Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined...
Reexamination Certificate
1998-07-07
2001-08-07
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
C435S235100, C514S730000
Reexamination Certificate
active
06270808
ABSTRACT:
The present invention relates to the field of anti-viral compounds, e.g. in the field of immune diseases, particularly acquired immune diseases, more in particular to infections with human immunodeficiency virus.
A new human immunodeficiency with opportunistic infections and malignicies was described in 1981. The causative agent was found to be a retrovirus, Human Immunodeficiency Virus (HIV). The pathogenesis of the disease begins with the alteration and eventual depletion of T4 lymphocyte cells, leading to a progressive destruction of the immune system.
HIV is a retrovirus, consisting of a capsulated RNA-genome surrounded by a double-layer core-membrane. An early finding was the variation of nucleotide sequences of certain parts of the genome, especially in the envelop gene (Shaw, et al, 1986).
the gag and pol genes of HIV are translated as two polyproteins (Pr55gag and Pr160gag-pol) (Jacks, et al, 1988). These are subsequently cleaved by the action of a virus-encoded protease into four structural gag proteins of the virion core (p17, p24, p7 and p6) (Veronese, F D., et al., 1987), and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H., and endonuclease).
Viral core-glycoprotein precursor gp160 component gp120 binds to CD-4, a cellular receptor of the susceptible T-cell subset with high affinity (Berman, et al, 1989). Therefore, viral recognition and fusion of CD-4 with gp120 is a pivotal occurrence in the infection cycle. It should be noted however, that other factors may play a role as well: Neutralizing antibodies to the V3 loop of gp120 prevent infection even though they do not inhibit binding to CD4 (Rusche, et al, 1985), and
Productive infection of monocytes, in absence of proliferation and DNA synthesis of T lymphocytes, can occur and serve as relative long-lived reservoirs of virus (Weinberg, et al, 1991).
Drug development against HIV infection sofar has aimed at preventing fusion between the CD4 membrane protein and viral gp120 and thus preventing mediation of HIV entry into the cytoplasm of T4 lymphocytes. Vaccination with recombinant gp120 (rgp120) led to protection of chimpansees from HIV infection (Berman P W., et al, 1990). Cytopathic fusion of CD-4 cells (syncytia) occurs on expression of gp120 at the plasma membrane. Soluble CD4 blocks secretion and surface expression of gp120 prevents such cell fusion (Buonocore & Rose, 1990).
Other attempts to develop drugs against infection with HIV have so far focused on the inhibition of HIV reverse transcriptase (RT) and HIV protease. RT is the target of AZT, the first anti-HIV drug in clinical use. A TIBO derivative RS2150 was found to inhibit 50% of HIV RT at a concentration of 3.1 &mgr;M and 4.9 &mgr;M respectively for different viral strains (Pauwels, et al, 1990).
HIV protease inhibition is for example disclosed in EP 541168, U.S. Pat. No. 5,196,438, and in WO092/08701. Inhibition of proteases results in the production of non-infectious virus particles and unprocessed gag and gag-pol precursor proteins (Kohn, et al, 1988: Loeb, et al, 1990). Recent disclosures aiming at inhibition of either HIV protease or HIV RT in patent literature are: WO 95/20384, WO 95/16688, WO94/06454, EP 0666755, EP 0666842, WO 95/14011, and many more.
A range of antigenically distinct HIVs have been isolated, (even within one individual) causing a major problem in drug development against HIV. Moreover, many drugs which seem promising in vitro suffer from the fact that the virus is capable of escaping their effect in vivo by quick mutagenesis.
The present invention provides a new group of substances capable of inhibiting Human Immunodeficiency Virus, which can thus be used to prevent or combat HIV infection and/or AIDS.
During laboratory testing of a newly discovered range of protease inhibitors from leeches (see EP 94117053.2 and EP 95103637.5) very potent elastase-, chymotrypsin-, trypsin-, plasmin- and thrombin inhibitors were defined. Since the proteolytic cleavage of the gag and env precursors in the replication cycle of HIV is an important step, it was investigated if the newly described inhibitors would have any effect on such virus replication. Development of inhibitors is a rationale in anti-viral drug development. The aim of such study was to evaluate the antiviral capacity of the protease inhibitors, which were the subject of EP 94117053.2 and EP 95103637.5, on primary cells.
It was concluded in the above-mentioned patent applications, that anti-viral capacity was indeed present in the described inhibitors. However, it has now been surprisingly found, that a farm more potent anti-viral substance or group of substances resided in the original leech and leech-head ethylalcoholic extractions. These substances or groups of substances containing such surprising activity are the subject of the present invention. We have purified and characterized the substances.
The present invention thus provides an isolate or compound having inhibitory activity towards virusses, e.g. towards a human immunodeficiency virus, obtainable from leeches or organisms associated with leeches of the phylum uniramia by solvent extraction techniques and having a molecular weight of about 300-600, preferably about 400-500 Dalton. Preferably the compound is obtainable from the subclass of euhirudinae, in particular it is obtainable from the suborder hirudiniformes, preferably the order of arhynchobelidae. Most preferred are compounds which are obtainable from the family hirudinidae, in particular those which are obtainable from the genus Limnatis, in particular the species
L. nilotica. L. nilotica
(Savigny, 1820, as following from Autrum 1936) is described as a “nasal leech” or horse leech (Mouquin-Tandon 1846). It was found to be present in the whole littoral area of the Mediterranean (Harant, 1928; Jarry, 1959). It lives in spring fountains and “oueds”, and feeds on cattle, dogs and man (Blaise, 1874/5; Neveu & Lemaire, 1938; Turner, 1969; Keegan, et al, 1970).
Amazingly, the feeding habits of this leech differ from other haemotophaguos leeches. It remains attached to its host (nasal—and laryngeal cavity) for prolonged periods (weeks to months). The animal feeds on its host repeatedly. We have observed that drinking cattle were infected with these leeches, which did not drop off while the cattle are drinking water. Only thick, fat, adult-size leeches do drop off at such occasions. Therefore, it is clear that this species of leech is mostly free of antigenic or immunogenic substances in its mucus or salivary gland productions. Reports of host animals dying from this species of leech mention anemia as the one common cause, but no direct antigenic effect has been described to our knowledge. In one aspect the invention thus provides compounds having HIV inhibiting activity obtainable from the leech Limnatis nilotica or fragments of derivatives of such compounds having similar activity.
Such compounds can be derived from all body parts and secretions of the leech, including saliva- and gut-, intestinal- and skin secretions and mucus.
The most suitable source for the compounds according to the invention is probably solvent extraction of the heads of the leeches, or compounds associated with these heads. As may be understood it is also possible that the actual source of the compounds according to the invention may not be the leech itself, but it may be derived from a leech associated material or organism, such as a bacterium.
These compounds obtainable from for instance a bacterium associated with the leeches are also a part of the present invention. The requirement is that they are obtainable by solvent extraction techniques when applied to the leeches, that they have the characteristics as disclosed herein, of which the anti-viral activity, and esp. the HIV-inhibition, is the most important characteristic. The compounds may even be the result of interaction between leech derived and other materials.
The present invention also provides a method for obtaining a compound having human immunodeficiency virus
Lankford , Jr. Leon B.
Merchant & Gould P.C.
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