 02/30866, or US 6,297,260, or WO 02/05318, or WO 02/92072, incorporated herein by reference, or such compounds and their derivatives known to a person skilled in the art.

Similarly, said COX 1 or COX 2 inhibitor may be an NO-donating COX 1 or COX 2 inhibitor.

According to one embodiment of the invention, the amount of nitrate is preferably between 1 and 1000 mg per unit dose.

When the nitrate, or a portion thereof, is present in the form of zinc nitrate (Zn(NO₃)₂' xH₂O), a suitable dose is a dose amounting to an intake of zinc of less than about 10 - 100 mg/day. Depending on the mode of administration, the disease or condition to be treated, and the age and state of health of the patient, different doses can be contemplated. Importantly, the nitrate can be present in the form several metal salts, such as a larger portion of sodium nitrate, which is less toxic even in high doses, and minor portions of e.g. zinc nitrate, magnesium nitrate etc.

When using NSAID compounds, or COX 1 or COX 2 inhibitor(s), the amount of NSAID(s) or COX 1 or COX 2 inhibitor(s) is preferably between 50 and 1500 mg per unit dose.

One preferred embodiment of the invention is a pharmaceutical composition wherein the pharmaceutically active agent is an anti-ulcer agent, chosen among cimetidin, famotidin, nizatidin, ranitidin, misoprostol, esomeprazol, lansoprazol, omeprazole, pantoprazol, rabeprazol, in the form of salts or enantiomers thereof, or mixtures thereof.

Another preferred embodiment of the invention is a pharmaceutical composition wherein said pharmaceutically active agent is a cholesterol-lowering agent, chosen among atorvastatin, cerivastatin, fluvastatin, pravastatin, simvastatin, rosuvastatin, in the form of salts or enantiomers thereof, or mixtures thereof.

In general, the invention comprises pharmaceutical compositions wherein said pharmaceutically active agent is an agent chosen among the examples given in Table 1.

In principle nitrate could be added to any pharmaceutically active compound where the beneficial effects of nitrate and its reaction products (e.g. NO) are wanted.

Consequently, the present invention makes available a method of treatment of a human patient, characterized in that a patient taking medication for the treatment of a disease or condition, is given nitrate before or simultaneously with said medication, the nitrate being administered in an amount and formulation such that the nitrate is absorbed before said medication and capable of preventing, alleviating, or treating the side effects of said medication, and/or enhancing the effects of the same.

The nitrate is preferably given in the form of a salt chosen among NaNO₃, KNO₃ and other water-soluble nitrate salts, such as metal or alkali metal nitrate salts, more preferably in the form of zinc nitrate (Zn(NO₃) - xH₂O). According to an alternative embodiment, the nitrate is given in the form of a plant extract.

The method or methods are applicable to the following disorders or conditions: a cardiovascular disorder, a gastrointestinal disorder, a respiratory disorder, an infectious disorder, an urological disorder, an inflammatory disorder or pain, a neoplastic disease, a degenerative disorder, or a combination thereof.

One particular application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of hypertension, hyperlipidemia, atherosclerosis, congestive heart disease, ischemic heart disease, myocardial infarction, and diabetes or a combination thereof. One specific application is in the treatment of hypertension in diabetic patients, where an increased release of vascular NO is believed to be highly beneficial.

Another application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of gastric and/or duodenal ulcer, drug-induced gastrointestinal damage, or a combination thereof.

Yet another application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of asthma, rhinitis, sinusitis, nasal polyposis, chronic obstructive pulmonary disorder, or a combination thereof.

Yet another application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of viral, fungal, and parasitic disorders, or a combination thereof.

Yet another application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of erectile dysfunction, overactive bladder, urinary tract infection, or a combination thereof. Yet another application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of rheumatoid inflammatory disorders, rheumatoid arthritis, pain, dermatitis, or a combination thereof.

Yet another application is the use of nitrate in the treatment of a disease or condition, said disease or condition being one of Alzheimer's disease, osteoporosis, dystrophy, or a combination thereof.

One specific application is in the treatment or prevention of cerebral vasospasm, in particular cerebral vasospasm in the wake of subarachnoidal hemorrhage.

The invention also includes the use of nitrate for the manufacture of a medicament for the treatment of one or more of the following disorders or conditions: a cardiovascular disorder, a gastrointestinal disorder, a respiratory disorder, an infectious disorder, an urological disorder, an inflammatory disorder or pain, a neoplastic disease, a degenerative disorder, or a combination thereof.

One particular application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of hypertension, hyperiipidemia, atherosclerosis, congestive heart disease, ischemic heart disease, myocardial infarction, and diabetes or a combination thereof. One specific application is the use of nitrate for the manufacture of a medicament for the treatment of hypertension in diabetic patients, where an increased release of vascular NO is believed to be highly beneficial.

Another application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of gastric and/or duodenal ulcer, drug-induced gastrointestinal damage, or a combination thereof.

Yet another application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of asthma, rhinitis, sinusitis, nasal polyposis, chronic obstructive pulmonary disorder, or a combination thereof.

Yet another application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of viral, fungal, and parasitic disorders, or a combination thereof. Yet another application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of erectile dysfunction, overactive bladder, urinary tract infection, or a combination thereof.

Yet another application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of rheumatoid inflammatory disorders, rheumatoid arthritis, pain, dermatitis, or a combination thereof.

Yet another application is the use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of Alzheimer's disease, osteoporosis, dystrophy, or a combination thereof.

One specific application is the use of nitrate for the manufacture of a medicament for the treatment or prevention of cerebral vasospasm, in particular cerebral vasospasm in the wake of subarachnoidal hemorrhage.

The present invention offers many advantages. Available drugs can be used without risk, or with reduced risk for serious side effects and/or the effect of the drugs enhanced without any modification of the active substance itself. Further, medical grade nitrate is easy to synthesize and available at a low cost. The same applies when nitrate from plant extracts is used. The development and testing of combinations according to the invention will be less time consuming than the development of new or modified chemical entities. It is also likely that the regulatory process for obtaining approval for such combinations will be significantly simplified.

Further, nitrate is believed to exert many beneficial effects through NO, which will be released systemically. Interestingly, it is believed that a composition and method according to the invention will compensate for possible "NO-deficiencies" and support the endogenous formation of NO in a patient, for example in patients with cardiovascular disorder. It is possible that this mechanism at least partially will be self-regulating, i.e. NO will be released in areas of poor circulation where blood pH is lowered.

It is noted that, in order to attain the beneficial effects of nitrate by dietary nitrate intake, impractically large amounts of nitrate containing food (e.g. vegetables) would have to be ingested ,

Examples

The present inventors investigated the generation of NO from nitrate/nitrite and its physiological effects in an animal model.

Example 1. Evidence that bacteria and nitrate/nitrite are reguired for generation of NO in the stomach

The present inventors compared gastric NO levels in conventional and germ-free rats. In some animals nitrate (NaNO₃) was given in the feeding tube one hour prior to NO measurements. Rats were anaesthetised and the stomach wall was punctured with a fine needle. NO-free gas was inflated in the stomach and incubated there for 15 seconds. After this period the gas was aspirated and immediately injected into a chemiluminescense NO analyser.

Gastric NO levels were >7000 ppb in conventional fasting rats and higher in rats pre- treated with nitrate (>30 000 ppb). In contrast, gastric NO was very low in germ free animals (<40 ppb) even after a nitrate load. This clearly shows that gastric NO production is dependent on bacteria. Moreover, it shows that ingestion of nitrate increases gastric NO production. The bacteria in the oral cavity initiate gastric NO production by reducing nitrate to nitrite, a reaction that cannot be catalysed by mammalian enzymes

Example 2. Effects of nitrite on gastric mucosal blood flow and mucus secretion

Rats were anesthetized and the gastric mucosa was exteriorized for intravital microscopy. Acid secretion was stimulated with pentagastrin (40μg ml^"1 h^"1) Gastric mucosal blood flow was measured with laser-Doppler flowmetry, before, during and after topical administration of 1mM NaNO₂ (nitrite) in HCI (10mM) for ten minutes followed by 5mM NaNO₂ in HCI (10mM). Mucus thickness was measured by means of a micropipette connected to a micromanipulator with a digmatic indicator. Total mucus thickness was measured before the outer, loosely adherent mucus layer was removed by suction leaving the inner, firmly adherent mucus layer. The inner mucus layer was measured and 1mM NaN0₂ in HCI (10mM) was applied for four consecutive 15min periods, during which the mucus accumulation was measured, followed by removal of the outer layer and the thickness of the inner mucus layer was measured.

The gastric mucosal blood flow increased dose-dependently by luminal NaNO₂ pH2 (155±12% by 1mM NaNO₂ and 186±20% by 5mM NaNO₂) meanwhile the mean arterial blood pressure was not altered. In control rats, gastric blood flow was not altered by luminal pH2. The mucus accumulation was significantly increased after 60min luminal NaNO₂ compared to the control group, and the mucus thickness of the inner, firmly adherent mucus gel was significantly thicker after 60min luminal NaN0₂ (89+6μm) compared to controls (55±1μm).

This study shows that nitrite given luminally increases both gastric mucosal blood flow and mucus thickness in rat. Nitrite in turn is derived from the reduction of salivary nitrate. This indicates that the dietary nitrate enhances two important gastric mucosal defense mechanisms.

Example 3. Nitrate intake increases nitrite levels in plasma

During the priority year, the present inventors have shown that oral nitrate intake, NaNO₃, 8 mg/kg results in significantly increase nitrite levels in plasma. In a study involving 9 healthy volunteers, an intake of nitrate 8 mg/kg resulted in a four-fold increase, from about 100 nM to about 400 nM nitrite in plasma. Example 4. The effect of salivary nitrate

During the priority year, the present inventors performed experiments using an in vivo rat model as a bioassay to test the effects of human saliva on gastric mucosal blood flow and mucus thickness. The results (J Clinical Investigation, January 2004 - in press) show that mucosal blood flow increased after luminal application of nitrate-rich saliva, whereas fasting saliva had no effects. Also, mucus thickness increased in response to nitrite-rich saliva. The effects of nitrite-rich saliva were similar to those of topically applied sodium nitrite. This supports a gastro protective role of salivary nitrate/nitrite. Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. —

References

1. Cantliffe, D. J., (1972) Nitrate accumulation in vegetable crops as affected by photoperiod and light duration. J.Amer.Soc.Hort.Sci. 97, 414-418.

2. Committee on Nitrite and alternative Curing Agents in Food. (1981) The health effects of Nitrate, Nitrite, and Λ/-Nitroso Compounds. Part 1 of a 2-Part Study page

5.41-5.52. National Academy Press, Washington D.C.

3. Hadara, M., Ishiwata, H., Nakamura, Y., Tanimura, A. & Ishidate, M., (1975) Studies on the in vivo formation of nitroso compounds 1. Changes of nitrite and nitrate concentration in human saliva after ingestion of salted Chinese Cabbage. J.Food.Hyg.SocAS 11

4. Spiegelhalder, B., Eisenbrand, G., Preussman, R., (1976) Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in-vivo formation of N- nitroso compounds. Foods Cosmet Toxicol 14:545-548.

5. Tannenbaum, S. R., Weisman, M., Fett, D., (1976) The effect of nitrate intake on nitrite formation in human saliva. Fd Cosmet Toxicol 14: 549-552.

6. Tannenbaum, S. R., Sinskey, A. J., Weisman, M. & Bishop, W., (1974) Nitrite in human saliva. Its possible relationship to nitrosamine formation. J.Nat.Cancer.inst. 53 79

7. Crampton, R. F., (1980) Carcinogenic dose-related response to nitrosamines. Oncology 37 251

8. Forman, D., Al-Dabbagh, S., Doll, R., (1985) Nitrate, nitrites and gastric cancer in Great Britain. Nature 313:620-625.

9. Knight, T. M., Forman, D., Pirastu, R., Comba, P., lannarilli, R., Cocco, P.L., Angotzi, G., Ninu, E., Schierano, S., (1990) Nitrate and nitrite exposure in Italian populations with different gastric cancer rates. Int J Epidemiol 19: 510-515.

10. Al-Dabbagh, S., Forman, D., Bryson, D., Stratton, I., Doll, R., (1986) Mortality of nitrate fertiliser workers. British Journal of Industrial Medicine. 43(8):507-15.

H .Vittozzi, L., (1992) Toxicology of nitrates and nitrites. Food Additives & Contaminants. 9(5):579-85, 12. Benjamin, N., O'Driscoll, F., Dougall, H., Duncan, C, Smith, , Golden, M., McKenzie, H., (1994) Stomach NO synthesis. Nature. 368(6471 ):502,

13. Lundberg, J. O. N., Weitzberg, E., Lundberg, J. M., Alving, K., (1994) Intragastric nitric oxide production in humans: measurements in expelled air. Gut 35: 1543-1546.

14. Herulf, M., et al., Scand J Gastroenterol, 1998:33.164-169.

15. Wallace, et a/., Nature Drug Rev, 2002: 1. 375-382.

16. Weitzberg, E. & Lundberg, J. O. N., NO Biol Chem 1998:2.1-7.

17.Wallace, J. L., Am J Med 2001.110: 19s-23s.

Claims

1. A pharmaceutical composition containing nitrate (NO₃ ^") in combination with a pharmaceutically active agent, characterized in that the amount of nitrate is sufficient to prevent, alleviate, or treat side effects associated with said active agent, and/or to enhance the effect of said active agent.

2. The pharmaceutical composition according to claim 1, wherein said nitrate is present in the form of a salt chosen among sodium, potassium, calcium, magnesium, lithium and other water-soluble nitrate salts.

3. The pharmaceutical composition according to claim 1 , wherein said nitrate is present in the form of zinc nitrate (Zn(NO₃)2^' xH₂O).

4. The pharmaceutical composition according to claim 1, wherein said nitrate is present in the form of a plant extract.

5. The pharmaceutical composition according to claim 1 , wherein said nitrate is combined with said pharmaceutically active agent in a galenical formulation such that the gastrointestinal tract is exposed first to the nitrate portion of said composition, and later to the pharmaceutically active agent.

6. The pharmaceutical composition according to claim 1 , wherein said nitrate is combined with said pharmaceutically active agent in a galenical formulation such that nitrate is released during gastric passage and the release of said active agent is delayed until after gastric passage.

7. The pharmaceutical composition according to claim 5 or 6, wherein an outer layer comprising nitrate surrounds said active agent.

8. The pharmaceutical composition according to claim 5 or 6, wherein said outer nitrate layer is coated.

9. The pharmaceutical composition according to claim 5 or 6, wherein said active agent is present in granulated form, the granules embedded in nitrate.

10. The pharmaceutical composition according to claim 5 or 6, wherein both said active agent and said nitrate are present in granulated form, the granules of the active substance being coated so that the release of the active substance is delayed.

11. The pharmaceutical composition according to any one of claims 1 - 10, wherein said pharmaceutically active agent is an NSAID compound.

12. The pharmaceutical composition according to claim 11 , wherein said NSAID compound is chosen among acetyl salicylic acid, indometacin, diclofenac, piroxicam, tenoxicam, ibuprofen, naproxen, ketoprofen, nabumetone, ketorolac, azapropazone, mefenamic acid, tolfenamic acid, sulindac, diflunisal, tiaprofenic acid, podophyllotoxin derivatives, acemetacin, aceclofenac, droxicam, oxaprozin, floctafenine, phenylbutazone, proglumetacin, flurbiprofen, tolmetin, and fenbufen, in the form of salts or enantiomers thereof, or mixtures thereof.

13. The pharmaceutical composition according to claim 11 , wherein said pharmaceutically active agent is acetylsalicylic acid.

14. The pharmaceutical composition according to any one of claims 1 - 10, wherein said pharmaceutically active agent is a COX 1 or COX 2 inhibitor.

15. The pharmaceutical composition according to claim 11 , wherein said COX 1 or COX 2 inhibitor(s) is chosen among rofecoxib, celecoxib, etodolac, etoricoxib, loxoprofen, lumiracoxib, nimesulide, meloxicam, valdecoxib, tilmacoxib, parecoxib sodium, flosulide, darbufelone mesylate, 2-benzyl-4-isopropoxy-5-(4- methanesulfonylphenyl)pyridazin-3-one and 1 ,2-diarylcyclopentones, in the form of salts or enantiomers thereof, or mixtures thereof.

16. The pharmaceutical composition according to claim 11 , wherein said NSAID is an NO-donating NSAID.

17. The pharmaceutical composition according to claim 14, wherein said COX 1 or COX 2 inhibitor is an NO-donating COX 1 or COX 2 inhibitor.

18. The pharmaceutical composition according to any one of claims 1 - 17, wherein the amount of nitrate is between 1 and 1000 mg per unit dose.

19. The pharmaceutical composition according to any one of claims 11 - 17, wherein the amount of NSAID(s) or COX 1 or COX 2 inhibitor(s) is between 50 and 1500 mg per unit dose.

20. The pharmaceutical composition according to any one of claims 1 - 10, wherein said pharmaceutically active agent is an anti-ulcer agent, chosen among cimetidin, famotidin, nizatidin, ranitidin, misoprostol, esomeprazol, lansoprazol, omeprazole, pantoprazol, rabeprazol, in the form of salts or enantiomers thereof, or mixtures thereof.

21. The pharmaceutical composition according to any one of claims 1 - 10, wherein said pharmaceutically active agent is a cholesterol-lowering agent, chosen among atorvastatin, cerivastatin, fluvastatin, pravastatin, simvastatin, rosuvastatin, in the form of salts or enantiomers thereof, or mixtures thereof.

22. The pharmaceutical composition according to any one of claims 1 - 10, wherein said pharmaceutically active agent is a drug chosen among the examples of Table !

23. A method of treatment of a human patient, characterized in that a patient taking medication for the treatment of a disease or condition, is given nitrate before or simultaneously with said medication, the nitrate being administered in an amount and formulation such that the nitrate is absorbed before said medication and capable of preventing, alleviating, or treating the side effects of said medication, and/or enhancing the effects of the same.

24. The method according to claim 23, wherein the nitrate is given in the form of a salt chosen among NaNO₃, KNO₃ and other water-soluble nitrate salts.

25. The method according to claim 23, wherein the nitrate is given in the form of zinc nitrate (Zn(NO₃)₂- xH₂O).

26. The method according to claim 23, wherein the nitrate is given in the form of a plant extract

27. The method according to claim 23, wherein said disease or condition is chosen among the following: a cardiovascular disorder, a gastrointestinal disorder, a respiratory disorder, an infectious disorder, an urological disorder, an inflammatory disorder or pain, a neoplastic disease, a degenerative disorder, or a combination thereof.

28. The method according to claim 23, wherein said disease or condition is one of hypertension, hyperiipidemia, atherosclerosis, congestive heart disease, ischemic heart disease, myocardial infarction, and diabetes or a combination thereof.

29. The method according to claim 23, wherein said disease or condition is one of gastric and/or duodenal ulcer, drug-induced gastrointestinal damage, or a combination thereof.

30. The method according to claim 23, wherein said disease or condition is one of asthma, rhinitis, sinusitis, nasal polyposis, chronic obstructive pulmonary disorder, or a combination thereof.

31. The method according to claim 23, wherein said disease or condition is one of viral, fungal, and parasitic disorders, or a combination thereof.

32. The method according to claim 23, wherein said disease or condition is one of erectile dysfunction, overactive bladder, urinary tract infection, or a combination thereof.

33. The method according to claim 23, wherein said disease or condition is one of rheumatoid inflammatory disorders, rheumatoid arthritis, pain, dermatitis, or a combination thereof.

34. The method according to claim 23, wherein said disease or condition is one of Alzheimer's disease, osteoporosis, dystrophy, or a combination thereof.

35. The use of nitrate for the manufacture of a medicament for the treatment of one or more of the following disorders or conditions: a cardiovascular disorder, a gastrointestinal disorder, a respiratory disorder, an infectious disorder, an urological disorder, an inflammatory disorder or pain, a neoplastic disease, a degenerative disorder, or a combination thereof.

36. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of hypertension, hyperiipidemia, atherosclerosis, congestive heart disease, ischemic heart disease, myocardial infarction, and diabetes or a combination thereof.

37. The use of nitrate for the manufacture of a medicament for the treatment of hypertension in diabetic patients, where an increased release of vascular NO is believed to be highly beneficial.

38. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of gastric and/or duodenal ulcer, drug-induced gastrointestinal damage, or a combination thereof.

39. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of asthma, rhinitis, sinusitis, nasal polyposis, chronic obstructive pulmonary disorder, or a combination thereof.

40. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of viral, fungal, and parasitic disorders, or a combination thereof.

41. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of erectile dysfunction, overactive bladder, urinary tract infection, or a combination thereof.

42. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of rheumatoid inflammatory disorders, rheumatoid arthritis, pain, dermatitis, or a combination thereof.

43. The use of nitrate for the manufacture of a medicament for the treatment of a disease or condition, said disease or condition being one of Alzheimer's disease, osteoporosis, dystrophy, or a combination thereof.

44. The use of nitrate for the manufacture of a medicament for the treatment or prevention of cerebral vasospasm, in particular cerebral vasospasm in the wake of subarachnoidal hemorrhage.