CA2158551A1 - Drugs for the treatment of retrovirus infections - Google Patents

Abstract

The invention concerns drugs for the treatment of retrovirus infections, the drugs containing bacteria-free and protein-free extracts of Escherichia coli, optionally with the addition of extracts of green or yellow leaves of Ginkgo biloba.

Description

The invention relates to drugs for treating retrovirus infections, and the use of bacteria-free and albumin-free extracts of Escherichia coli to manufacture drugs for the treatment of retrovirus infections Retroviruses are spherical enveloped RNA viruses, two subfamilies of which are pathogenic to humans and animals. Retroviruses are characterized by reverse transcriptase, which is an RNA-dependent DNA polymerase. During replication, the single-strand viral RNA
is transcribed by the reverse transcriptase into a double-strand DNA intermediate stage that is incorporated as a provirus into the genome of the host cell. The formulation of RNA-containing virus particles is initiated from the provirus by a regular transcription, and these particles leave the host cell after budding. Retrovirus cause leukemias, autoimmune diseases, and immunosuppressions, and in particular in humans they cause AIDS and ARS (AIDS-related syndrome), which is the precursor to full-blown AIDS. Up to now, it has not been possible to cure retrovirus infections in humans since every attempt to develop a vaccine against the virus, made up to now, has failed because the virus mutates so quickly. Therapeutically, medications that inhibit reverse transcriptase have been used thus far; these generally involve substituted deoxynucleosides of guanosine, cytidine and thymidine. The best known agent so far is AZT, but it, too, can do no more than markedly delay the symptoms of a retrovirus infection; it cannot cure the disease.

Quite surprisingly, it has now been found that bacteria-free and albumin-free extracts of Escherichia coli, known per se, optionally in combination with extracts of Ginkgo biloba, are capable of retarding the development of retrovirus infections to a very marked extent, without displaying the side effects, known thus far, of the nucleoside therapeutic agents. The coli extracts can be used together with the Ginkgo extracts as is, although it is also possible to combine these extracts with nucleoside therapy; it is then possible to reduce the dose of nucleosides and hence to lessen the anticipated side effects.

Extracts from the leaves of Ginkgo biloba have long been used in therapy for problems that involve peripheral and cerebral arterial circulation. Processes for preparing Ginkgo extracts from green leaves are known, for instance, from German Patent 17 67 098, German Patent 21 17 429, European Patent Disclosures EP-A
0 324 197 and EP-A 0 330 567, and Published, Non-Examined German Patent Disclosure DE-OS 39 40 091. Another process for extraction from yellow autumn leaves of Ginkgo biloba is described in European Patent Disclosure EP-B 0 352 146.

The leaves of Ginkgo biloba contain a number of different compounds, with so-called Ginkgo flavonoglycosides predominating.
A typical example of this class of substances is 5,7,3'4'-tetrahydroxy-flavono-3-0-.alpha.-rhamnopyranosyl-4-0-.beta.-D-(6'''-transcumaroyl) gylcopyranoside. They contain smaller amounts of terpene lactones, such as the Ginkgolids, which have been described in detail, for instance, by Okabe et al, J.Chem. Soc.
(1967), 2201-2206. A close relative of terpene lactones is bilobalide, the use of which for treating neuropathies and similar diseases is known from US Patent 4,571,407.

Ginkgo biloba leaves also contain so-called Ginkgolic acids, which are 6-alkyl-salicylic acids that have alkyl radicals with from 13 to 19 carbon atoms and from 0 to 3 double bonds. The corresponding substituted phenol, which is known to be allergizing, can be made from the Ginkgolic acids, either biogenetically or by processing the leaves. The preparation processes for the extracts therefore differ essentially in that an attempt is made to eliminate the undesired ingredients or concomitant substances as far as possible, especially if the extracts are intended for late use in injectable form. However, one skilled in the art has a number of methods available for removing undesirable ingredients; such methods are described in the know literature. As a rule, the Ginkgo extracts currently available on the market are well tolerated even in injectable form; such products have been put on the market, for example, by Dr. Wilmar Schwabe GmbH & Co., in D-7500 Karlsruhe, Federal Republic of Germany, under the tradename "Tebonin?."

European Patent Disclosure EP-B 0 352 146 suggests that extracts from the yellow leaves of Ginkgo biloba would be able to reduce an elevated level of gamma globulins in mammals, which can also be observed in AIDS patients. However, this publication provides no further details as to whether or not this extract can actually be used against AIDS, and with what success.

Bacteria-free and albumin-free filtrates of Escherichia coli cultures have already been used therapeutically for a long time, on the one hand for disorders connected with motility or permeability of the intestinal mucus membrane, but also against inflammations such as Crohn's disease, or after antibiotic therapy, chemotherapy or radiation therapy. Another possible application for coli extracts is against allergies such as hay fever, uticaria, eczema, food allergies, and asthma. Finally, a further therapeutic application has been found against acne and migraine; moreover, as is described in German Patent 32 16 298, these extracts are anabolically effective and lead to stimulation of the immune defenses that are not pathogen-specific.
Preparation processes have been described in German/Patent 38 16 298, as well as other sources. The bacteria-free and albumin-free extracts of Escherichia coli contain relatively short-chain oligopeptides and, as shown by the tests reported in Drug Research 23, 829-830 (1973), a polysaccharide component made predominantly of glucose, galactose, and xylose, and a slight proportion of fatty acids. It is striking that the quantitative analyses of these peptides performed thus far have confirmed the absence of aromatic amino acids, with the exception of histidine.

Cell-free and albumin-free extracts of Escherichia coli cultures of this kind can be administered orally or by injection.
Corresponding preparations are on the market, for instance from Laves Arzneimittel GmbH, Barbarastr.14, 3003 Ronnenberg, Federal Republic of G5rmany, under the tradename "Colibiogen?."

At present, the action of coli extracts in retrovirus infections, especially in combination with Ginkgo extracts, cannot as yet be explained scientifically in unequivocal terms. However, clinical tests have shown quite clearly that the patients' general condition improves, and the proportion of T4 lymphocytes is maintained--or typically even increases markedly--with use of this medication. According to a recently developed working hypothesis, the observed failure of the specific immune defense 2158~51 is suspected to be due to the fact that infections with retroviruses cause a drop in T4 lymphocytes and later a drop in T8 lymphocytes, as well. It has been hypothesized that an attendant overproduction of "unoriented" immunoglobulins by B-lymphocytes then contributes to weakening the specific immune defense and to its collapse. Clinical observations give grounds to assume that the active ingredients in the coli and Ginkgo extracts reduce or prevent the increasing production of "unoriented" immunoglobulins.

It has also been found that when coli and Ginkgo extracts are administered, the dose of retrovirus-specific drug can be reduced, and this substantially reduces the danger of side effects, particularly secondary anemias. The dose reduction is apparently only due in part to a better response to nucleoside therapeutic agents. The extent to which the combination addresses other biochemical mechanisms as well has not yet been determined.

The drugs according to the invention can be administered orally, although it is preferred that they be administered by injection.
It has been found that coli and Ginkgo extracts can be administered together with the nucleoside therapeutic agents but, on the other hand, there are also advantages if the coli or Ginkgo extract is used prior to the administration of the nucleoside therapeutic agent, and specifically if it is administered a few hours to one to two days earlier. Thus, the present invention also relates to a so-called kit that contains the individual active ingredients for oral or parenteral administration in spatially separate form. The dose for the dose unit with the coli extract is albumin-free and cell-free products 21~551 of metabolism of approximateiy 4 to 9 X10l2 microorganisms, while for the Ginkgo extract, the dose unit contains approximately 50 to 200 mg of dry extract. The daily dose must be adjusted individually for each patient. No side effects have yet been observed, even in the case of long-tern treatment.

The invention will be described in detail below on the basis of the examples that follow:

Example 1 The coli extract was prepared by the processes described in detail in German Patent 38 16 2g8, in which bouillon cultures were innoculated with a strain of the desired E. coli serotype and incubated for at least five days at 37C. It is preferred that the 02:Kl:K6 serotype be used. Bacteria-free and albumin-free filtrates are then prepared from this bouillon culture, with approximately 4 to 9 x 1012 colony-forming units per milliliter, in a manner known per se, under sterile conditions; they can either be further processed in a manner known per se to make injection solutions, or used as is for orally administered medication. The extracts can also be freed of water by gentle vacuum extraction and made into capsules or tablets in a manner known per se.

2~ 58~1 Example 2 In order to prepare the Ginkgo extract, green or yellow leaves of Ginkgo biloba are finely comminuted and extracted with aqueous acetone or ethanol in a manner known per se; this is done, in particular, by using a mixture of water with acetone or ethanol in a proportion of 50:50, for a period of several hours at temperatures between approximately 40 and 60. After extraction, liquid is pressed out of the leaves and can be subjected once again to further extraction. In a first purification step, the aqueous extract so obtained is extracted with a solvent, preferably cyclohexane, that is immiscible with water. The aqueous-ketonic phase is then further processed, and the nonpolar phase is discarded. The aqueous-ketonic phase is then concentrated to approximately half its volume, and the resultant precipitate is filtered off. The filtered solution is mixed in a known manner with ammonium sulfate and extracted with butanol.
After drying over sodium sulfate, the extract so obtained is concentrated at reduced pressure and then extracted to exhaustion with ethanol. This residue is then concentrated in a vacuum for drying.

On average, which can vary up or down by approximately 10~
depending on the origin and age of the leaves, this extract, which can now be packaged in the usual way, contains approximately 25% Ginkgo flavonoglycosides and approximately 5 terpene lactones.

21SB5:~ 1 Example 3 Clinical tests conducted up to now show that with daily oral administration of a teaspoon of coli extract by mouth or an injection of coli extract, patients with ARS display marked improvement in their general well-being within two days, along with a rise in the T4 lymphocyte population. If, in addition to the coli extract, one ampule of the Ginkgo extract, with a dry extract content of 50 mg, was administered, then an increase in the T4 lymphocyte count to over 400 per microliter was attainable, even in severe cases. If coli and Ginkgo extracts are given in addition to the therapy with nucleoside therapeutic agents, markedly fewer subjective and objective side effects were observed, so that for the first time, it has now become possible to reduce the dose during long-term therapy.

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Claims (16)

1. A drug for treating retrovirus infections, characterized in that it contains bacteria-free and albumin-free extracts, known per se, of Escherichia coli.

2. A drug as defined in Claim 1, characterized in that in addition, it contains extracts of the green or yellow leaves of Ginkgo biloba.

3. A drug as defined in Claim 1 or Claim 2, characterized in that the dose unit of the coli extract contains metabolic products from approximately 4 to 9 x 1012 microorganisms.

4. A drug as defined in Claim 2 or Claim 3, characterized in that the dose unit of the Ginkgo extract contains 50 to 200 mg of an extract of dry leaves.

5. A drug as defined in Claim 2 to Claim 4, characterized in that the proportion of Ginkgo flavonoglycosides to terpene lactones in the dry extract is approximately 5:1.

6. A drug as defined in Claim 2 to Claim 5, characterized in that the extracts are contained in spatially separate form.

7. A drug as defined in Claim 1 to Claim 6, characterized in that it additionally contains a chemotherapeutic agent against retrovirus infections.

8. A drug as defined in Claim 1 to Claim 7, characterized in that it is in injectable form.

9. The use of bacteria-free and albumen-free extracts of Escherichia coli to manufacture drugs for treating retrovirus infections.

10. Use as defined in Claim 9, charactrized in that the drugs also contain extracts from the green or yellow leaves of Gingko biloba.

11. Use as defined in Claim 9 or Claim 10, characterized in that the dose unit of the Escherichia coli contains metabolic products of approximately 4 to 9 X 1012 microorganisms

12. Use as defined in Claim 10 or Claim 11, characterized in that the dose unit of a Gingko extract contains 50 to 200 mg of an extract from dry leaves.

13. Use as defined in Claim 10 to Claim 12, characterized in that the proportion of Gingkoflavonoglycosides to terpene lactones in the dry extract is 5:1.

14. Use as defined in Claim 10 to Claim 13, characterized in that the drugs contain the E. coli or Gingko biloba extracts in spatially separated form.

15. Use as defined in Claim 9 to Claim 14, characterized in that the drugs additionally contain a chemotherapeutic agent against retrovirus infections.

16. Use as defined in Claim 9 to Claim 15, characterized in that the drug is in injectable form.