Drug – bio-affecting and body treating compositions – Inorganic active ingredient containing – Nitrates or nitrites or nitric acid or nitrogen oxides
Reexamination Certificate
1999-06-11
2004-03-23
Pak, John (Department: 1616)
Drug, bio-affecting and body treating compositions
Inorganic active ingredient containing
Nitrates or nitrites or nitric acid or nitrogen oxides
C424S400000, C424S408000, C424S482000, C514S474000, C514S568000, C514S557000, C514S574000, C514S858000, C514S859000, C514S865000, C514S887000, C514S931000, C514S932000, C514S933000, C514S934000, C422S029000
Reexamination Certificate
active
06709681
ABSTRACT:
The present invention relates in one aspect to acidified nitrite as an antimicrobial agent, and to a complex of nitrogen oxides arising from the interaction of nitrite and acid as an antiviral composition for the treatment of viral diseases of the skin by topical application thereto. Such nitrogen oxides include in particular NO which is of importance particularly if acidified.
An active entero-salivary circulation in man provides a continuous flow of nitrate into the mouth where it is rapidly reduced to nitrite by bacteria on the tongue. The effect of salivary nitrate excretion is to provide a precursor for the generation of nitrogen oxides by the break down of the nitrite.
In brief we have found that exposure of a yeast,
Candida albicans
and the bacterium
E coli
to concentrations of nitrite in saliva together with acid conditions similar to those found in the stomach for one hour caused a dose-dependent reduction in their survival. It is apparent therefore that the generation of nitrogen oxides and/or nitrous acid in the mouth and in the gastrointestinal tract, particularly the upper gastrointestinal tract, from acidified nitrite is preventative of microbial infection.
In the mouth bacteria rapidly reduce nitrates to nitrites. Once swallowed the acid conditions of the stomach protonate the nitrite to form nitrous acid (pKa approx 3.5). The nitrous acid in turn dissociates to form oxides of nitrogen as shown below.
NO
2
−
+H+=HNO
2
(1)
2HNO
2
=H
2
O+N
2
O
3
(2)
N
2
O
3
=NO+NO
2
(3)
N
2
O
3
+C
2
H
8
O
6
=2NO+H
2
O+C
6
H
6
O
6
(4)
Endogenous and dietary nitrate is actively concentrated by salivary glands to more than 10 times the concentration in plasma and secreted in saliva. Thus the saliva provides a continuous source of nitrate to the upper gastrointestinal tract. Oral conversion of nitrate to nitrite is rapid and is restricted to the surface of the tongue in man and to the posterior third of the tongue in the rat.
The function of the entero-salivary circulation of nitrate is not known but it may well be that gastric acid by itself is not always sufficient to destroy many ingested micro-organisms and that the primary role of salivary nitrate secretion and conversion to nitrite is as a precursor for nitrogen oxides in the lumen of the stomach which will kill swallowed micro-organisms.
The above identified mechanism is also applicable to the destruction of micro-organisms on an in the skin. For example athlete's foot or tidea pedis.
In WO 95/22335 we have disclosed a pharmaceutical composition comprising a pharmaceutically acceptable source of nitrites and a pharmaceutically acceptable acidifying agent, inter alia for the direct treatment of disease by topical application. These compounds have a direct effect on the organism concerned but the precise mode of action is not known.
U.S. Pat. No. 4,595,591 reveals a composition comprising an aqueous solution of nitric acid and nitrous acid at a pH below 1 preferably with a organic acid and copper and cadmium ions for the treatment of superficial lesion of the skin, for example tumorous growths.
U.S. Pat. No. 5,648,101 provides a vaso-active composition comprising NO adapted for delivery to a body site inter alia by means of a cream or ointment. The NO is generated from an admixture of ferrous sulphate, an organic acid and an inorganic nitrite and caused to be reactive in the presence of moisture adjacent or at the site. Acidification is not discussed.
WO 96/02268 reveals the inhibition of a virus by nitric oxide (NO
2
) derived from a complex unstable organic molecule, but the advantages of reduction of pH at the environment of use have not been appreciated, neither have the beneficial effects the chemical release of the NO and NO
2
moieties immediately adjacent to the environment of use, been realized.
WO 93/25213 reveals a composition comprising nitrous oxide contained in a dermatological composition comprising as an essential feature a fatty acid or a lower alkyl ester thereof, pH values, particularly at the environment of use, are not mentioned.
All are single formulations which are admixed well prior to application to the environment of use so that NO and NO
2
all escape prior to use and hence have a very limited utility.
We have now found inter alia that nitrite at concentrations of up to 4% in an inert carrier cream or ointment when mixed with an organic acid such as salicylic acid reacts to produce oxides of nitrogen which are effective in killing infectious organisms on the skin including fungi, yeast, bacteria and viruses. The combination of nitrite and acid causes mild erythema (redness) of the skin due to release of nitric oxides at the environment of use but this causes no significant inflammation.
We have also found that as far as viruses, as opposed to bacteria for example, are concerned, that the above nitrogen oxide complex, comprising for example NO and/or NO
2
while it may effect replication to a degree, more importantly modifies the virally infected cells such that the immune system can recognize the viral particles. Inter alia, this is supported by the fact that the complex is less effective in immunosuppressed hosts. Generally the greater the percent of nitric oxide (NO) the better the immuno-potentiation. If possible up to 100% NO can be used.
It is thought, although more work is required, that smaller molecules, particularly NO and NO
2
penetrate the skin by direct diffusion or via the seat glands or hair follicles through the epidermis to the sweat cells. It has been found that although the healthy keratinocytes find the oxides of nitrogen toxic they do not die as they are relatively resistant to its effects. However, the surprising clinical results in our examples lead us to believe that virally infected cells are more susceptible to these effects, leading to destruction of the virally infected cells via a combination of toxicity leading to programmed cell death and potentiation of the immune response to the presence of the virus.
The above identified mechanism is also useful in the sterilisation of objects such as dentures by utilising a sterilizing nitrate solution. Conventional solutions which are effective in sterilising dentures often taste unpleasant due to chlorine-based disinfectants. A combination of nitrite and acid results in a antimicrobial solution which has little or no taste. Other objects such as contact lenses may be sterilised in the same way.
Gastroenteritis continues to be a major problem in rearing pigs and other farm animals. Enteropathogenic
Escherichia coli
(especially those bearing the K88 antigen) are particularly implicated. Although gastric acidity is thought to be one of the main host defence systems which provides a barrier to orally-acquired infection, this is clearly ineffective in preventing organisms from reaching the more distal intestine in these animals.
The role of NO as a compound which inhibits viral replication in vitro has been disclosed by J. B. Mannick; 63
rd
Forum in Immunology, and papers in Intervirology 1995; 38: 206-213, Trends in Microbiology 1995; 3: 81-82, Science 1993; 261: 1445-1448, and The Journal of Clinical Investigation 1993; 91: 2446-2452. The above papers disclose the effects of NO on various viruses, for example herpes simplex virus, vaccinia virus and vesicular stomatitis virus. Exogenous NO donors such as S-nitroso-N-acetyl penicillamine(SNAP)or SIN-1 were used in vitro to determine the role of NO as an antiviral compound. Application of exogenous NO to cell-lines infected with the virus under test resulted in inhibition of the viral DNA replication. The exact mechanism of the inhibition seemed to differ depending on the virus involved. For example in the case of vaccinia virus it is thought that the NO may inhibit replication by binding to non-haem iron or thiol groups that are essential for the catalytic activity of enzymes involved in vaccinia replication. In this in vitro model the antiviral effects of
Benjamin Nigel
Dougall Hamish
Ormerod Anthony
Aberdeen University
Fish & Richardson P.C.
Pak John
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