Title of Invention

A SYNERGISTIC ANTIBIOTIC PHARMACEUTICAL COMPOSITION HAVING ENHANCED BIOACTIVITY

Abstract The present invention relates to a synergistic antibiotic pharmaceutical composition having enhanced bioactivity. A pharmaceutical composition with lysergol as bioactive enhancer and bioavailability facilitator for broad-spectrum antibiotics, the present invention has direct implication in reducing the dosage of antibiotics while increasing the efficiency of absorption of nutritional elements. The said composition comprises; a) an antibiotic compound of the kind such as herein described 0.2-20 µg/ml; b) an effective amount lysergol l-10µg/ml; and c) and remaining optionally pharmaceutically acceptable additives. .
Full Text TECHNICAL FIELD
The invention relates to a synergistic antibiotic pharmaceutical composition having enhanced bioactivity. The present invention has direct implication in reducing the dosage of antibiotics while increasing the efficiency of absorption of nutritional elements. The present invention also provides a method of treatment for bacterial infections.
BACKGROUND ART
The consumption of antibiotics and drugs by man is increasing at an alarming rate. Out of the total drugs and chemicals, 20%-50% of that use is unnecessary depending on the class of antibiotic. In addition, indiscriminate use of antibiotics promotes antibiotic resistance leading to multiple drug resistance and makes it difficult to control the diseases. Really speaking, the infected individuals consume much more amount of antibiotics in the given dosage that is actually required to control a given population of parasite in the body. This may be due to (i) reduced absorption in the gut membrane when taken orally (ii) restrictive uptake by the target microbe or (iii) operation of efflux pump leading to indiscriminate extrusion of the antibiotics or therapeutic molecules. So the major amount of the drugs we apply are wasted and only a minor percentage is being targeted to the infective microbes. In addition, the unutilized drug / antibiotic amount remains as a load in the body and environment acting as a selection pressure facilitating emergence of drug resistance in parasites and their predominance, ultimately leading to failure of antibiotics against resistant infections. This also is responsible for side effects, illness and reduction in life expectancy. One of the ways, which has been feasible to reduce drug dosage, has been synergism between two therapeutic agents. However, if both have the antibiotic property, still the problem of continued selection pressure on microbes is likely to continue. So, we thought of searching only those molecules, which by them are not microbicidal but when present with a drug or active molecule, enhance its activity and availability (bioenhancers). This way these molecules by their presence will not exert any selection pressure for mutants to emerge resistant against them and on the other hand could reduce the dosage of antibiotics or drugs so that their ill effects are minimized and the resistance development process will be substantially delayed
already available. The seeds of Ipomoea muricata are commonly known as 'Kaladana' in trade and are being used as purgative in Pakistan and India. The seeds are a good source of clavine alkaloids. The seeds are reported to contain 0.49% of total alkaloid, out of which lysergol constitutes 53% and chanoclavine 37%. Lysergol is used as hypotensive, psychotrophic, analgesic, immunostimulant, analeptic and uterus and intestine stimulating drug. Also, the compound is available commercially (Sigma Chemicals, USA).
The compound lysergol is chemically known as 9,10-Didehydro-6-methylergoline-8-□-methanol.
Ergotamine and all compounds either structurally and/or pharmacologically similar to it like lysergol are 5HT agonist vasoactive agents (United States Patent 6,077,539) that means ensure normal blood flow in blood vessels in a therapeutically effective amount. This compound also is reportedly psychoactive causing nausea, which may be experienced during first hour. This compound also has hallucination and anti-tension properties.
Great emphasis now is being laid towards quality assurance of crude drugs from plants sources widely used in the Indian system of medicine. The scientific study of traditional medicines, derivation of drugs through bioprospection and systematic conservation, domestication and cultivation of the concerned medicinal plants has assumed great importance in the present day context when more and more people prefer safe and effective medicines at affordable price for curing their ailments. The present invention enlarges the scope and use of the natural plant compound lysergol in therapeutical applications. OBJECTS OF THE INVENTION
Main object of the present invention is to provide a synergistic antibiotic pharmaceutical composition having enhanced bioactivity.
Another object of the present invention is to provide a synergistic antibiotic pharmaceutical composition for the treatment of bacterial infections. Yet another object of the present invention is to provide an antibiotic pharmaceutical composition having reduced concentration of antibiotic compounds. Further object of the present invention is to provide an antibiotic pharmaceutical composition to prevent antibiotic drug resistance.
SUMMARY OF THE IVNENTION
The invention provides a synergistic pharmaceutical composition with lysergol as bio-
erihancer of antibiotic action on the target. The molecule of invention helps in the
absorption of antibiotics across the cell membrane in animal cells for action against
gram positive and negative bacteria. The present invention also provides a method of
treatment for bacterial infections.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a synergistic antibiotic pharmaceutical
composition having enhanced bioactivity, said composition comprising:
a) an antibiotic compound of the kind such as herein described 0.2-20 µg/ml;
b) an effective amount lysergol l-10µg/ml; and
c) and remaining optionally pharmaceutically acceptable additives.
An embodiment of the present invention, wherein the antibiotic is selected from the
group consisting of rifampicin, tetracycline and ampicillin.
Yet another embodiment of the present invention, wherein the preferable amount of
lysergol is l0µg/ml.
Still another embodiment of the present invention, wherein the enhanced activity of
antimicrobial effect is in the range of 2-12 folds.
Yet another embodiment of the present invention, wherein said composition is
effective against broad-spectrum microbes both gram positive and negative, selected
from the group consisting E.coli, Bacillus subtilis and Mycobacterium smegmatis and
other similar microbes.
Further embodiment of the present invention, wherein lysergol is isolated from a
genera of lower fungi consisting of Claviceps, Pencillium and Rhizopus and from the
plants selected from Rivea corymbosa and ipomoea violace.
Yet another embodiment of the present invention, wherein lysergol enhances the
transport of antibiotics across the intestinal gut and cell membrane for better efficacy
on the target site.
Still another embodiment of the present invention, wherein the reduced dosage of
antibiotics and the enhanced bioactivity of the composition reduces the ill effects of
antibiotics.
Yet another embodiment of the present invention, wherein the resistance to antibiotics
is substantially reduced due to reduced concentration of antibiotics.


The present invention also provides a method of treating bacterial infection, wherein
administering to subject an effective amount of synergistic pharmaceutical
composition, said composition comprising:
(a) an antibiotic compound;
(b) an effective amount lysergol 2-10ng/ml; and
(c) optionally pharmaceutically acceptable additives.
An embodiment of the present invention, a method wherein the antibiotic is selected
from the group consists of rifampicin, tetracycline and ampicillin.
Yet another embodiment of the present invention, a method wherein the preferable
dosage of lysergol is lOug/ml.
Still another embodiment of the present invention, a method wherein the enhanced
activity of antimicrobial effect is in the range of 2-12 folds.
Further embodiment of the present invention a method wherein said composition is
effective against broad-spectrum microbes both gram positive and negative, selected
from the group consisting E.coli, Bacillus subtilis and Mycobacterium smegmatis and
other similar microbes.
Yet another embodiment of the present invention, a method wherein lysergol is
isolated from genera of lower fungi consisting of Claviceps, Pencillium and Rhizopus
and from higher plants selected from Rivea corymbosa and ipomoea violace.
Still another embodiment of the present invention, a method wherein lysergol
enhances the transport of antibiotics across the intestinal gut and cell membrane for
better efficacy on the target site.
Further embodiment of the present invention, a method wherein the reduced dosage of
antibiotics and the enhanced bioactivity of the composition reduces the ill effects of
antibiotics.
Still another embodiment of the present invention, a method wherein the resistance to
antibiotics is substantially reduced due to reduced concentration of antibiotics.
Yet another embodiment of the present invention, a method wherein the subject is
selected from mammals and humans.
The invention is further explained in the form of following embodiments.
1. Assay for bio-enhancement of anti-infective agents
(a) The minimum inhibitory concentration (MIC) of antibiotic is determined
against Escherichia coli (ATCC 10536), Bacillus subtilis (ATCC 6051)
and Mycobacterium smegmatis (ATCC 14468) in broth and disc diffusion
assay.
(b) The antibiotics at concentrations 1/4, 1/3, 1/2 and equal to MIC are added
alone and in combination with the test compound at varying
concentrations on disc and in broth to evaluate the comparative inhibition.
(c) These combinations showing significant advantage or higher activity than
antibiotic alone in terms of enhanced inhibition of bacterial growth (large
inhibition zone in disc diffusion and effectivity of lower concentration in
broth assay) are picked up for future testing.
(d) In broth assay the activity is quantified by counting number of viable cells
in a given treatment and converted in fold enhancement by combination
compared to antibiotic / drug alone in the killing percentage of cells.
(e) The pretreatment assay followed to determine whether the compound is
required along with antibiotic to enhance its activity or even its
withdrawal after treatment or prior to antibiotic treatment would benefit.
For this, the cells are treated with compound for 4 to 8 hours and then
washed free of it by centrifugation and washing in sterile water. This is
followed by treatment with antibiotic as in steps b to d.
Process for the isolation of lysergol.
The seeds of Ipomoea muricata are powdered and defatted with hexane and then
extracted with methyl alcohol. The alcoholic extract is dried and extracted with 5-
10% Hcl solution. The acidic extract is then converted to basified upto pH 9.0 and
extracted with chloroform and butanol successively. The crude alkaloid obtained in
chloroform and butanol extract is further purified by column chromatography to yield
lysergol in maximum yield upto 0.2%.
Bioactivity is experimented with the killing activities of different antibiotics against
the bacteria singly and in combination with the test compound Lysergol following the
method described above. These experiments are being described in the following
examples. When the bacteria are grown in presence of the compound as such no
significant killing is observed. In all the experiments the Lysergol concentration is
kept at 10 ng/ml, unless it is specifically mentioned.
The invention is further explained in the form of examples. However, these examples
should not be considered as limiting the scope of the invention.
Example 1
Lysergol mediated enhancement in the killing action of antibiotics against Gram
negative bacterium Escherichia coli.
Example 2.
Lysergol mediated enhancement in the killing action of antibiotics against Gram
positive bacterium Bacillus subtilis.
It is calculated as = Survival fraction of viable cells upon treatment with antibiotic
and lysergol in combination / Survival fraction of viable cells upon treatment with
antibiotic alone
Example 3
Lysergol mediated enhancement in the killing action of antibiotics against bacterium
Mycobacterium smegmatis
It is calculated as = Survival fraction of viable cells upon treatment with antibiotic
and lysergol in combination / Survival fraction of viable cells upon treatment with
antibiotic alone
From the above experiments it is deduced that the potency of the antibiotic is
increased against both Gram positive and negative bacteria when applied along with
the compound lysergol.
Example 4
Lysergol mediated enhancement in the killing action of antibiotic against bacteria in
disc diffusion assays.
In other observations the compound lysergol enhances the transport of antibiotics e.g.
Rifampicin, Tetracycline across the gut as well as artificial membrane. We performed
the experiments in U-shaped tubes with joint in between, where the freshly isolated
gut membrane is fixed. In control tube only antibiotic solution (4ml @ Img/ml
solution) is poured in the left arm where as in the right arm only water is poured. In
the other tube in addition to the antibiotic solution lysergol (4ug @ lug/ml) is added.
Then changes in absorbency in the right arm of both the tubes are noted at 340nm (for
rifampicin) and 223nm (for tetracycline). The enhancement in transport is
approximately 2.96 to 8.53 folds. This in-turn has immense importance for absorption
of the drugs, Pharmaceuticals, nutraceutical and other related compounds and ions by
the cells.
ADVANTAGES
1. The main advantage of the present invention is the reduction of antibiotic dosage
by means of synergistic composition.
2. Reduction in antibiotic dosage resulting in prevention of antibiotic drug
resistance.
3. Incorporation of bioactive enhancer in the antibiotic composition, which non-toxic
to animals and humans.








WE CLAIM:
1. A synergistic antibiotic pharmaceutical composition having enhanced bioactivity,
said composition comprising:
c) an antibiotic compound of the kind such as herein described 0.2-20 µg/ml;
d) an effective amount lysergol l-10 µg/ml; and
c) and remaining optionally pharmaceutically acceptable additives.
2. A pharmaceutical composition as claimed in claim 1, wherein the antibiotic is selected from the group consisting of rifampicin, tetracycline and ampicillin.
3. A pharmaceutical composition as claimed in claim 1, wherein the preferable amount of lysergol is 10 µg/ml.
4. A pharmaceutical composition according to claim 1, wherein the enhanced activity of antimicrobial effect is in the range of 2-12 folds over antibiotic compounds used singly.
5. A pharmaceutical composition according to claim 1, wherein said composition is effective against broad-spectrum microbes both gram positive and negative, selected from the group consisting E.coli, Bacillus subtilis and Mycobacterium smegmatis and other similar microbes.
6. A pharmaceutical composition according to claim 1, wherein lysergol is isolated from genera of lower fungi consisting of Claviceps, Pencillium and Rhizopus and from the plants selected from Rivea corymbosa and ipomoea violace.
7. A pharmaceutical composition according to claim 1, wherein lysergol enhances the transport of antibiotics across the intestinal gut and cell membrane for better efficacy on the target site.
8. A pharmaceutical composition according to claim 1, wherein the resistance to antibiotics is substantially reduced due to reduced concentration of antibiotics.
9. A synergistic antibiotic pharmaceutical composition having enhanced bioactivity substantially as herein described with reference to examples accompanying this specification.

Documents:

2564-DELNP-2004-Abstract-(02-04-2009).pdf

2564-DELNP-2004-Abstract-(19-03-2009).pdf

2564-delnp-2004-abstract.pdf

2564-DELNP-2004-Claims-(02-04-2009).pdf

2564-DELNP-2004-Claims-(19-03-2009).pdf

2564-delnp-2004-claims.pdf

2564-delnp-2004-complete specification (granted).pdf

2564-DELNP-2004-Correspondence-Others-(02-04-2009).pdf

2564-DELNP-2004-Correspondence-Others-(19-03-2009).pdf

2564-delnp-2004-correspondence-others.pdf

2564-DELNP-2004-Description (Complete)-(02-04-2009).pdf

2564-DELNP-2004-Description (Complete)-(19-03-2009).pdf

2564-delnp-2004-description (complete).pdf

2564-DELNP-2004-Form-1-(19-03-2009).pdf

2564-delnp-2004-form-1.pdf

2564-delnp-2004-form-18.pdf

2564-DELNP-2004-Form-2-(19-03-2009).pdf

2564-delnp-2004-form-2.pdf

2564-DELNP-2004-Form-3-(19-03-2009).pdf

2564-delnp-2004-form-3.pdf

2564-delnp-2004-form-5.pdf

2564-DELNP-2004-Others-Document-(02-04-2009).pdf

2564-DELNP-2004-Others-Document-(19-03-2009).pdf

2564-DELNP-2004-PCT-210-(02-04-2009).pdf

2564-DELNP-2004-PCT-409-(02-04-2009).pdf

2564-DELNP-2004-Petition-137-(19-03-2009).pdf


Patent Number 233906
Indian Patent Application Number 2564/DELNP/2004
PG Journal Number 18/2009
Publication Date 01-May-2009
Grant Date 20-Apr-2009
Date of Filing 01-Sep-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 JAI SHANKAR ARYA CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
2 SANTOSH KUMAR SRIVASTAVA CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
3 AJIT KUMAR SHASANY CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
4 TIRUPPADIRIPULIYUR RANGANATHAN KUMAR CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
5 MAHENDRA PANDURANG DAROKAR CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
6 SUSHIL KUMAR CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
7 SUMAN PREET SINGH KHANUJA CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS P.O.CIMAP, PICNIC SPOT ROAD LUCKNOW
PCT International Classification Number A61K 31/495
PCT International Application Number PCT/IB02/01125
PCT International Filing date 2002-03-25
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA