FIELD OF THE INVENTION
The present invention relates to a method for the treatment of tuberculosis in a
subject by administering to the subject a therapeutically effective amount of the compound
of Formula 1 (as described herein), or a stereoisomer, a tautomer, a pharmaceutically
acceptable salt, or a derivative thereof. The present invention, also relates to a
pharmaceutical composition comprising the compound of Formula 1 as an active ingredient
for use in the treatment of tuberculosis.
BACKGROUND OF THE INVENTION
Tuberculosis (TB) is a common lethal infectious disease caused by various strains
of mycobacteria, particularly Mycobacterium tuberculosis (MTB). The distribution of
tuberculosis is not uniform across the globe. Approximately fifty million people are
infected with TB worldwide. Of those infected, about ten million people may develop
active disease and there will be close to three million deaths annually due to tuberculosis.
Tuberculosis can affect any organ of the body and is manifested in several different
forms, but the primary site of infection is the lung. Tuberculosis of the lungs usually
spreads through the air when people who have an active MTB infection cough, or sneeze.
The classic symptoms are a chronic cough with blood-tinged sputum, fever, night sweats,
and weight loss. Diagnosis relies on radiology (commonly chest X-rays), a tuberculin skin
test, blood tests, as well as microscopic examination and microbiological culture of body
fluids.
Effective tuberculosis treatment involves use of antibiotics. Unusual structure and
chemical composition of the mycobacterial cell wall makes many antibiotics ineffective
and hinders the entry of the anti-tubercular drugs. Instead of the short course of antibiotics
typically used to cure other bacterial infections, TB requires much longer period of
treatment (around six to twenty four months) to entirely eliminate mycobacteria from the
body. The two antibiotics most commonly used for the treatment of tuberculosis are
isoniazid and rifampicin.
The DOTS (Directly Observed Treatment Short-course) strategy of tuberculosis
treatment recommended by WHO (the World Health Organisation) was based on clinical
trials done in the 1970s by Tuberculosis Research Centre, Chennai, India.
Multidrug resistant tuberculosis (MDR-TB) is a form of tuberculosis that is
resistant to at least two of the front-line antitubercular drugs, isoniazid and rifampicin.
MDR-TB is treated with second line antitubercular drugs like fluoroquinolones, paraaminosalicyclic
acid, thioacetazone and aminoglycosides such as amikacin, kanamycin or
capreomycin. The treatment of MDR-TB is associated with prolonged illness and
disability. The average recommended duration of treatment is two years. Second line
antitubercular drugs are generally considered to be less effective, show adverse reactions,
and poor bioavailability in the lungs, which is detrimental for disease eradication (Eur.
Respir. J., 2002, Supplement 36, 66S-77S).
Thus, in spite of the recent development, there is still a need to provide drugs to
effectively treat patients who are infected with Mycobacterium tuberculosis, especially
with multi drug resistant Mycobacterium tuberculosis. Anti-tuberculosis compounds from
natural sources have an enormous potential for the development as new drugs.
Fusaricidins are known antibiotics that have a ring structure composed of six amino
acid residues in addition to 15-guanidino-3-hydroxypentadecanoic acid (GHPD). Various
analogs of Fusaricidins (LI-F03, LI-F04, LI-F05, LI-F06, LI-F07 and LI-F08) were isolated
from culture broth of Paenibacillus polymyxa and have been characterised (The Journal of
Antibiotics, 1987, 40, 1506-1514; Biochemical and Biophysical Research
Communications, 2008, 365, 89-95). Fusaricidin A, B, C and D were isolated from culture
broth of Bacillus polymyxa KT-8, and were reported to be active against fungi and grampositive
bacteria (The Journal of Antibiotics, 1996, 49, (2), 129-135; The Journal of
Antibiotics, 1997, 50, (3), 220-228).
The inventors of the present invention have found that the compound of Formula 1
(Fusaricidin B) is active against both, Mycobacterium tuberculosis and multidrug resistant
Mycobacterium tuberculosis, and hence, is useful in the treatment of TB and MDR-TB.
SUMMARY OF THE INVENTION
The present invention relates to a method for the treatment of tuberculosis in a
subject comprising administering to the subject, a therapeutically effective amount of the
compound of Formula 1 (as described herein), or a stereoisomer, a tautomer, a
pharmaceutically acceptable salt or a derivative thereof.
The present invention also relates to the compound of Formula 1, or a stereoisomer,
a tautomer, a pharmaceutically acceptable salt or a derivative thereof, for use in the
treatment of tuberculosis.
The present invention also relates to a method of inhibiting growth of
Mycobacterium tuberculosis organism, comprising contacting the Mycobacterium
tuberculosis organism in vitro or ex vivo with the compound of Formula 1, or a
stereoisomer, a tautomer, a pharmaceutically acceptable salt or a derivative thereof, in an
amount sufficient to inhibit the growth of Mycobacterium tuberculosis organism.
The present invention also relates to a pharmaceutical composition, comprising the
compound of Formula 1, or a stereoisomer, a tautomer, a pharmaceutically acceptable salt
or a derivative thereof, and at least one pharmaceutically acceptable carrier, for use in the
treatment of tuberculosis.
The present invention also relates to a use of the compound of Formula 1, or a
stereoisomer, a tautomer, a pharmaceutically acceptable salt or a derivative thereof, for the
manufacture of a medicament for the treatment of tuberculosis.
DETAILED DESCRIPTION OF THE INVENTION
Fusaricidins are antibiotics which have a ring structure composed of six amino acid
residues in addition to 15-guanidino-3-hydroxypentadecanoic acid (GHPD) isolated from
fermentation of Paenibacillus sp., and have excellent antifungal activity.
In one aspect, the present invention provides Fusaricidins having the following
general formula, for use in the treatment of tuberculosis.
wherein,
GHPD 15-guanidino-3-hydroxypentadecanoic acid,
L-Thr L-Threonine,
D-Ala D-Alanine,
D-allo-Thr D-a//o-Threonine,
Xi, X2 and X3 are amino acids as described in Table 1
Table 1
Ref: Biochemical and Biophysical Research Communications, 2008, 365, 89-95.
In an embodiment, the present invention provides Fusaricidin B represented by
Formula 1 (referred to herein as the compound of Formula 1),
L - Val ( 2 ) D - Val ( 1 )
Formula 1
for use in the treatment of tuberculosis.
In an embodiment, the present invention relates to a method for the treatment of
tuberculosis in a subject comprising administering to the subject, a therapeutically effective
amount of the compound of Formula 1 (as described herein), or a stereoisomer, a tautomer,
a pharmaceutically acceptable salt or a derivative thereof.
The compound of Formula 1 has the molecular formula C42H76N10O11 (molecular
weight 896). The compound of Formula 1 was characterised by the physico-chemical and
spectral properties, such as mass spectrum (MS) and nuclear magnetic resonance (NMR)
spectroscopic data as discussed herein below. The compound has been identified as
Fusaricidin B by comparing the spectral data with the data reported in the literature
(Journal of Antibiotics, 1997, 50 (3), 220 - 228).
The compound of Formula 1 is found to be active against Mycobacterium
tuberculosis. Accordingly, the compound of Formula 1 is useful in the treatment of
tuberculosis.
The present invention encompasses within its scope, use of the compound of
Formula 1 i.e. Fusaricidin B that is obtained by a synthetic method and/or from natural
sources including microorganisms; for the treatment of tuberculosis.
One microorganism from which the compound of Formula 1 is isolated, belongs to
eubacteria (PM0834793/MTCC5620) (herein after referred to as culture no. PM0834793),
which is isolated from the soil sample collected from the land near the hot-springs located
in Uttarkashi district of Uttranchal State, India.
A process for the production of the compound of Formula 1 from the culture no.
PM0834793 comprises the steps of:
(a) growing the culture no. PM0834793;
(b) isolating the compound of Formula 1 from the culture broth; and
(c) purifying the compound of Formula 1.
The step (c) involving purification of the compound of Formula 1 is carried out by
using purification procedures generally used in the related art.
Definitions:
For the purpose of the disclosure, listed below are definitions of various terms used
to describe the present invention. These definitions apply to the terms as they are used
throughout the specification and the appended claims (unless they are otherwise limited in
specific instances) either individually or as part of a larger group. They should not be
interpreted in the literal sense. They are not general definitions and are relevant only for
this application.
In the specification where the term "compound of Formula 1" is used alone,
wherever appropriate, it is deemed to include a stereoisomer, or a tautomer or a
pharmaceutically acceptable salt and a derivative such as an ester or an ether of the
compound of Formula 1. The compound of Formula 1 refers to Fusaricidin B. For the
purpose of the present invention, Fusaricidin B refers to a synthetically prepared
Fusaricidin B and/or that obtained from other natural sources including microorganisms.
The term "pharmaceutically acceptable" refers to a compound or a carrier or an
additive or a salt that is not biologically or otherwise undesirable. In other words a
pharmaceutically acceptable compound or a salt must not cause any undesirable biological
effects or interact in an undesirable manner with any of the other ingredients of the
pharmaceutical composition in which it is contained.
The term "pharmaceutically acceptable carrier" as used herein means a diluent,
excipient, encapsulating material or formulation auxiliary, which is non-toxic, and inert,
which does not have undesirable affect on a subject, preferably a mammal, more preferably
a human, and is suitable for delivering an active agent to the target site without affecting
the activity of the agent.
The term, "therapeutically effective amount" as used herein means an amount of the
compound of Formula 1 or a composition comprising said compound, sufficient to
significantly induce a positive modification in the condition to be treated, but low enough
to avoid side effects, if any (at a reasonable benefit/risk ratio), within the scope of sound
medical judgment. The requirement of therapeutically effective amount of the compound of
Formula 1 or the composition containing said compound, for the treatment will vary with
the severity of tuberculosis being treated, the age and physical condition of the subject
(patient), the duration of the treatment, the nature of concurrent therapy, the particular
pharmaceutically acceptable carrier utilized, and like factors.
The term "in an amount sufficient" as used herein in relation to inhibiting the
growth of Mycobacterium tuberculosis organism, means an amount of the compound of
Formula 1 or a composition comprising said compound, sufficient to significantly induce
inhibition of the growth of Mycobacterium tuberculosis organism.
The term 'treating", "treat" or "treatment" as used herein have their ordinary or
customary meanings, and include alleviating the infection or disease, slowing the
progression of, attenuation or cure of the existing disease, in this case tuberculosis.
The term "subject" as used herein refers to an animal, preferably a mammal, and
most preferably a human.
The term "mammal" used herein refers to warm-blooded vertebrate animals of the
class mammalia, including humans, characterized by a covering of hair on the skin and, in
the female, milk-producing mammary glands for nourishing the young. The term mammal
includes without limitations animals such as cat, dog, horse, rabbit, bear, fox, wolf,
monkey, deer, mouse, pig as well as human.
The term "pharmaceutically acceptable salt(s)" is meant to include salt(s) of the
compound of Formula 1, which are prepared with acids or bases. Examples of
pharmaceutically acceptable base addition salts include sodium, potassium, calcium,
magnesium, ammonium or an organic base salt. Examples of pharmaceutically acceptable
organic base addition salts include those derived from organic bases such as lysine,
arginine or guanidine. Examples of pharmaceutically acceptable acid addition salts include
those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid
or sulfuric acid, as well as the salts derived from organic acids such as acetic acid,
propionic acid, oxalic acid, maleic acid, benzoic acid, succinic acid, fumaric acid, phthalic
acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid or methanesulfonic
acid.
As used herein, the term "mutant" refers to an organism or cell carrying a mutation,
which is an alternative phenotype to the wild-type.
As used herein, the term "variant" refers to an individual organism that is
recognizably different from an arbitrary standard type in that species.
As used herein, the term "nutrient" refers to a substance that provides nourishment
for growth or metabolism.
Preliminary identification of culture no. PM0834793, from which the compound of
Formula 1 (Fusaricidin B) is produced, is performed by examination of its colony
characteristics. The colonies are picked up with sterile needle by viewing the colony
morphology through stereo microscope (SM). Culture no. PM0834793 has been identified
as a microorganism belonging to eubacteria.
Culture no. PM0834793 has been deposited with Microbial Type Culture Collection
(MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh - 160 036, India, a
World Intellectual Property Organization (WIPO) recognized International Depository
Authority (IDA). The culture no. PM0834793 has been given the accession number
MTCC5620.
In addition to the specific microorganism described herein, it should be understood
that mutants of PM0834793, such as those produced by the use of chemical or physical
mutagens including X-rays, U.V. rays etc. and organisms whose genetic makeup has been
modified by molecular biology techniques, may also be cultivated to produce the
compound of Formula 1.
The screening for suitable mutants and variants which can produce the compound
according to the invention can be confirmed by HPLC, NMR, MS and/or determination of
biological activity of the active compounds accumulated in the culture broth, for example
by testing the compounds for anti-tuberculosis activity or by a combination thereof.
The medium and/or nutrient medium used for isolation and cultivation of culture
no. PM0834793, from which the compound of Formula 1 is produced, preferably contains
sources of carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for
example, one or more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol,
lactose, or galactose. A preferred carbon source is soluble starch and glucose. The sources
of nitrogen are, for example, one or more of soyabean meal, casein, peanut meal, yeast
extract, beef extract, peptone, malt extract, corn steep liquor, gelatin, tryptone or casamino
acids. Preferred nitrogen source is casein, peptone, beef extract and yeast extract. The
nutrient inorganic salts are, for example, one or more of sodium chloride, potassium
chloride, calcium chloride, manganese chloride, magnesium chloride, strontium chloride,
cobalt chloride, potassium bromide, sodium fluoride, sodium hydrogen phosphate,
potassium hydrogen phosphate, dipotassium hydrogen phosphate, disodium phosphate,
calcium carbonate, sodium bicarbonate, sodium silicate, sodium nitrate, ammonium nitrate,
potassium nitrate, sodium sulphate, ammonium sulphate, ammonium heptamolybdate,
ferric citrate, copper sulphate, magnesium sulphate, ferrous sulphate, zinc sulphate or boric
acid. Calcium carbonate, sodium chloride and potassium nitrate are the preferred inorganic
salts.
Typically, culture no. PM0834793 was purified and maintained on slants of
agarified soyabean casein digest medium (SCDM); [Hi Media, Mumbai, Maharashtra,
India Catalogue no. MO11-500G].
For long term preservation, the culture is preserved in glycerol vials with
preservation media composed of 20 % glycerol, 0.3 % tryptone and yeast extract and is
stored at -80 °C.
Seed culture cultivation of culture no. PM0834793 may be carried out at a
temperature ranging from 27 °C to 33 °C and a pH of about 6.0 to 8.5, for 40-55 h at 240-
260 rpm (revolutions per minute). Typically, culture no. PM0834793 seed is cultivated at
29 °C -31 °C and a pH of about 7.0-7.8, for 45- 50 h at 245-255 rpm.
The production of the compound of Formula 1 may be carried out by cultivating
culture no PM0834793 by fermentation at a temperature ranging from 27 °C to 33 °C and a
pH of about 6.0 to 8.5, for 40-55 h at 240- 260 rpm. Typically, culture no. PM0834793 is
cultivated at 29 °C-31 °C and pH 7.0-7.8 for 45-50 h at 240- 250 rpm.
The production of the compound of Formula 1 may be carried out by cultivating
culture no. PM0834793 in a suitable nutrient broth under conditions described herein. The
progress of fermentation and production of the compound of Formula 1 may be detected by
high performance liquid chromatography (HPLC) and by measuring the bioactivity in antiinfective
screening test models using organisms such as Staph aureus 209P, Escherichia
faecium VRE 323, Candida albicans, Escherichia coli 20732, and Aspergillus fumigatus.
Fermentation is a process of growing microorganisms for the production of various
chemical or pharmaceutical compounds. Microbes are normally incubated under specific
conditions in the presence of nutrients. Whole broth is obtained after completing the
process of fermentation. The whole broth is subjected to centrifugation which results in
formation of cell mass and culture filtrate, which can be processed further by processes
described herein.
The compound of Formula 1 present in the culture broth may be isolated using
different extraction methods and chromatographic techniques.
Thus, the compound of Formula 1 may be recovered from the culture filtrate by
extraction with a water immiscible solvent such as petroleum ether, dichloromethane,
chloroform, ethyl acetate, diethyl ether or butanol, or by hydrophobic interaction
chromatography using polymeric resins such as "Diaion HP-20®" (Mitsubishi Chemical
Industries Limited, Japan), "Amberlite XAD®" (Rohm and Haas Industries, USA) or
adsorption on activated charcoal. These techniques may be used repeatedly, alone or in
combination.
The active material may be recovered from the cell mass by treatment with a water
miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or iso-propanol or by
extraction with a water immiscible solvent such as petroleum ether, dichloromethane,
chloroform, ethyl acetate or butanol.
One other option to recover the active material involves extracting the whole broth
with a solvent selected from petroleum ether, dichloromethane, chloroform, ethyl acetate,
methanol, acetone, acetonitrile, n-propanol, iso-propanol, or butanol.
Typically, the active material is obtained from the whole broth using methanol for
extraction, followed by concentration and chromatography using Diaion HP-20.
Concentration and lyophilization of the eluates of Diaion HP-20 column, give the active
crude material.
According to the present invention, the compound of Formula 1 may be recovered
from the crude material by fractionation using any of the following techniques: dilution
with water and extraction with a water immiscible solvent such as petroleum ether,
dichloromethane, chloroform, ethyl acetate, diethyl ether or butanol; normal phase
chromatography (using alumina or silica gel as stationary phase; eluents such as petroleum
ether, ethyl acetate, dichloromethane, acetone, chloroform, methanol, or combinations
thereof; and additions of amines such as NEt3) ; reverse phase chromatography (using
reverse phase silica gel such as dimethyloctadecylsilylsilica gel, (RP-18) or
dimethyloctylsilyl silica gel (RP-8) as stationary phase; and eluents such as water, buffers
(for example, phosphate, acetate, citrate (pH 2-8)), and organic solvents (for example
methanol, acetonitrile, acetone, tetrahydrofuran, or combinations of these solvents)); gel
permeation chromatography (using resins such as Sephadex LH-20® (Pharmacia Chemical
Industries, Sweden), TSKgel® Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in
solvents such as methanol, chloroform, acetone, ethyl acetate, or their combinations); or by
preparative high performance liquid chromatography (HPLC) (using a eluent system made
up of two or more solvents such as water, trifluoroacetic acid, methanol, ethanol, isopropanol,
n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride,
chloroform, ethyl acetate, petroleum ether, benzene, and toluene). These techniques may be
used repeatedly, alone or in combination. Typically, pure compound is obtained by dilution
with water and extraction with a water immiscible solvent; followed by chromatography
over normal phase silica gel; followed by gel permeation chromatography using Sephadex
LH-20® and chromatography using reverse phase silica gel (RP-18).
As used herein, the term "stereoisomer" is a general term used for all isomers of the
compound of Formula 1, that differ only in the orientation of their atoms in space. The term
isomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers
(racemates or racemic mixtures) and isomers of compounds with more than one chiral
center that are not mirror images of one another (diaisomers). The compound of the present
invention may have asymmetric centers and occur as racemates, racemic mixtures,
individual diaisomers, or enantiomers, or may exist as geometric isomers, with all isomeric
forms of said compound being included in the present invention.
As used herein, the term "tautomer" refers to the coexistence of two (or more)
compounds that differ from each other only in the position of one (or more) mobile atoms
and in electron distribution, for example, keto-enol tautomers.
The compound of Formula 1, or a stereoisomer, or a tautomer thereof may be
converted into their pharmaceutically acceptable salts, which are all contemplated by the
present invention.
The salts may be prepared by standard procedures known to one skilled in the art,
for example, salts like sodium and potassium salts, may be prepared by treating the
compound of Formula 1, or a stereoisomer, or a tautomer thereof, with a suitable sodium or
potassium base, for example sodium hydroxide or potassium hydroxide. Similarly, salts
like hydrochloride and sulphate salts, may be prepared by treating the compound of
Formula 1, or a stereoisomer, or a tautomer, thereof, with a suitable acid, for example
hydrochloric acid, or sulphuric acid.
The derivative of the compound of Formula 1 may be an ester or ether of the
compound of Formula 1. The ester and ether of the compound of Formula 1 can be
prepared by following the methods described in the literature (Advanced Organic
Chemistry, 1992, 4th Edition, J . March, John Wiley & Sons).
The compound of Formula 1 has activity against Mycobacterium tuberculosis
organism, which is sensitive or multi drug resistant such as M. tuberculosis H37Rv; M.
tuberculosis Clinical isolate - S (Streptomycin), H (Isoniazid or Isonicotinyl hydrazine), R
(Rifampicin) and E (Ethambutol) - resistant.
The term "Mycobacterium" or "Mycobacterium species" refers to Gram-positive,
non-motile, pleomorphic rods related to the actinomyces. MDR-TB (multi-drug resistant
tuberculosis) describes strains of tuberculosis that are resistant to at least the two first-line
TB drugs, isoniazid and rifampicin.
Embodiments:
In an embodiment, the present invention relates to a method of treating tuberculosis
in a subject, comprising administering to the subject a therapeutically effective amount of
the compound of Formula 1, or a stereoisomer, a tautomer, a pharmaceutically acceptable
salt, or a derivative thereof.
In an embodiment, the present invention relates to a method of treating tuberculosis
in a subject, comprising administering to the subject in need thereof a therapeutically
effective compound of Formula 1, or a stereoisomer, a tautomer, a pharmaceutically
acceptable salt, or a derivative thereof.
In an embodiment, the present invention relates to a method of inhibiting growth of
the Mycobacterium tuberculosis organism, comprising contacting the Mycobacterium
tuberculosis organism with the compound of Formula 1, or a stereoisomer, a tautomer, a
pharmaceutically acceptable salt, or a derivative thereof in an amount sufficient to inhibit
growth of the organism.
The method of inhibiting growth of the Mycobacterium tuberculosis organism, may
be in vitro or ex vivo, comprising contacting the organism in vitro or ex vivo with the
compound of Formula 1, or a stereoisomer, a tautomer, a pharmaceutically acceptable salt,
or a derivative thereof, in an amount sufficient to inhibit the growth of the organism.
In an embodiment, the present invention relates to a method of inhibiting growth of
Mycobacterium tuberculosis organism, comprising contacting the Mycobacterium
tuberculosis organism in vitro with the compound of Formula 1, or a stereoisomer, a
tautomer, a pharmaceutically acceptable salt, or a derivative thereof, in an amount
sufficient to inhibit the growth of the organism.
In an embodiment, the present invention relates to a method of inhibiting growth of
Mycobacterium tuberculosis organism comprising contacting the Mycobacterium
tuberculosis organism ex vivo with the compound of Formula 1, or a stereoisomer, a
tautomer, a pharmaceutically acceptable salt, or a derivative thereof, in an amount
sufficient to inhibit the growth of the organism.
The compound of Formula 1, or a stereoisomer, a tautomer, a pharmaceutically
acceptable salt, or a derivative thereof, may be administered to animals, such as mammals,
including humans, to a subject who is diagnosed with tuberculosis, and in the form of a
pharmaceutical composition.
Accordingly, the present invention also relates to the compound of Formula 1, or a
stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a derivative thereof, for use
in the production of medicaments for the treatment of tuberculosis.
Accordingly, the present invention also relates to use of the compound of Formula
1, or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a derivative thereof,
for the treatment of tuberculosis.
Tuberculosis for the treatment of which the compound of Formula 1, or a
stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a derivative thereof is
provided, is caused by Mycobacterium tuberculosis organism. The Mycobacterium
tuberculosis organism is a sensitive Mycobacterium tuberculosis organism or a multi drug
resistant Mycobacterium tuberculosis organism or a combination thereof.
Pharmaceutical Compositions:
The present invention further relates to a pharmaceutical composition comprising a
therapeutically effective amount of the compound of Formula 1, or a stereoisomer, a
tautomer, a pharmaceutically acceptable salt, or a derivative thereof, together with a
pharmaceutically acceptable carrier. The effective amount of the compound of Formula 1,
or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a derivative as the
active ingredient in the pharmaceutical compositions ranges from about 10 mg to 1000 mg.
The present invention further relates to a pharmaceutical composition which
contains a therapeutically effective amount of the compound of Formula 1, or a
stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a derivative thereof,
together with a pharmaceutically acceptable carrier, wherein the said pharmaceutical
composition is adapted for use in the treatment of tuberculosis.
The present invention also relates to a method for the manufacture of a medicament
containing the compound of Formula 1, or a stereoisomer, a tautomer, a pharmaceutically
acceptable salt, or a derivative thereof, for use in the treatment of tuberculosis.
The compound of Formula 1 or a stereoisomer, a tautomer, a pharmaceutically
acceptable salt, or a derivative thereof, may be administered orally, nasally, topically,
subcutaneously, intramuscularly, intravenously, or by other modes of administration.
Pharmaceutical compositions which contain the compound of Formula 1, or a
stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, with other
pharmaceutically active substances may be prepared by mixing the compound of Formula 1
with one or more pharmacologically acceptable auxiliaries and/or excipients such as,
wetting agents, solubilisers such as surfactants, vehicles, tonicity agents, fillers, colorants,
masking flavors, lubricants, disintegrants, diluents, binders, plasticizers, emulsifiers,
ointment bases, emollients, thickening agents, polymers, lipids, oils, cosolvents,
complexation agents, or buffer substances, and converting the mixture into a suitable
pharmaceutical form such as, for example, tablets, coated tablets, capsules, granules,
powders, creams, ointments, gels, syrup, emulsions, suspensions, or solutions suitable for
parenteral administration.
Examples of auxiliaries and/or excipients that may be used in preparation of
pharmaceutical composition include: cremophor, poloxamer, benzalkonium chloride,
sodium lauryl sulphate, dextrose, glycerin, magnesium stearate, polyethylene glycol,
starch, dextrin, lactose, cellulose, carboxymethylcellulose sodium, talc, agar-agar, mineral
oil, animal oil, vegtetable oil, organic and mineral waxes, paraffin, gels, propylene glycol,
benzyl alcohol, dimethylacetamide, ethanol, poly glycols, Tween 80, solutol HS 15, and
water. It may also be possible to administer the active substance as such, without vehicles
or diluents, in a suitable form, for example, in capsules.
Formulations for parenteral administration can be in the form of aqueous or non
aqueous isotonic sterile injection solutions, suspensions or fat emulsions. The parenteral
form used for injection must be fluid to the extent that easy syringability exists. These
solutions or suspensions can be prepared from sterile concentrated liquids, powders or
granules.
Excipients used in parenteral preparations also include, without limitation,
stabilizing agents (e.g. carbohydrates, amino acids and polysorbates, such as 5 % dextrose),
solubilizing agents (e.g. cetrimide, sodium docusate, glyceryl monooleate,
polyvinylpyrolidone (PVP) and polyethylene glycol (PEG)), surfactants (e.g. polysorbates,
tocopherol PEG succinate, poloxamer and CremophorTM), buffers (e.g. acetates, citrates,
phosphates, tartrates, lactates, succinates, amino acids and the like), antioxidants and
preservatives (e.g. BHA, BHT, gentisic acids, vitamin E, ascorbic acid, sodium ascorbate
and sulfur containing agents such as sulfites, bisulfites, metabisulfites, thioglycerols,
thioglycolates and the like), tonicity agents (for adjusting physiological compatibility),
suspending or viscosity agents, antibacterials (e.g. thimersol, benzethonium chloride,
benzalkonium chloride, phenol, cresol and chlorobutanol), chelating agents, and
administration aids (e.g. local anesthetics, anti-inflammatory agents, anti-clotting agents,
vaso-constrictors for prolongation and agents that increase tissue permeability), and
combinations thereof. Parenteral formulations using hydrophobic carriers include, for
example, fat emulsions and formulations containing lipids, lipospheres, vesicles, particles
and liposomes. Fat emulsions include in addition to the above-mentioned excipients, a lipid
and an aqueous phase, and additives such as emulsifiers (e.g. phospholipids, poloxamers,
polysorbates, and polyoxyethylene castor oil), and osmotic agents (e.g. sodium chloride,
glycerol, sorbitol, xylitol and glucose). Liposomes include natural or derived phospholipids
and optionally stabilizing agents such as cholesterol.
In an embodiment, the parenteral unit dosage form of the compound of Formula 1
can be a ready-to-use solution of the compound of Formula 1 in a suitable carrier in sterile,
hermetically sealed ampoules or in sterile pre-loaded syringes. The suitable carrier
optionally comprises any of the above-mentioned excipients.
Alternatively, the unit dosage of the compound of Formula 1 of the present
invention can be in a concentrated liquid, powder or granular form for ex tempore
reconstitution in the appropriate pharmaceutically acceptable carrier, such as sterile water,
at the time of delivery. In addition to the above-mentioned excipients, powder forms
optionally include bulking agents (e.g. mannitol, glycine, lactose, sucrose, trehalose,
dextran, hydroxyethyl starch, ficoll and gelatin), and cryo or lyoprotectants.
In intravenous (IV) use, a sterile formulation of the pharmaceutical compositions of
the present invention and optionally one or more additives, including solubilizers or
surfactants, can be dissolved or suspended in any of the commonly used intravenous fluids
and administered by infusion. Intravenous fluids include, without limitation, physiological
saline, phosphate buffered saline, 5 % dextrose in water or Ringer's TM solution.
In intramuscular preparations, a sterile formulation of the pharmaceutical
compositions of the present invention can be dissolved and administered in a
pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5 %
dextrose in water. A suitable insoluble form of the pharmaceutical compositions may be
prepared and administered as a suspension in an aqueous base or a pharmaceutically
acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
The method of administration which is suitable in a specific case depends on the
type of Mycobacterium tuberculosis organism to be treated and on the stage of the
respective infection or disease caused by the Mycobacterium tuberculosis organism.
Further, the method of administration can be optimized by a medical practitioner using
methods known in the art.
The dose of the compound of Formula 1 may vary depending upon the physical
characteristics of the subject, the severity of the subject's symptoms, the formulation and
the means used to administer the active ingredient i.e. the compound, and the method being
practiced. The specific dose for a given subject is usually set by the judgment of the
attending physician or medical practitioner. However, according to the present invention
the dose of the compound of Formula 1 typically ranges from about 0.5 mg/kg body weight
to about 200 mg/kg body weight or the dose of the compound of Formula 1 may range
from about 1 to about 100 mg/kg or the dose of the compound of Formula 1 may range
from about 5 to about 50 mg/kg, regardless of the formulation. In some situations, a dose of
the compound of Formula 1 greater than 200 mg/kg body weight may be required for the
treatment of tuberculosis.
Suitable frequencies of administration may vary based on whether administration is
for the purposes of treatment or prophylaxis of tuberculosis (the Mycobacterium
tuberculosis infection). Administration frequencies of doses for the treatment of a subject
having a Mycobacterium tuberculosis infection, prophylaxis or prevention of
Mycobacterium tuberculosis infection include 4, 3, 2 or once daily, every other day, every
third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days,
every nine days, every ten days, bi-weekly, monthly or bi-monthly. In other embodiments,
the course of treatment may require the administration of many doses over many days, such
as administration of 4, 3, or 2 doses or a single dose daily over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15 or more days.
Depending on the means of administration, the dosage may be administered as an
oral formulation for example, as a capsule, or slowly over a period of time, such as with an
intravenous administration. For slower means of administration, the administering period
can be a matter of minutes, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more minutes, or a period of hours, such
as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more hours.
A pharmaceutical composition, containing the compound of Formula 1, can also be
administered to a subject, in particular a human, with another therapeutically active agent
or a pharmaceutical composition containing the therapeutically active agent. The
therapeutically active agent that may be used in conjunction with the compound of Formula
1 may be an anti tuberculosis agent such as isoniazid and rifampicin, known to be useful in
treating infections caused by Mycobacterium tuberculosis.
The following are provided as illustrative examples of the present invention and do
not limit the scope thereof.
The following terms/abbreviations/chemical formulae are employed in the
examples:
Example 1
Isolation of culture no. PM0834793 from soil sample collected from Uttarkashi
(a) Composition of the isolation medium:
Starch Casein Digest Medium, (SCDM) [contains % (w/v): Soluble starch 1 g,
casein 0.03 g, potassium nitrate 0.2 g, sodium chloride 0.2 g, dipotassium hydrogen
phosphate 0.2 g, magnesium sulfate heptahydrate 0.005 g, calcium carbonate 0.002 g,
ferrous sulphate heptahydrate 0.001 g, agar 1.6 g in 100 mL demineralised water, pH
adjusted to 8.0 before autoclaving]. The pH of the medium was adjusted to 8.0 to support
growth of actinomycetes and baciUary forms. 50 g/mL of amphotericin B and g/ of
nalidixic acid were incorporated into the isolation media to avoid fungal and gram negative
bacterial growth respectively.
(b) Procedure:
The soil sample was collected from a land near hot-springs in Uttarkashi District of
Uttaranchal State, India, in a sterile container and was stored at 2 °C - 4 °C throughout the
journey back to Piramal Life Sciences Limited, Goregaon, Mumbai, India. The suspension
of soil ( 1 g) in saline (9 mL) was diluted to 10 . 100 of this dilution was spread on the
isolation media. The isolation plates were incubated at 30 °C for 30 days and were
observed periodically macro and microscopically. Colonies were picked up with sterile
needle by viewing the colony morphology. PM0834793 was one such eubacteria colony
isolated by this procedure.
Example 2
Purification and preservation of producer strain culture No. PM0834793
(a) The isolate was purified and maintained on slants of SCDM containing 1.6 % agar
agar. The SCDM was procured from Hi Media, Mumbai, Maharashtra, India, [Catalogue
no.MOll-500G]
(b) The culture was preserved in triplicates in glycerol vials with preservation media
composed of 20 % glycerol, 0.3 % tryptone and yeast extract and stored at -80 °C.
Example 3
Fermentation of the culture no. PM0834793 in shake flasks
(a) Composition of seed medium [SCDM] :
As culture PM0834793 belongs to eubacteria class, SCDM was used as seed
medium.
(b) The above medium was distributed in 40 mL amounts in 500 mL capacity
Erlenmeyer flasks. Each flask was inoculated with a loopful of the well-grown slant culture
(culture no. PM0834793). The flasks were incubated at 30 °C for 48 h on shaker at 250
rpm to obtain the seed culture.
(c) Composition of the production medium [SM12 (1)]:
SM12 (1) [contains % (w/v): Glucose 5 g, yeast extract 1.1 g, sodium chloride 0.25
g, calcium carbonate 0.5 g, peptone 0.4 g, beef extract 0.4 g; volume made using water and
pH adjusted to 7.2 using 1.0 M NaOH].
2 % (v/v) of the seed culture was aseptically transferred to 200 mL of the
production media in 1000 mL capacity Erlenmeyer flasks.
(d) Fermentation parameters:
The flasks were incubated on shaker at 30 °C and 250 rpm for 48 h. The bioactivity was
tested for anti-infective activity in agar diffusion assay in the test models S. aureus 209P,
E.faecium VRE 323, C. albicans, and E. coli 20732.
Example 4
Isolation and purification of the compound of Formula 1
Step 1
The culture broth (60 L) was stirred with methanol (1:1) for 1 h and was filtered.
The filtrate was concentrated to remove methanol. The aqueous portion was subjected to
Diaion HP-20 resin (Mitsubishi Chemical Industries Limited, Japan) chromatography. The
column was sequentially eluted with water (12 L) and mixture of water and methanol (2:8,
12 L). The water-methanol elutate was concentrated and lyophilized to obtain crude
extract. Yield: 5 1 g.
Step 2
The crude extract (as obtained in step 1) was further suspended in water (240 mL)
and extracted with n-butyl alcohol (240 mL). Organic layer was separated and solvent was
evaporated to obtain n-butyl alcohol fraction (38 g) which was processed by column
chromatography (silica gel, 400 g, 100-200 mesh, solvent: methanol in chloroform). The
fraction containing compound of Formula 1 was eluted with 20 % methanol in chloroform,
which was concentrated to obtain the semi pure compound. Yield: 11 g.
Step 3
The semi pure compound (as obtained in step 2) was further purified by using
Sephadex LH-20 resin chromatography using methanol as eluating solvent Active eluates
were pooled and concentrated to obtain the semi pure compound. Yield: 1.7 g.
Step 4
The semi pure compound (as obtained in step 3) was further purified using reverse
phase silica gel (RP-18) cartridge ( 1 g, Phenomenex, 8B - S100 - JDG Starta-X - 33 
polymeric sorbent), eluted with acetonitrile and water gradient ( 1 to 100 % acetonitrile in
water) and the fractions were monitored by bioactivity. The active fractions were pooled,
solvent was evaporated to obtain semi pure compound of Formula 1. Yield: 390 mg.
Step 5
Further purification was carried out by reverse phase preparative HPLC.
Preparative HPLC conditions:
Column : RP-18 column (Waters Xbridge 250 X 10 mm, 5 micron);.
Eluent : Acetonitrile: 0.05 % aqueous trifluoroacetic acid (30:70);
Flow rate : 6 niL/min;
Detection (UV): 220 nm;
Retention time: 9 to10 min;
Active fractions were pooled and concentrated to obtain pure compound of
Formula 1. Yield: 30 mg.
Compound obtained in step 5, is referred as compound of Formula 1.
Physical & spectral properties of the compound of Formula 1:
Appearance : White powder
HPLC :Column: Lichrosphere RP-18, 125 x 4 mm, 5 micron;
Solvent system: Acetonitrile : 0.1 % aqueous trifluoroacetic acid;
(2 % to 100 % in 15 min (gradient mode))
Flow rate: 1 niL/min; Detection (UV): 220 nm;
Retention time: 8.54 min.
LC-MS : (m/z: 896.7 (M+H) +
Molecular weight: 896
Molecular formula: C42 H7 N10 O
H NMR(DMSO-d , 500 MHz): 8.36 (d), 7.91 (d), 7.52, 7.39 (d), 7.30 (d), 7.25
(s), 6.87 (s), 5.53 (d), 5.34 (m), 5.01 (brs), 4.89, 4.46 (m), 4.42 (d), 4.12 (dd), 4.09
(dd), 4.01 (dd), 3.94 (m), 3.91 (m), 3.80 (brs), 3.08 (dd), 2.38 (m), 2.14 (m), 2.0
(m), 1.99 (m), 1.84 (m), 1.44 (t), 1.38 (brs), 1.24 (br), 1.19 (d), 1.17 (d), 1.10 (d),
0.88 (d), 0.86 (d), 0.81 (d), and 0.75 (d).
The compound of Formula 1 was characterised to be Fusaricidin B by comparing
physical and spectral data with that reported in the literature (Journal of Antibiotics, 1997,
50(3), 220 - 228).
Example 5
Biological evaluation of the compound of Formula 1
In-vitro assay
The activity against Mycobacterium tuberculosis organism can be determined by
using BACTEC MGIT 960 System from Becton Dickinson (BD), USA. The BACTEC 960
instrument is an automated system that exploits the fluorescence of an oxygen sensor to
detect growth of mycobacteria in culture as described in a reference, Journal of Clinical
Microbiology, 2006, 44 (3), 811-818. It is specially designed to accommodate
Mycobacteria Growth Indicator Tube (MGIT) that contains a fluorescent compound
embedded in silicone on the bottom of a tube. The fluorescent compound is sensitive to the
presence of oxygen dissolved in the broth. The initial concentration of the dissolved
oxygen quenches the emission of fluorescence from the compound, and little fluorescence
can be detected. Later, actively growing and respiring microorganisms consume oxygen,
which allows compound to fluoresce.
Two strains, Mycobacterium tuberculosis complex (Non-MDR strain) and
Mycobacterium tuberculosis complex (MDR strain) are used in the study. These clinical
strains are maintained at Super Raligare Laboratories Ltd, India. The terms resistance and
susceptible are designated based on the interpretation of BACTEC-960.
If the test compound added to the medium is bacteriostatic or bactericidal to the test
mycobacteria, it would inhibit growth of the mycobacteria and therefore, there would be
little or no oxygen consumption, and ultimately there would be little or no fluorescence of
the sensor. Whereas the growth control will grow uninhibited and will have increasing
fluorescence. Growth is monitored by the BACTEC 960 instrument which automatically
interprets results as resistant (R) or susceptible (S).
To determine the 1 % proportion of resistance, the bacterial inoculum used in the
control vial is 100 fold less than that used for the drug-containing vial.
Method: Two MGIT tubes were inoculated with the test culture. A known
concentration of a test compound was added to one of the MGIT tubes, and growth was
compared with the MGIT tube without the test compound (growth control).
Results were interpreted as per guidelines of MGIT and were given in Table 2 and
Table 3.
Table 2
Activity of the compound of Formula 1 against Mycobacterium tuberculosis
S: Sensitive, R : Resistant
Table 3
Activity of Rifampicin against Mycobacterium tuberculosis
S : Sensitive, R : Resistant
Conclusion:
The minimum concentration of the compound of Formula 1 required to exhibit its
effect against Non-MDR-TB strain was between 0.05 and 0.1 g/mL and against MDR-TB
strain was between 1 and 2 / . Thus, by this method MICs against drug-susceptible
and MDR-TB isolates were, respectively, 0.05-0.1 g/mL and 1.0-2.0 g/mL for
compound of Formula 1.
It should be noted that, as used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless the content clearly
dictates otherwise. It should also be noted that the term "or" is generally employed in its
sense including "and/or" unless the content clearly dictates otherwise.
The invention has been described with reference to various specific and preferred
embodiments and techniques. However, it should be understood that many variations and
modifications may be made while remaining within the spirit and scope of the invention.
We Claim:
1. A method of treating tuberculosis in a subject comprising administering to the
subject a therapeutically effective amount of the compound of Formula 1,
L - Val ( 2 ) D - Val ( 1 )
or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof.
2. The method according to claim 1, wherein the tuberculosis is caused by
Mycobacterium tuberculosis organism.
3. The method according to claim 2, wherein Mycobacterium tuberculosis organism is
sensitive Mycobacterium tuberculosis organism.
4. The method according to claim 2, wherein Mycobacterium tuberculosis organism is
multi drug resistant Mycobacterium tuberculosis.
5. A method of inhibiting growth of Mycobacterium tuberculosis organism
comprising contacting the Mycobacterium tuberculosis organism in vitro or ex vivo with
the compound of Formula 1 as defined in claim 1, or a stereoisomer, a tautomer, or a
pharmaceutically acceptable salt thereof in an amount sufficient to inhibit growth of the
organism.
6. The method according to claim 5, wherein the Mycobacterium tuberculosis
organism is contacted with the compound of Formula 1 in vitro.
7. The method according to claim 5, wherein the Mycobacterium tuberculosis
organism is contacted with the compound of Formula 1 ex vivo.
8. A method of treating tuberculosis in a subject comprising administering to the
subject with a pharmaceutical composition comprising the compound of Formula 1 as
defined in claim 1, or a stereoisomer, or a tautomer, or a pharmaceutically acceptable salt
thereof.
9. The method of treating tuberculosis according to claim 1, wherein the compound of
Formula 1 is isolated from a microorganism belonging to eubacteria (culture no.
PM0834793).
10. A process for the production of the compound of Formula 1 as defined in claim 1,
from the culture no. PM0834793, wherein the process comprises the steps of:
(a) growing the culture no. PM0834793;
(b) isolating the compound of Formula 1 from the culture broth; and
(c) purifying the compound of Formula 1.
11. Use of compound of Formula 1 as defined in claim 1, or a stereoisomer, or a
tautomer, or a pharmaceutically acceptable salt, or a derivative thereof, for the treatment of
tuberculosis.
12. The use according to claim 11, wherein the tuberculosis is caused by
Mycobacterium tuberculosis organism.
13. The use according to claim 12, wherein Mycobacterium tuberculosis organism is
sensitive Mycobacterium tuberculosis organism.
14. The use according to claim 12, wherein Mycobacterium tuberculosis organism is
multi drug resistant Mycobacterium tuberculosis.
15 Use of compound of Formula 1 as defined in claim 1, or a stereoisomer, a tautomer,
a pharmaceutically acceptable salt, or a derivative thereof, for the production of
medicaments for the treatment of tuberculosis.