WO1998047495A1

WO1998047495A1 - New composition

Info

Publication number: WO1998047495A1
Application number: PCT/SE1998/000301
Authority: WO
Inventors: Kjell Alving; Jan M. Lundberg; Jon Lundberg; Eddie Weitzberg; Peter Wiklund
Original assignee: Nitricare Kb
Priority date: 1997-02-21
Filing date: 1998-02-20
Publication date: 1998-10-29
Also published as: AU6128398A; SE9700617D0

Abstract

Prevention and/or treatment of bacterial infections is achieved through the administration of a pharmaceutical composition which includes and/or enhances the formation of NO in body fluids, such as urine and vaginal secretions. Said composition comprises at least one pharmaceutically suitable acidifying agent and a reduction agent. Preferably said composition further comprises a source of nitrate. The pharmaceutical composition can be administered e.g. in the form of a lozenge, an effervescent tablet to be dissolved in water, a solution etc.

Description

New composition

The present invention relates to the prevention and treatment of bacterial infections in mammals by inducing and/or enhancing the endogenous production of nitric oxide (NO) and/or other reactive nitrogen oxides in body fluids. The present invention also relates to a pharmaceutical composition and the use of said composition in the prevention and treatment of bacterial infections in mammals.

Background of the invention

The role of NO as an indicator of inflammatory diseases in various organs has recently been the subject of both scientific articles and patent applications i.a. by the present inventors, e.g. SE 9404161-3 (which concerns the diagnosis of inflammatory conditions in the intestines), WO 95/02181 (which concerns the determination of NO levels in exhaled air for purposes of diagnosing inflammatory conditions in the upper airways) and SE 9601369-3 (which concerns the determination of NO levels in gas samples taken from the urogenital organs for purposes of diagnosing inflammatory conditions in said organs).

Even though the presence of NO in inflammation has been described in numerous studies, the role of NO and possibly other reactive nitrogen oxides in inflammation is far from settled. When produced in excess, NO or its reaction products may be cytotoxic to host cells. On the other hand, increased NO production might enhance local host defence mechanisms: it is well-known that this gas has bacteriostatic, antiviral and anti-tumour properties. Furthermore, it has been suggested that NO may serve protective functions by scavenging other, more toxic, radicals.

Nitric oxide is normally produced enzymatically by constitutive NO synthases in, e.g. nerves and endothelial cells; these enzymes yield the relatively small amounts of NO involved in physiological regulation of nerve transmission and vascular tone. In contrast, the inducible NO synthase found, e.g. in activated white blood cells produces NO at a high rate. It was recently shown that nitric oxide is also produced through a second route, i.e. through reduction of nitrite. Nitrite is present in body fluids such as saliva and urine, in amounts depending on the individual's diet and health. For example, a diet rich in nitrate (such as certain vegetables) will result in high nitrate levels in saliva and urine. The bacteria normally present in the oral cavity will reduce salivary nitrate to nitrite. Further reduction of nitrite to nitric oxide occurs normally in the acidic environment of the stomach (Lundberg et al. 1994, Gut, 35: 1543-46).

In urine, nitrate is normally not further reduced to nitrite, since no bacteria are present in the urine of healthy subjects. However, urine from patients with urinary tract infec- tions may contain considerable amounts of nitrite as a result of bacterial nitrate reductase activity and detection of nitrite is routinely used in the diagnosis of bacterial cystitis. There is today a global overuse of certain antibiotics resulting in increasing development of bacterial resistance to these drugs. Moreover, many of the antibiotics used in clinical praxis have troubling side effects such as gastrointestinal disturbances, secondary fungal infections and allergic reactions. There is therefore a growing need for the development of new antibiotic agents with different mechanisms of action and without effects on the normal bacterial flora, e.g. the bacterial flora of the gastrointestinal tract.

Closest prior art Nigel Benjamin et al. have in WO 95/22335 disclosed the use of acidified nitrate as an anti-microbial agent and further described a dosage form for use in the treatment of bacterial, viral or fungal conditions. An important characteristic of that disclosure is the presence of an acidifying agent, adapted to reduce the pH at the environment of use to below pH 4. According to an embodiment, the acidifying agent is salicylic acid. In the experiments presented by Benjamin et al. no significant effect is achieved at pH 4, 5 or 6. This is also in line with experiments by the present inventors, showing that only minute amounts of NO is formed above pH 5 i saline (Lundberg et al. 1994, Gut, 35: 1543-46).

Summary of the invention The present inventors have now surprisingly found, that bacterial infections in body fluids can be prevented and/or treated at physiological pH by the administration of a suitable reduction agent and a suitable acidifying agent. Preferably said agents are administered in connection with, but not necessarily simultaneously or conjunctly with a nitrate source. The inventive composition induces and/or significantly enhances the formation of nitric oxide and/or other reactive nitrogen oxides in nitrite-containing body fluids, in particular in urine. The present invention is described in closer detail in the following description and examples, to be read as exemplifying and not limiting the scope of the invention, as set forth in the appended patent claims.

Short description of the drawings The present invention will be described with reference to the following drawings, in which:

Fig. la shows urinary nitric oxide release from control urine (open bars), control urine with 100 μM sodium nitrite added (striped bars) and infected urine (containing 8-400 μM nitrite, hatched bars), Fig. lb shows nitric oxide release from control urine (100 μM nitrite added) at different pH values, with and without 10 mM ascorbic acid (filled and striped bars, respectively), and

Fig. 2 shows the growth of E. coli following exposure to acidified nitrite-containing urine. The experiments were performed using different concentrations of nitrite (μM) at pH 5.0 (a in the figure) and with different pH using a fixed concentration of nitrite (100 μM, b in the figure, filled bars). Optical density in figure b was measured at 4 h.

Fig. 3 shows the growth of E. coli in human urine with and without the addition of the different components of the invention, Fig. 4 shows the effect of ingested ammonium nitrate and vitamin C on bacterial growth in human urine.

Description of the invention The present invention introduces a new concept of prevention and/or treatment of bacterial infections in body fluids, according to which the local production of NO and/or other reactive nitrogen oxides in body fluids, such as urine, vaginal fluid and cerebrospinal fluid is induced and/or enhanced by the administration of a new pharmaceutical composition.

The present invention mainly concentrates on the treatment of manifest infections, in particular infections in the urinary tract, such as lower urinary tract infection, infectious cystitis etc. The invention is naturally also useful in the long-term or short-term prevention of infections, given that the dosage, route of administration and composition is adjusted as necessary.

The present invention concerns a composition, comprising at least a reducing agent and an agent, capable of acidifying the urine. The present invention also comprises a method for treating bacterial infections, in particular infections in the urogenital tract and especially in the urinary tract. Said method comprises the steps of acidifying the body fluid in question, e.g. the urine, the administration of a reducing agent and, optionally, the administration of a nitrate source.

Examples of urine acidifiers include, but are not limited to, the following: ammonium chloride, ammonium nitrate, metheneamine hippurate (sold under the trade mark Hiprex^®), metolazone (sold under the trade mark Zaroxolyn^®), diazoxide (sold under the trade mark Hyperstat^®) and L-arginine hydrochloride. Further, preliminary tests show that L- arginine hydrochloride may have the additional effect of potentiating the enzymatic synthesis of reactive nitrogen oxides in the bladder. Examples of reducing agents include, but are not limited to, the following compounds or groups of compounds: ascorbyl palmitate, vitamin C, vitamin E, manganese, selenium, beta-carothene, pro-anthocyanidins, polyp henols, urat, co-enzyme Q10, thioles and bioflavonoids. According to a preferred embodiment of the invention, the pharmaceutically suitable reducing agent is ascorbic acid. This compound has several advantages, for example that it is freely soluble in water and thus will be distributed to all body fluids, and that any excess amounts are excreted in the urine; it is non-toxic and it's physiological effect and behaviour are well documented. Additionally, ascorbic acid also functions as a weak acidifying agent. Various sources of nitrogen can be contemplated. Preferably the source of nitrogen is simultaneously a source of nitrate or nitrite. An example of a suitable source of nitrogen are various nitrate salts, for example sodium nitrate, NaNO₃. Using nitrate and nitrite compounds, the toxicity of these compounds must be considered. The MDL values (orally in rats) are 200 mg kg and 330 mg/kg for sodium nitrate and sodium nitrite, respectively. By using nitrate as a source of nitric oxide in urine, it is assured that no nitric oxide is generated in the urine unless bacteria are present. Thus urinary NO production only occurs if bacterial in urine first convert urinary nitrate to nitrite. By this mechanism urinary NO production is auto-regulated in vivo and bacteria normally present in e.g. the gastrointestinal tract are not affected since the higher pH of the intestines prevent further reduction of nitrite to bacteriostatic NO.

Preferably the inventive composition is administered orally, in any of the following forms; a solution, a syrup, a chewable tablet, a tablet to be swallowed as a whole, a capsule to be swallowed, a water-soluble lozenge and a effervescent tablet to be dissolved in water. In the manufacturing of any of the administration forms above, care has to be taken in choosing the necessary excipients, such as tabletting agents, braking agents, lubricants etc. Any excipients must be chosen among agents accepted for use in pharmaceutical preparations or as food additives. Care has also to be taken in choosing the excipients so, that they do not react with or catalyse reactions between the constituents of the pharmaceutical preparation. In some cases, it may be suitable to employ microencapsulation techniques for ensuring the stability of the product.

The inventive composition is specially useful in the treatment of manifest urinary infections, such as urinary tract infections in post operative patients, catherized patients, cystectomized patients and patients with a urinary diversion. In the latter group of patients, where the bladder has been operatively removed and replaced with a section of the intestines, converted to a reservoir for urine, infections are a serious problem. The present invention is highly useful in the treatment of such conditions and the inventive composition can be administered either orally or as a solution for flushing the surgically constructed reservoir. It is also contemplated by the present inventors, that the inventive pharmaceutical composition is administered as a prophylactic regimen to catherized patients or any patient, susceptible for infections in the urinary tract. Obviously, the inventive composition can be administered in conjunction with other treatments for bacterial infections in the urinary tract, potentiating these treatments.

When the pharmaceutical composition according to the invention is to be directed specifically towards the prevention and/or treatment of infections in the urinary tract, the inclusion of a diuretic in said composition is possible. Examples of conventionally used diuretics, potentially suitable in this application, are thiazide compounds, sulphonamid derivatives, phenoxi acetic acid derivatives, so called loop-diuretics and aldosterone antagonsists. In choosing the type of diuretic, attention has to be paid to the general condition of the patient, possible interaction with other, simultaneously administered pharmaceuticals etc.

One specific compound, simultaneously functioning as a source of nitrate, urinary acidifier and diuretic, is ammonium nitrate, NH NO₃. According to one preferred embodiment of the invention, ammonium nitrate is included in the composition. This is specially preferred in compositions intended for the prevention and/or treatment of bacterial infections in the urinary tract.

According to one embodiment of the invention, the pharmaceutical composition is delivered in the form suitable for sublingual or buccal administration, e.g. as a lozenge or tablet. Said lozenge or tablet comprises, in addition to the constituents described above, a water-soluble, direct compressible excipient and minor amounts of a lubricant. This compressible excipient should be palatable or tasteless and water-soluble and accepted for use in pharmaceutical preparations or as a food additive. Suitable excipients are those, conventionally used, for example dextrin compounds, various sugars and sugar alcohols, such as lactose, sorbitol, mannitol and xylitol. Said lubricant is chosen among conventional lubricants, such as magnesium stearate. The excipient and lubricant have to be chosen so, and employed in such amounts, not to endanger the stability of the product, that is not to react with or catalyse a reaction between the constituents or substantially shorten the shelf-life of the pharmaceutical composition.

The finding that the inventive concept was effective in urine was highly surprising, in particular the finding that the bacteriostatic effects were significant at a pH above 4. Firstly, o it is generally known that urinary infections are difficult to treat without resorting to antibiotics, recur easily and can constitute a practically chronic condition in some patients. Secondly, it is well known in the art that urine is difficult to acidify. This makes the skilled person reluctant to believe the approach of folk medicine that the mere acidification of urine would constitute an efficient cure. Thirdly, comparative experiments made by the present inventors show that acidification, intake of vitamin C and nitrate, separately have very limited if any effect. This is in line with the general experience in the art, that the components of the invention, when taken separately lack effect or have only limited effect. The dramatic reduction of bacterial growth resulting from the combined use of the three components is therefore unexpected.

Examples Example 1: Urine was collected from 10 healthy controls (age 26-42 years) and 8 patients (age 41-70 years) with bacteriuria as confirmed by urinary cultures. Nitrite concentration in infected urine was measured with capillary electrophoresis, and nitric oxide release was studied after adjusting pH to 4.5 or 5.0 with 1 M HCl. In control urine, nitric oxide formation was measured at different pH values (4-8) and with varying amounts of nitrite (10-500 μM). It was studied whether nitric oxide release from acidified nitrite-containing urine would be influenced by the addition of ascorbate at a concentration (10 raM) resembling that found in urine (Brandt et aL, Am J Clin Pathol. 68(1977) 592-594) after daily ingestion of 1-2 gram. Similar measurements were also performed in urine from 5 healthy controls before and after ingestion of vitamin C (2 grams/day) for two days. In all experiments, urinary samples (10 ml) were incubated in a closed syringe at 37°C with a head space of 50 ml. After 30 min the head space gas was removed and immediately injected into a chemiluminescence nitric oxide analyser (Eco Physics, Switzerland). Ambient nitric oxide levels were below 4 parts per billion (ppb) in all experiments.

A reference strain, Escherichia coli (E. coli) ATCC 25922 was grown in Mueller- Hinton broth for 6 h at 37°C resulting in 3 x 10⁸ CFU/ml. The strain was diluted to a bacterial density of approximately 10⁶ CFU/ml in acidified urine with or without the addition of nitrite and kept for 2 h at 37°C in a closed tube. Bacterial growth was measured continuously in control urine medium for 10 h by vertical photometry in a computerised incubator for bacteria, Bioscreen C (Labsystems, Helsinki, Finland).

Basal nitric oxide formation was low both in control urine and in infected urine. In contrast, large amounts of nitric oxide were generated from infected urine (containing 8-400 μM nitrite) when the urine was acidified to pH 4.5 or 5.0 and from acidified non-infected urine if nitrite was added (Fig. la). Urinary nitric oxide release was strongly pH dependent in the presence of nitrite and was greatly enhanced by the addition of ascorbic acid (Fig. lb). Also, nitric oxide formation increased with higher nitrite concentrations in urine. At pH 5, 50 parts per billion (ppb) nitric oxide was released from 10 μM nitrite, whereas 100, 250 and 500 μM yielded 400, 1500 and 4000 ppb nitric oxide, respectively. After ingestion of vitamin C urinary nitric oxide release (at pH 5.0, 100 μM nitrite) increased seven-fold compared to the control situation.

Example 2: The addition of nitrite to acidified urine (pH 5.0) dose-dependently reduced the growth of E. coli (Fig. 2a). The inhibition of bacterial growth was greater at lower urinary pH when using a fixed concentration of 100 μM nitrite (Fig. 2b).

Example 3: Escherichia coli (E. coli, strain ATCC 25922) was grown in Mueller- Hinton broth for 6 hours at 37 °C. The strain was then diluted to a bacterial density of approximately 10⁶ CFU/ml in human urine. The bacteria were kept in a closed tube for 2 hours at variable pH with or without the addition of nitrate 10 mM and ascorbate 10 mM. Bacterial growth was then measured continuously in control urine medium for 20 hours by vertical photometry in a computerised incubator for bacteria, Bioscreen C (Labsystems, Helsinki, Finland). The growth rate is expressed as the time (hours) to reach 50% of the maximal growth found in control urine at pH 6 without addition of nitrate or ascorbate. Note that the growth inhibition by acidification, nitrate or ascorbate was markedly enhanced after combining acidification with a nitrate source and a reducing agent (ascorbate). Thus neither the combination of acidification and nitrate nor the combination of acidification and ascorbate showed a growth inhibitory effect of similar potency as was seen after the combination of acidification, nitrate and ascorbate. Cf. figure 3.

Example 4: Urine was collected from a healthy male control. Urine was also collected after a ingestion of ammonium nitrate 4.5 gram daily for 3 days with or without simultaneous ingestion of 3 gram vitamin C (ascorbate).To study the growth inhibitory effect of ammonium nitrate and vitamin C, Escherichia coli (E. coli, strain ATCC 25922) was grown in Mueller-Hinton broth for 6 hours at 37 °C. The strain was then diluted to a bacterial density of approximately 10⁶ CFU/ml in the urine collected from the healthy control and were kept in a closed tube for 2 hours. Bacterial growth was then measured continuously in control urine medium (at pH 6 without nitrate and vitamin C) for 20 hours by vertical photometry in a computerised incubator for bacteria, Bioscreen C (Labsystems, Helsinki, Finland). Cf. figure 4. (A) The growth in control urine at pH 5, (B) the growth in urine (pH 5) after daily ingestion of ammonium nitrate 4.5 gram. (C) daily ingestion of ammonium nitrate 4.5 gram and 3 gram vitamin C (ascorbate). Note the growth inhibitory effect after ingestion of ammonium nitrate (B) and the additive effect of simultaneous ingestion of vitamin C (C).

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.

Claims

I. A pharmaceutical composition for the treatment and/or prevention of bacterial infections in the urogenital tract, characterized in that said composition comprises an acidifying agent and a reducing agent.

2. A pharmaceutical composition according to claim 1, characterized in that said composition further comprises a source of nitrogen, capable of forming nitrite or nitrate.

3. A pharmaceutical composition according to any one of claim 1-2, characterized in that said acidifying agent is one chosen from the following: ammonium chloride, ammonium nitrate, metheneamine hippurate, metolazone, diazoxide and L-arginine hydrochloride.

4. A pharmaceutical composition according to any one of claim 1-3, characterized in that said reducing agent is one chosen from the following: ascorbyl palmitate, vitamin C, vitamin E, manganese, selenium, beta-carothene, pro-anthocyanidins, polyphenols, urat, co-enzyme Q10, thioles and bioflavonoids.

5. A pharmaceutical composition according to any one of claim 1-4, characterized in that said source of nitrogen is a nitrate salt, for example NaNO₃.

6. A pharmaceutical composition according to claim 1, characterized in that said composition further comprises a diuretic.

7. A pharmaceutical composition according to claim 6, characterized in that said diuretic is one chosen from the group comprising NHUCL and NH4NO3.

8. A lozenge comprising an acidifying agent and a reducing agent.

9. A lozenge according to claim 8, further comprising a source of nitrate.

10. An effervescent tablet comprising an acidifying agent and a reducing agent.

I I . An effervescent tablet according to claim 10, further comprising a source of nitrate.

12. A solution comprising an acidifying agent and a reducing agent.

13. A solution according to claim 12, further comprising a source of nitrate.

14. A method for the treatment of bacterial infections in the urogenital tract in a human patient, characterized in that an agent capable of acidifying the local environment is administered to said patient in conjunction with a reducing agent.

15. A method for the treatment of bacterial infections in the urinary tract in a human patient, characterized in that an agent capable of acidifying urine is administered to said patient in conjunction with a reducing agent.