MICROPARTICLE FORMULATION FOR PULMONARY DRUG DELIVERY OF ANTI-INFECTIVE MOLECULE FOR TREATMENT OF INFECTIOUS DISEASES
FIELD OF THE INVENTION
The present invention relates to a biodegradable, inhalable microparticle formulation
comprising a compound obtained by fermentation of a microorganism of the
Streptomyces species (PM0626271/MTCC5447), as described in PCT application
publication WO 201 1027290, hereinafter referred to as compound of formula I, and a
biodegradable lipid for drug delivery wherein the ratio of drug (compound of formula
I) to lipid is 1: 1 5 to 1:25. The present invention also relates to a method of treatment
of pulmonary tuberculosis, multi drug resistant tuberculosis (MDRTB), methicillin
resistant Staphylococcus aureus (MRSA) pneumonias and methicillin sensitive
Staphylococcus aureus (MSSA) pneumonias by administering therapeutically
effective amount of the formulation to a mammal in need thereof. The present
invention also relates to the use of the microparticle formulation for the treatment of
pulmonary tuberculosis, MDRTB, MRSA pneumonias and MSSA pneumonias.
BACKGROUND OF THE INVENTION
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 affecting the lungs is
known as pulmonary tuberculosis. Pulmonary tuberculosis is the most predominantly
occurring form of tuberculosis (Tuberculosis, 2005, 85, 227-234). The current
chemotherapeutic regimen for treating pulmonary tuberculosis consists of co
administration of front-line antitubercular drugs (isoniazid, rifampicin, ethambutol,
and/or pyrizinamide) for a period of four months followed by two months of treatment
with isoniazid, rifampicin, and/or ethambutol, but depending upon the type of
tuberculosis, the treatment can be further extended upto a period ranging from 9
months to 2 years. This current chemotherapeutic regimen is given in the form of
once a day oral dosing, which is associated with poor plasma half-life (International
Journal of Pharmaceutics, 2004, 276, 4 1-49) and a plethora of dose related adverse
effects (Journal of Antimicrobial Chemotherapy, 2004 54, 761 -766). These adverse
effects are attributed to an undesirable biodistribution profile. Moreover, in respect of
the orally administered drugs it has been observed that only a small fraction of the
drug reaches the site of action i.e. the lungs and is cleared within hours
(Tuberculosis, 2005, 85, 227-234). The above problems are associated with poor
patient compliance and result in the development of multidrug resistant tuberculosis
(MDRTB).
MDRTB is a form of tuberculosis that is resistant to at least two of the best
antitubercular drugs, isoniazid and rifampicin (Multidrug resistant tuberculosis fact
sheet, Center for Disease Control, 2008). MDRTB is treated with second line
antitubercular drugs like fluoroquinolones, aminoglycosides like amikacin,
kanamycin, capreomycin, para-aminosalicyclic acid and thioacetazone (Treatment of
drug resistant tuberculosis, fact sheet, Center for Disease Control, 2007). The
second line tuberculosis drugs are associated with dose related side effects, poor
bioavailability in the lungs, which is detrimental for disease eradication.
Analogous to the problem of pulmonary tuberculosis, the problems of poor
bioavailability of drugs and higher dose induced adverse effects are also
encountered in the treatment of MRSA and MSSA pneumonias. Nosocomial
pneumonias and ventilator-associated pneumonias resulting from MRSA are
associated with high mortality rates (International Journal of Antimicrobial Agents,
2007, 30, 19-24), the reason for the aforesaid being inadequate treatment.
First line drugs that are used to treat MRSA pneumonia include vancomycin and
linezolid. Vancomycin, the drug of choice for treating MRSA pneumonia, is
associated with unsatisfactory pharmacokinetic profile in the lung tissue and has
lung concentrations, which are just 20 % of the plasma concentrations (Antimicrob.
Agents Chemother., 1999, 37, 281 -286). Moreover, long-term administration of
vancomycin is associated with nephrotoxicity, which is a dose-limiting factor (Clin.
Microbiol Infect., 2006, 12, 92-95). Linezolid, which is accepted for therapy in MRSA
pneumonia, exhibits good oral bioavailability (administered as 600 mg oral twice
daily) but is associated with gastrointestinal adverse effects, thrombocytopenia, and
reversible anemia (Clinical Infect. Dis., 2003, 37, 1609-1 6 16). On rare occasions,
administration of linezolid is also associated with optic and peripheral neuropathy (J
Antimivrob. Chemother., 2004, 53, 1114-1 115).
Beta lactam agents (such as ampicillin and cepholosporins) are very effective
against MSSA pneumonia as first line of therapy. Though vancomycin is considered
as next line of therapy, it is not as effective as the beta lactam agents in infections
caused by MSSA. Also vancomycin is excreted in the urine by glomerular filtration
and is not metabolized. Lung tissue penetration of vancomycin is also relatively poor
(US Respiratory Disease, 2006, 62-64). In summary, the therapy for MRSA/MSSA
pneumonia has several drawbacks such as poor pulmonary bioavailability of drugs,
drug dosage induced toxicity, etc.
To overcome the problems associated with the current standard treatment regimen
and patient non-compliance, it is essential to develop a drug delivery system that
directly reaches the site of action, has the potential to target the lung macrophages
where mycobacteria reside and reduce drug associated systemic toxicity.
SUMMARY OF THE INVENTION
The present invention relates to a biodegradable, inhalable microparticle formulation
comprising compound of formula I (as described herein) obtained by fermentation of
a microorganism of the Streptomyces species (PM0626271/MTCC5447), and a
biodegradable lipid for drug delivery wherein the ratio of drug (compound of formula
I) to lipid is from 1: 1 5 to 1:25.
The present invention also relates to the process for preparation of the microparticle
formulation.
The present invention further relates to the method of treatment of pulmonary
tuberculosis, MDRTB, MRSA pneumonias and MSSA pneumonias by administering
a therapeutically effective amount of the microparticle formulation to a mammal in
need thereof.
The present invention also relates to a method of delivering the microparticle
formulation to a mammal in need thereof, wherein the formulation is administered by
inhalation or intratracheal instillation for pulmonary delivery.
The present invention further relates to the use of the microparticle formulation
comprising compound of formula I and a biodegradable lipid for drug delivery
wherein the ratio of drug (compound of formula I) to lipid is 1: 1 5 to 1:25 for the
treatment of pulmonary tuberculosis, MDRTB, MRSA pneumonias and MSSA
pneumonias.
The present invention further relates to the use of the microparticle formulation
comprising compound of formula I and a biodegradable lipid for drug delivery
wherein the ratio of drug (compound of formula I) to lipid is 1: 1 5 to 1:25 for the
manufacture of a medicament for the treatment of pulmonary tuberculosis, MDRTB,
MRSA pneumonias and MSSA pneumonias.
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention in detail, it has to be understood that this
invention is not limited to particular embodiments. It is also to be understood that the
terminology used herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
As used in the specification and claims, the singular forms "a", "an" and "the" include
plural references unless the context clearly indicates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of the ordinary skill in the art to
which the invention belongs.
Definitions
Entrapment efficiency: Entrapment efficiency is the fraction of drug associated and
physically entrapped in the microparticle formulation relative to the initial total
amount of drug in the solution.
Drug: A drug is defined as any substance intended for use in the diagnosis, cure,
relief, treatment or prevention of disease or intended to affect the structure or
function of the body. As used herein compound of formula I is a drug.
Mass balance: A mass balance (also called a material balance) is an application of
conservation of mass to the analysis of physical systems. By accounting for material
entering and leaving a system, mass flows can be identified which might have been
unknown, or difficult to measure without this technique.
Osmolality: Osmolality is a measure of solute concentration, defined as the number
of osmoles (mOsm) of solute per kilogram of solvent (mosmol/kg or mOsm/kg).
Phase Transition: A phase transition is the transformation of a thermodynamic
system from one phase or state of matter to another. A phase of a thermodynamic
system and the states of matter have essentially uniform physical properties. During
a phase transition of a given medium certain properties of the medium change, often
discontinuously, as a result of some external condition, such as temperature,
pressure, and others. The measurement of the external conditions at which the
transformation occurs, is termed as the phase transition point.
Aerosolization: Aerosolization is the production of an aerosol - a fine mist or spray
containing minute particles.
Nebulization: Nebulization involves the process of transforming liquid medications
into faster-acting inhaled mists. Nebulization is used to treat respiratory conditions,
such as asthma or cystic fibrosis. Nebulizers effectively deliver medicine directly into
an individual's respiratory tract so that it can reach the lungs quickly. A non-limiting
example of nebulizer is a machine equipped with a compressor and a mouthpiece or
face mask.
Particle Size: Particle size is a notion introduced for comparing dimensions of solid
particles, liquid particles (droplets). For droplets and aerosols, terms such as
"aerodynamic diameter" and "mass median aerodynamic diameter (MMAD) are
used. The definitions are given below.
Aerodynamic diameter: The diameter of a unit-density sphere having the same
terminal settling velocity as the particle in question. It is used to predict where in the
respiratory tract such particles will deposit.
Mass Median Aerodynamic Diameter: The geometric mean aerodynamic
diameter. Fifty per cent of the particles by weight will be smaller than the MMAD,
50 % will be larger.
During particle sizing experiment, the suspensions contain innumerable number of
particles of varying sizes in motion. When the particle-sizing machine analyzes these
particles, it forms a particle distribution curve, which covers the entire particle size
range starting from the smallest particle, which could be 1 nm to the largest, which
could be 100 microns. In the particle size distribution curve, a cumulative frequency
is calculated for the particles. D 0 refers to that particular particle diameter where
10 % of the particles in the suspension have a smaller diameter or equal diameter as
that of the particular particle diameter.
D50 : Similar to the D 0, D50 is the cut off diameter for 50 % of the particle population
in the formulation and refers to that particular particle diameter where 50 % of the
particles in the suspension have a smaller diameter or equal diameter as that of the
particular particle diameter.
D90 : D90 is the cut off diameter for 90 % of the particle population in the formulation
and refers to that particular particle diameter where 90 % of the particles in the
suspension have a smaller diameter or equal diameter as that of the particular
particle diameter.
Entrapped drug retention on nebulization: During nebulization process, due to the
force induced by the nebulizer some liposomes rupture and the drug leeches out of
the formulation and gets retained in the nebulization cup itself. The drug which does
not leech out of the formulation during the nebulization process is the actual amount
of drug retained in the formulation during nebulization and is designated as
"entrapped drug retention". The drug which is lost to/leeched out during the
nebulization process is recovered from the nebulization cup. The nebulization cup is
washed with a suitable solvent (in this case methanol) and drug retained in the cup is
quantified by HPLC or LC-MS.
Liquid Crystalline Phase: It is a distinct phase of matter observed between the
crystalline (solid) and isotropic (liquid) states.
Rippled Gel Phase: It is a metastable state between liquid crystalline phase and gel
phase.
Intratracheal Instillation: It is a method of drug administration wherein the drug is
administered through an endotracheal tube or by percutaneous injection into the
trachea for the delivery of drugs into the lungs.
Ventilatory support: In medicine, mechanical ventilation is a method to
mechanically assist or replace spontaneous breathing. This is achieved by attaching
an endotracheal tube of the disease afflicted patient to the ventilator which is
designed for the aforesaid purpose. Ventilators work by altering the patient's airway
pressure through an endotracheal or tracheostomy tube. Patients with fulminant
pneumonia including MRSA pneumonia are subjected to tracheostomy so that their
tissue oxygenation is maintained.
Biodegradable lipid: Biodegradable lipid refers to a lipid which is amenable to
chemical degradation in vivo (in the human body) either by enzymatic action or by
innate occurring biological processes in which the lipid molecule is broken down to
its basic constitutive components.
Therapeutically effective amount: Therapeutically effective amount refers to the
amount of drug enough to treat and eliminate the infectious organism of interest in
the in vivo conditions. The therapeutic amount of compound of formula I present in
the microparticle formulation is in the range of 1 % to 5 % (w/w).
Non-invasive method of treatment: Non-invasive method of treatment refers to
methods like nebulization or intratracheal instillation in pre-tracheotomy individuals.
The procedure is painless and doesn't require additional medical interventions which
include administration of anesthetic agents to relive pain or its associated
components.
The present invention relates to a microparticle formulation comprising compound of
formula I, and a biodegradable lipid for drug delivery wherein the ratio of drug
(compound of formula I) to lipid is from 1:15 to 1:25 and the microparticle formulation
is a biodegradable and inhalable formulation.
According to one aspect of the invention, compound of formula I constitutes 1 % to
5 % (w/w) of the formulation.
The compound of formula I is structurally represented by the following formula:
Formula I
The microorganism, which may be used for the production of the compound of
formula I is a strain of Streptomyces species (PM0626271/ MTCC 5447), herein after
referred to as culture no. PM0626271 , isolated from a soil sample collected from
Schirmacher Oasis in Antarctic region. Culture no. PM0626271 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) and has accession
number MTCC 5447.
The compound can be produced from culture no. PM0626271 , its mutants and
variants, comprising the steps of: growing the culture no. PM0626271 under
submerged aerobic conditions in a nutrient medium containing one or more sources
of carbon and one or more sources of nitrogen and optionally nutrient inorganic salts
and/or trace elements; isolating the compound of formula I, from the culture broth;
and purifying the compound of formula I, using purification procedures generally
used in the art.
In addition to the specific microorganism described herein, it should be understood
that mutants of the microoganism, 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.
The medium and/or nutrient medium used for isolation and cultivation of culture no.
PM0626271 , which produces the compound of formula I, 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. Preferred carbon sources are soluble starch and
glucose. The sources of nitrogen are, for example, one or more of soybean meal,
peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor,
gelatin, or casamino acids. Preferred nitrogen sources are peptone and yeast
extract. The nutrient inorganic salts are, for example, one or more of sodium
chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride,
strontium chloride, cobalt chloride, potassium bromide, sodium fluoride, sodium
hydrogen phosphate, potassium hydrogen phosphate, dipotassium hydrogen
phosphate, magnesium phosphate, calcium carbonate, sodium bicarbonate, sodium
silicate, ammonium nitrate, potassium nitrate, ferrous sulphate, sodium sulphate,
ammonium sulphate, magnesium sulphate, ferric citrate, boric acid or trace salt
solution such as copper sulphate, manganese chloride or zinc sulphate. Calcium
carbonate, sodium chloride, and magnesium chloride are the preferred nutrient
inorganic salts.
The maintenance of culture no. PM0626271 may be carried out at a temperature
ranging from 22 °C to 36 °C and a pH of about 7.5 to 8.0. Typically, culture no.
PM0626271 is maintained at 25 °C to 27 °C and a pH of about 7.4 to 7.8. The wellgrown
cultures may be preserved in the refrigerator at 4°C to 8°C.
Seed culture cultivation of culture no. PM0626271 may be carried out at a
temperature ranging from 25 °C to 36 °C and a pH of about 7.5 to 8.0 for 66 hours to
75 hours at 200 rpm (revolutions per minute) to 280 rpm. Typically, culture no.
PM0626271 seed is cultivated at 29 °C to 3 1 °C and a pH of about 7.4 to 7.8, for 72
hours at 230 rpm to 250 rpm.
The production of the compound of formula I may be carried out by cultivating culture
no PM0626271 by fermentation at a temperature ranging from 26°C to 36 °C and a
pH of about 6.5 to 8.5, for 24 hours to 96 hours at 60 rpm to 140 rpm and 100 Ipm
(liter per minute) to 200 Ipm aeration. Typically, culture no. PM0626271 is cultivated
at 30 °C to 32 °C and pH 7.4 to 7.8 for 40 hours to 96 hours at 90 rpm and 110 Ipm
aeration.
The progress of fermentation and production of the compound can be detected by
high performance liquid chromatography (HPLC) and by measuring the bioactivity of
the culture broth against Staphylococci and/or Enterococci species by the known
microbial agar plate diffusion assay method. The preferred culture is Staphylococcus
aureus E71 0, which is a strain resistant to methicillin, a b-lactam antibiotic reported
in the literature, and Enterococcus faecium R2 (VRE) which is resistant to
vancomycin. In the resulting culture broth, the compound may be present in the
culture filtrate as well as in cell mass and can be isolated using known separation
techniques such as solvent extraction and column chromatography. The compound
of formula I can be recovered from the culture filtrate by extraction at a pH of about 5
to 9 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
U.S.A.), activated charcoal, or by ion exchange chromatography at pH 5 to 9. The
active material can be recovered from the cell mass by extraction with a water
miscible solvent such as methanol, acetone, acetonitrile, n-propanol, or iso-propanol
or with a water immiscible solvent such as petroleum ether, dichloromethane,
chloroform, ethyl acetate or butanol. One other option is to extract 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 extracted with ethyl acetate from the whole broth.
Concentration and lyophilization of the extracts gives the active crude material.
The compound of formula I can be recovered from the crude material by fractionation
using any of the following techniques: normal phase chromatography (using alumina
or silica gel as stationary phase; and eluents such as petroleum ether, ethyl acetate,
dichloromethane, acetone, chloroform, methanol, or combinations thereof); reverse
phase chromatography (using reverse phase silica gel such as dimethyloctadecylsilyl
silica gel, (RP-1 8) or dimethyloctylsilyl silica gel (RP-8) as stationary phase; and
eluents such as water, buffers [for example, phosphate, acetate, citrate (pH 2 to 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 Sephadex ® G-1 0 and G-
25 in water); or by counter-current chromatography (using a biphasic eluent system
made up of two or more solvents such as water, methanol, ethanol, iso-propanol, npropanol,
tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform,
ethyl acetate, petroleum ether, benzene, and toluene). These techniques may be
used repeatedly, alone or in combination. A typical method is chromatography over
normal phase using silica gel.
The compound of formula I and isomers thereof, can be converted into their
pharmaceutically acceptable salts and derivatives, like esters and ethers, which are
all contemplated by the present invention.
The biodegradable lipid used in the formulation is dipalmitoylphosphatidylcholine
(DPPC), which is a naturally occurring phospholipid of the endogenous lung
surfactant system. Other non-limiting examples of biodegradable lipids that can be
used in combination with DPPC include DPPG (Dipalmitoyl phosphatidyl glycerol),
DPPE (dipalmitoylphoshatidylethanolamine), cholesterol, phosphatidyl inositol, and
phosphotidyl serine.
In another aspect of the invention, the size of the microparticles of the formulation
ranges between 0.5 microns and 10 microns.
In yet another aspect of the invention, 90 % of the microparticles of the formulation
are of size less than 10 microns.
In yet another aspect of the invention, the formulation is an aqueous liposomal
dispersion.
In an aspect of the invention, the pH of the formulation is from 6 to 7.
In another aspect, the osmolality of the formulation is from 300 mOsmol/kg to 400
mOsmol/kg. In yet another aspect of the invention, the phase transition temperature
of the formulation is from 4 1 C to 43 C. In another aspect of the invention, the
formulation can be aerosolized to a mass median aerodynamic diameter of 1 mi to
10 mi by using a nebulizer.
The types of nebulizers which can be used include but are not limited to Jet
nebulizers, Ultrasonic wave nebulizers and Vibrating Mesh nebulizers.
The present invention also relates to the process for preparation of the microparticle
formulation.
In an aspect of the invention, the process for preparation of the formulation involves
use of "Solvent evaporation method" which includes the following steps:
(a) dissolving compound of formula I and DPPC ( 1 : 1 5 to 1:25 ratio)
in 3 ml_ to 15 ml_ chloroform to obtain a solution;
(b) adding 20 ml_ to 45 ml_ of methanol to the solution of step (a)
and mixing well to obtain homogeneous solution;
(c) adding 20 ml_ to 50 ml_ of simulated lung fluid (SLF) to the
solution of step (b);
(d) evaporating the solvents;
(e) making up the volume obtained in step (d) to 30 ml_ with SLF
and centrifuging at 15000 G TO 35000 G, at 4 C for ten minutes
to obtain a pellet;
(f) resuspending the pellet obtained in step (e) in SLF to obtain a
suspension of concentration 0.5 img/mL to 10 img/mL;
(g) filtering the suspension obtained in step (f) through a 0.5 mi - 5
mi polycarbonate filter to obtain uniform particle size of the
microparticles formed.
The "Solvent evaporation method" used herein is a modification of the method
reported in US Patent 4,877,561 .
In an embodiment of the invention, in the step (a) of the process for preparation of
the microparticle formulation, the compound of formula I and DPPC are dissolved in
a 1:20 ratio.
In another embodiment of the invention, in the step (a) of the process for preparation
of the microparticle formulation, the compound of formula I and DPPC are dissolved
in 5 to 10 mL of chloroform to obtain a solution.
In an embodiment of the invention, in the step (b) of the process for preparation of
the microparticle formulation, 30 to 40 ml_ of methanol is added.
In another embodiment of the invention, in the step (c) of the process for preparation
of the microparticle formulation, 25 to 35 ml_ of SLF is added. In another
embodiment of the invention, in the step (e) of the process for preparation of the
microparticle formulation, centrifugation is performed at 20,000 to 30,000 G.
In another embodiment of the invention, in the step (f) of the process for preparation
of the microparticle formulation, the pellet is resuspended in SLF to obtain a
suspension of concentration 1 to 5 img/mL.
In another embodiment of the invention, in the step (g) of the process for preparation
of the microparticle formulation, the suspension obtained in step (f) is filtered through
2 mi to 5 mi polycarbonate filter.
In another aspect of the invention, the process for the preparation of the formulation
is a "Solvent free lipid self assembly method" which includes the following steps:
(i) adding 20 mL to 45 mL of SLF to a mixture of compound of
formula I and DPPC ( 1 : 1 5 to 1:25 ratio);
(ii) subjecting the mixture of step (i) to 100 rpm to 200 rpm rotation
at 42 C to 45 C for one hour to obtain a suspension;
(iii) centrifuging the suspension obtained in step (ii) at 15000 G -
35000 G at 4 C for ten minutes to obtain a pellet;
(iv) resuspending the pellet obtained in step (iii) in SLF to obtain a
suspension of concentration 0.5 img/mL to 10 img/mL; and
(v) filtering the suspension obtained in step (iv) through 0.5 mi - 5
mi polycarbonate filter to obtain uniform particle size of the
microparticles formed.
In an embodiment of the invention, in the step (i) of the process for preparation of the
microparticle formulation, 30 mL to 40 mL of SLF is added to a mixture of compound
of formula I and DPPC.
In another embodiment of the invention, in the step (i) of the process for preparation
of the microparticle formulation, the compound of formula I and DPPC are dissolved
in 1:20 ratio.
In an embodiment of the invention, in the step (iii) of the process for preparation of
the microparticle formulation, the suspension obtained in step (ii) is centrifuged at
20,000 G to 35,000 G at 4 C for ten minutes to obtain a pellet.
In another embodiment of the invention, in the step (iv) of the process for preparation
of the microparticle formulation, the pellet obtained in step (iii) is resuspended in SLF
to obtain a suspension of concentration 1 mg/mL to 5 img/mL
In another embodiment of the invention, in the step (v) of the process for preparation
of the microparticle formulation, the suspension obtained in step (iv) is filtered
through 2 mi to 5 mi polycarbonate filter.
The present invention further relates to the use of the formulation in a method of
treatment of pulmonary tuberculosis, MDRTB, MRSA pneumonias and MSSA
pneumonias by administering therapeutically effective amount of the formulation to a
mammal in need thereof.
The present invention further relates to the use of the microparticle formulation
comprising compound of formula I and a biodegradable lipid for drug delivery
wherein the ratio of drug to lipid (compound of formula I) is 1: 1 5 to 1:25 for the
manufacture of a medicament for the treatment of pulmonary tuberculosis, MDRTB,
MRSA pneumonias and MSSA pneumonias.
In an aspect of the invention, the method of treatment targets alveolar macrophages,
which can harbour the mycobacteria and methicillin resistant as well as methicillin
sensitive Staphylococcus aureus.
The present invention also relates to a method of delivering the microparticle
formulation to a mammal in need thereof, wherein the formulation is administered by
inhalation or intratracheal instillation for pulmonary delivery.
In an aspect of the invention, the method of delivering the microparticle formulation is
by inhalation.
In another aspect of the invention, the method of inhalation is nebulization in which
the compound of formula I is entrapped in the microparticles.
In respect of the microparticle formulation of the present invention it has been
observed that when administered by inhalation significant concentration of the
compound of formula I contained in the formulation is detected in the lungs of mice.
However, no significant concentration of compound of formula I is detected in the
lungs of mice that received the unformulated compound of formula I. This is
indicative of increased bioavailability of the compound of formula I in the
microparticle formulation. Further, it has been observed that the drug (compound of
formula I) is retained in the lungs over a period of 24 hours when the microparticle
formulation of the present invention is administered by inhalation.
In another aspect of the invention, the retention of the entrapped compound of
formula I is greater than 30 %. Particularly, retention of the entrapped compound of
formula I range from 30 % to 70 %.
The dosage of compound of formula I for inhalation ranges between 0.05 and 10
mg/kg body weight/day.
In another aspect of the invention, the method of delivering the microparticle
formulation is intratracheal instillation in a patient on ventilatory support system.
In another aspect of the invention, administration by nebulization helps reduce the
amount of compound of formula I required for the treatment of pulmonary
tuberculosis, MDRTB, MRSA pneumonias and MSSA pneumonias.
In yet another aspect of the invention, the method helps compound of formula I
reach the lungs.
The efficacy of the microparticle formulation has been established by biological
assays which are described in detail in subsequent examples. These examples are
herein provided for the purpose of illustration only and are not intended to limit the
scope of the invention.
EXAMPLES
The following terms/abbreviations/chemical formulae are employed in the
Examples:
NaCI Sodium chloride
CaCI2 Calcium chloride
NaOH Sodium hydroxide
SLF Simulated lung fluid
HPLC High Performance Liquid Chromatography
DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DMSO Dimethyl Sulfoxide
RB flask Round bottomed flask
rpm Rotations per minute
DSC Differential Scanning Calorimeter
TSI Twin Stage Impinger
MSSA Methicillin Sensitive S.aureus
MRSA Methicillin Resistant S.aureus
VRE : Vancomycin Resistant Enterococci
TSA : Tryptose Soya Agar
CFU : Colony Forming Units,
HBSS : Hanks Buffered Salt Solution
MTS : (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)
-2-(4-sulfophenyl)-2H-tetrazolium)
GC-HS : Gas Chromatograph with Head Space Attachment
CPCSEA : Committee for the Purpose of Control and Supervision
of Experiments on Animals
IAEC : Institutional Animal Ethics Committee
API : Active Pharmaceutical Ingredient
Example 1
Isolation of culture no. PM0626271 from soil collected from Antarctic region
a) Composition of the isolation medium:
Modified artificial sea water agar: Peptone 1.5 g, yeast extract 0.5 g, ferric chloride
0.007 g, 1.0 L water (750 ml_ artificial sea water + 250 ml_ demineralised water),
agar powder 15.0 g, final pH (at 25 °C) 7.4 to 7.8.
Composition of the artificial seawater: Sodium chloride 24.6 g, potassium chloride
0.67 g, calcium chloride.2H2O 1.36 g, magnesium sulphate.7H2O 6.29 g, magnesium
chloride. 6H2O 4.66 g, sodium bicarbonate 0.1 8 g, demineralised water 1.0 L, final pH
(at 25 °C) 7.8 to 8.2.
b) Procedure:
From Schirmacher Oasis region in Antarctica area, surface level soil was collected
and was stored at -20 °C throughout the journey to Piramal Life Sciences Limited,
Goregaon, Mumbai, India. The sample was stored at -20 °C to -22 °C and later
thawed to room temperature (25±2°C) for isolation of the microbes. The soil sample
(~1 g) was suspended in 25 mL of sterile 1 % peptone water in a 100 mL sterilized
flask. The flask was vortexed for 30 seconds. Serial dilutions up to 10 5 were
prepared in sterile 1 % peptone water. 100 m of 10 5 dilution was surface spread on
modified artificial seawater agar. The plate was incubated at room temperature
(25±2°C) till colonies were observed. After incubation for one and a half month, the
colony which appeared on this medium was streaked on petri plates containing
actinomycete isolation agar [Hi Media] prepared in 75 % artificial sea water
[Accumix Tm] (AS-AIA). The isolate was purified and was provided culture ID number
PM0626271 . The culture no. PM0626271 was thus isolated from amongst the
growing microorganisms as single isolate.
Example 2
Purification of culture no. PM0626271
a) Composition of the purification medium (Actinomycete Isolation Agar, agarified by
1.5 % agar agar):
Glycerol 5.0 mL, sodium caseinate 2.0 g, L-asparagine 0.1 g, sodium propionate 4.0
g, dipotassium phosphate 0.5 g, magnesium sulphate 0.1 g, ferrous sulphate 0.001
g, 1.0 L water (750 mL Artificial Sea Water + 250 mL demineralised water), agar
powder 15.0 g, final pH (at 25 °C) 7.4 to 7.8.
Composition of the artificial seawater: Sodium chloride 24.6 g, potassium chloride
0.67 g, calcium chloride. 2H2O, 1.36 g, magnesium sulphate. 7H2O 6.29 g,
magnesium chloride. 6H2O 4.66 g, sodium bicarbonate 0.1 8 g, demineralized water
1.0 L, final pH (at 25 °C) 7.8 to 8.2.
b) Procedure:
The culture no. PM0626271 was streaked on Actinomycete Isolation Agar
(containing 75 % artificial sea water salts) petriplate. The petriplate was incubated for
10 days at 25 °C. One of the isolated colonies from the petriplate was transferred to
fresh slants of Actinomycete Isolation Agar prepared in 75 % artificial seawater. The
slants were incubated for 10 days at 25 °C.
Example 3
Maintenance of producer strain - culture no. PM0626271
a) Composition of the medium (Actinomycete Isolation Agar):
Glycerol 5.0 mL, sodium caseinate 2.0 g, L-asparagine 0.1 g, sodium propionate 4.0
g, dipotassium phosphate 0.5 g, magnesium sulphate 0.1 g, ferrous sulphate 0.001
g, 1.0 L water (750 mL artificial sea water + 250 mL demineralised water), agar
powder 15.0 g, final pH (at 25 °C) 7.4 to 7.8.
Composition of the artificial sea water: Sodium chloride 24.6 g, potassium chloride
0.67 g, calcium chloride. 2H2O 1.36 g, magnesium sulphate. 7H2O 6.29 g, magnesium
chloride. 6H2O 4.66 g, sodium bicarbonate 0.1 8 g, demineralized water 1.0 L, final pH
(at 25 °C) 7.8 to 8.2.
b) After dissolving the ingredients thoroughly by heating, the resultant solution was
distributed in test tubes and sterilized at 12 1 °C for 30 minutes. The test tubes were
cooled and allowed to solidify in a slanting position. The agar slants were streaked
with the growth of culture no. PM0626271 by a wire loop and incubated at 27 °C to
29 °C until a good growth was observed. The well-grown cultures were stored in the
refrigerator at 4°C to 8°C.
Example 4
Fermentation of the culture no. PM0626271 in shake flasks
a) Composition of seed medium [AS-274 ( 1 )]:
Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast extract 7.5 g, calcium
carbonate 2.0 g, sodium chloride 5.0 g, volume made with 750 ml_ artificial sea water
and 250 ml_ demineralised water.
b) The above medium was distributed in 40 ml_ amounts in 500 ml_ capacity
Erlenmeyer flasks and autoclaved at 12 1 °C for 30 minutes. The flasks were cooled
to room temperature (25 °C ± 2°C) and each flask was inoculated with a loopful of the
well-grown producing strain (culture no. PM0626271 ) on the slant and shaken on a
rotary shaker for 72 hours at 230 rpm to 250 rpm at 30 °C ±°C to give seed culture.
c) Composition of the production medium [AS 36P ( 1 )]:
Soluble Starch 20 g, glucose 15 g, yeast extract 2 g, peptone 3 g, calcium carbonate
2 g, ammonium sulfate 0.5 g, corn steep liquor 2 g, sodium chloride 2 g, magnesium
phosphate 5 g, cobalt chloride 1ml_/L from stock of 1 g/L, trace salt solution 1 mL/L,
volume made to 1 L using with 75 % artificial sea water and 25 % demineralized
water.
d) 40 ml_ of the production media in 500 ml_ capacity Erlenmeyer flasks was
autoclaved at 12 1 °C for 30 minutes, cooled to 29 °C to 30 °C and seeded with 5 %
(v/v) of the seed culture mentioned in Example 4b.
e) Fermentation parameters:
The production flasks were incubated on shaker at 29 °C and 220 rpm for 96 hours.
The production flasks were harvested and the whole broth from each media flask
was extracted with equal volume of methanol under shaking condition for one hour at
29 °C and centrifuged at 3500 rpm for half an hour. The supernatant was used for
antibacterial agar well diffusion assay for monitoring of the activity.
Example 5
Preparation of seed culture in shake flasks for fermentation
a) Composition of the medium [AS-274 ( 1 )]:
Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast extract 7.5 g, calcium
carbonate 2.0 g, sodium chloride 5.0 g, volume made with 750 ml_ Artificial Sea
Water and 250 ml_ demineralised water.
b) The above medium was distributed in 200 ml_ amounts in 1000 ml_ Erlenmeyer
flasks and autoclaved at 12 1°C for 30 minutes. The flasks were cooled to room
temperature (25+2 °C) and each flask was inoculated with a loopful of the well-grown
producing strain (PM0626271 ) on the slant and shaken on a rotary shaker for 70
hours to 74 hours at 230 rpm to 250 rpm at 29°C to 30°C to obtain the seed culture.
Example 6
Cultivation of the culture no PM0626271 in fermenter
a) Composition of the production medium:
Artificial Sea Water (artificial sea water salt 28.32 g) (75 %), soluble starch 20 g,
glucose 15 g, yeast extract 2 g, peptone 3 g, calcium carbonate 2 g, ammonium
sulphate 0.05 g, corn steep liquor 2 g, sodium chloride 2 g, magnesium phosphate 5
g, cobalt chloride (cobalt chloride 1 g demineralized water 1.0 L) 1 mL/L, trace salt
solution (copper sulphate 7 g, ferrous sulphate 1 g, manganese chloride 8 g, zinc
sulphate 2 g, demineralized water 1.0 L) 1 mL/L, demineralized water 1.0 L, pH 6.5
to 7.5 (before sterilization).
b) 100 L of the production medium in 150 L fermenter along with 30 mL of
desmophen as an antifoaming agent was sterilized in situ for 30 minutes at 12 1°C,
cooled to 29°C to 30°C and seeded with 2.5 L to 3.5 L of the seed culture obtained
above (Example 5).
c) Fermentation parameters: The fermentation was carried out at temperature 29°C
to 30°C, agitation 100 rpm, aeration 60 Ipm and harvested at 70 hours to 74 hours.
The production of the compound of formula I in the fermentation broth was detected
qualitatively by testing the bioactivity against S. aureus E71 0 (MRSA strain) and/or
Enterococcus faecium R2 (VRE) using the agar well diffusion method. The harvest
pH of the culture broth was 7.5 to 8.0. After the harvest, whole broth was subjected
to solvent extraction.
Example 7
Isolation and purification of the compound
The whole broth ( 10 L batch) was extracted using ethyl acetate ( 1 : 1 ) . The organic
and aqueous layers were separated. The organic layer was processed to evaporate
the solvent to obtain crude ethyl acetate extract ( 1 .5 g). The crude extract was
further processed by flash chromatography (silica gel, 30 g, solvent:
methanol/chloroform step gradient, flow: 15 imL/minute). The active compound eluted
with 1 % methanol to 5 % methanol in chloroform, which was concentrated to obtain
the semipure compound (250 mg). Further purification was carried out by repeated
normal phase preparative HPLC.
Preparative HPLC conditions:
Column : Eurospher silica ( 10m, 20x250 mm)
Eluent : methanol: chloroform (5:95)
Flow rate : 20 imL/minute
Detection (UV) : 245 nm
Retention time : (5 to 6 minutes)
Purity of fractions was checked by bioassay against E. faecium R2 and/or S. aureus
3066 and/or analytical HPLC. The eluates were pooled and concentrated under
reduced pressure to remove the solvent to obtain the compound.
Analytical HPLC conditions:
Column : Eurospher RP-1 8, (3m, 4.6 x 125 mm)
Solvent system : Gradient (0 % acetonitrile to 100 % in 15
minutes against water, followed by
100 % acetonitrile for 5 minutes)
Flow rate : 1 imL/minute
Detection (UV) : 245 nm
Retention time : compound of formula I ( 12 to 13 minutes)
Physical & Spectral properties of the compound of formula I:
Appearance : White powder
Melting point : 240 °C (decomposes)
Solubility : Soluble in chloroform, ethyl acetate, methanol
and insoluble in water
HR-ESI 1650.4858 (M+H)
Molecular weight (ESI) 1650.5 (M+H)
Molecular formula 71H83N1 9O1 8S5
IR (KBr) 3386, 2927, 1648, 1507, 1206, 756, 666 cm 1
H NMR refer to Table 1
3C NMR refer to Table 2
Table 1
H NMR of the compound of formula I in CDCI3: CD3OD (4:1 ) at 500 MHz
Peak d Peak d Peak d
1 0.7(d, 3H) 18 3.67(d, 1H) 35 6.91 (s, 1H)
2 0.74(d, 3H) 19 3.7 (q, 1H) 36 6.94(s, 1H)
3 0.95(d, 3H) 20 4.33(d, 1H) 37 7.2(s, 1H)
4 1.04(s, 3H) 2 1 4.33(d, 1H) 38 7.43(s, 1H)
5 1.08(d, 3H) 22 4.62((q, 1H) 39 7.45(s, 1H)
6 1.2(d, 3H) 23 4.86(dd, 1H) 40 7.65(s, 1H)
7 1.28(d, 3H) 24 5.1 9 (s, 1H) 4 1 7.87(s, 1H)
8 1.34(d, 3H) 25 5.1 9(s, 1H), 42 8.05(s, 1H)
5.67 (s, 1H)
9 1.37(m, 1H) 26 5.2(t, 1H) 43 8.1 7(s, 1H)
10 1.5(d, 3H) 27 5.6(d, 1H) 44 8.2 (s, 1H)
11 1.6(d, 3H) 28 5.62(s, 1H), 45 8.5 (s, 1H)
6.44(s, 1H)
12 2.1 (m, 1H) 29 5.65(d, 2H) 46 8.67 (s, 1H)
13 2.2 (m, 1H) 30 5.72(s, 1H), 47 8.99 (s, 2H)
6.61 (s, 1H)
14 2.2(m, 1H) 3 1 6.1 (q, 1H) 48 9.72 (s, 1H)
3.99(m, 1H)
15 2.8(d, 1H) 32 6.25(m, 1H) 49 9.8 (s, 1H)
16 3.05(t, 1H) 33 6.28(d, 2H)
3.5(t, 1H)
17 3.49(d, 2H) 34 6.8(d, 1H)
Table 2
3C NMR of the compound of formula I in CDCI3: CD3OD (4:1 ) at 500 MHz
Signal d Signal d Signal d
1 12.1 1 25 64.45 49 144.61
2 13.74 26 64.73 50 148.1 7
3 14.1 27 65.69 5 1 148.45
4 14.8 28 65.95 52 15 1.89
5 16.48 29 70.27 53 152.76
6 17.1 1 30 77.1 54 155.45
7 17.27 3 1 10 1.45 55 157.9
8 17.55 32 10 1.45 56 159.0
9 20.9 33 102.6 57 160.04
10 23.1 3 34 116.52 58 160.36
11 27.56 35 120.63 59 16 1.01
12 27.56 36 12 1.59 60 163.75
13 29.04 37 123.28 6 1 164.46
14 33.24 38 123.87 62 164.5
15 46.1 7 39 123.87 63 166.6
16 50.14 40 125.48 64 167.1 3
17 5 1.3 4 1 126.03 65 167.95
18 53.91 42 126.69 66 168.47
19 53.91 43 128.24 67 168.67
20 55.8 44 130.34 68 170.35
2 1 57.32 45 130.98 69 170.35
22 58.8 46 13 1. 14 70 17 1.63
23 62.42 47 132.39 7 1 172.0
24 62.73 48 14 1.85
Example 8
Estimation of solubility of compound of formula I in SLF (pH 7.4)
Materials used:
NaCI RFCL Limited, India
CaCI2 RFCL Limited, India
NaOH RFCL Limited, India
Procedure:
SLF was prepared by method based on Respiratory Physiology & Neurobiology,
2008, 162, 73-79.
Preparation of SLF
9 g of sodium chloride, 0.220 g of calcium chloride and 6.5 g of lactose were
dissolved in 1000 mL of water. The pH of the resultant solution was adjusted to 7.4.
Solubility of compound of formula I in SLF was measured at two temperatures, 45 C
and 60 C. For this, 1 mg of compound of formula I was added to 3 mL of SLF (in
duplicates) and after vortexing for few seconds was incubated in water bath set at
the required temperatures. Solubility was measured at one hour intervals for three
hours. The solution was filtered through 0.22 mi filter and the filtrate was injected on
HPLC. Solubility was calculated with respect to appropriate calibration curve.
Results obtained are given in Table 3.
Table 3
Conclusion:
Compound of formula I exhibits poor solubility in SLF at 45 C and 60 C. The
solubility is also not time dependent. Compound of formula I is a water insoluble
compound, thus solubility was measured in the buffer SLF.
Example 9
Preparation of the microparticle formulation of the compound of formula I
Materials used:
DPPC Avanti Polar Lipids, Canada
Methanol RFCL Limited, India
Chloroform RFCL Limited, India
DMSO RFCL Limited, India
NaCI RFCL Limited, India
CaCI2 RFCL Limited, India
NaOH RFCL Limited, India
Lactose monohydrate Signet Chemical Corporation Private Ltd, USA
Glassware Merck Limited, India
Glass beads N.M. Enterprises, India
Polycarbonate membrane ISOPORE™, Millipore, USA
filters
Polypropylene filter SWINNEX®, Millipore, USA
holders
Gas tight glass syringe Hamilton Company, USA
with metal leur lock
pH meter Eutech instruments, USA
Procedure:
Method A
Solvent Evaporation Method
The assay was carried out based on the reference US 4877561 .
The compound of formula I and DPPC, in ratios of 1: 1 , 1: 10 and 1:20 w/w, were
dissolved in 5 mL chloroform in a glass beaker. 30 mL of methanol was added and
the mixture was transferred to a 250 mL round bottomed flask. 30 mL of SLF was
poured into the mixture slowly. The solvents were evaporated by using a rotary
evaporator (Buchi GMBH, Switzerland). The water bath was set at 45 C and rotation
was set at 100 rpm. The vacuum controller was set to a pressure of 400 mBar.
Evaporated solvents were collected in a glass solvent collector. The remaining
solution in the RB flask turned milky indicating formation of liposomes. Volume of the
suspension from the RB flask was made up to 30 mL with SLF, centrifuged at
25,000G at 4 C for 10 minutes. The pellet obtained was resuspended in SLF,
vortexed and filtered through 1.2 mi polycarbonate filter eleven times to ensure
uniform particle size and stored at 4 C. The microparticle formulation was analyzed
by optical microscopy and electron microscopy.
Method B
Solvent free lipid self assembly method
The aim is to develop a solvent free manufacturing process for the formulation.
20 mg of compound of formula I and 20 mg of DPPC ( 1 :20 w/w) were added to a RB
flask containing 20 to 30 glass beads and 30 mL of SLF. The mixture was subjected
to rotation of 100 rpm at a temperature of 45 C for one hour. The formulation
obtained was subjected to centrifugation and filtration as described in Method A.
The formulation was analyzed by optical microscopy.
Observations:
For microparticles in formulation prepared by Method A.
(i) Formulation of microparticles containing compound of formula I
and DPPC in the ratio of 1: 1 w/w and 1: 1 0 w/w exhibited nonhomogenous,
unstable suspension with drug aggregates when
observed under optical microscope. The microparticles settled at
the bottom of the tube.
(ii) Formulation of microparticles containing compound of formula I
and DPPC in the ratio of 1:20 w/w exhibited homogenous
suspension with no drug aggregates when observed under
optical microscope. The microparticles did not settle down in the
tube.
(iii) Formulation of microparticles containing compound of formula I
and DPPC in the ratio of 1:20 w/w exhibited microparticles of
size 2 mi to 3 mi when observed under electron microscope.
Result:
Formulation of microparticles containing compound of formula I and DPPC is
optimally formed using drug: lipid ratio of 1:20 w/w.
Example 10
Determination of entrapment efficiency and mass balance of compound of formula I
in the formulation of Example 9 Method A and formulation of Example 9 Method B.
Materials used:
Methanol : RFCL Limited, India
DMSO : RFCL Limited, India
Glassware : Merck Limited, India
Autopipettes : Eppendorf GMBH, Germany
Procedure:
The entrapment efficiency of compound of formula I in the formulation of Example 9
Method A and Example 9 Method B were analyzed by HPLC (Agilent, USA). The
HPLC conditions are as follows:
Column : Lichrosphere® 100, RP-1 8e, 150 x 4.6 mm, 5 mi
Mobile phase : (a) 0.01 M ammonium acetate + 0.5 % triethylamine in
1000 mL water; pH adjusted to 6.5 with glacial
acetic acid
(b) acetonitrile
Composition : (a) : (b) : : 50 : 50
Run time : 5 minutes
Column temperature: 25 C
Injection volume : 20 m
Retention time : ~ 3.5 minutes
Solvent : 20 % DMSO in methanol
Compound of formula I was dissolved in 20 % DMSO to obtain 200 mg/mL
concentration and was used as reference standard. 100 m of formulation of
Example 9 Method A and 100 m of formulation of Example 9 Method B separately
were pipetted into 2 mL centrifuge tubes and 900 m of 20 % DMSO was added to
each tube. The tubes were vortexed which led to the rupture of microparticle matrix
and release of entire content of compound of formula I entrapped within the matrix.
The samples injected independently on the HPLC were as follows: reference
standard, formulation of Example 9 Method A (after rupture of the microparticle
matrix) and formulation of Example 9 Method B (after rupture of the microparticle
matrix). Mean of the peak areas was considered for calculation.
The entrapment efficiency was determined by the formula:
W
% entrapment = x 100
w
wherein:
W = amount of drug (compound of formula I) associated/ entrapped within the
microparticle
w = initial amount of compound of formula I
For the mass balance calculation, compound of formula I was recovered from the
glassware, filters, all the other labware which was used in the formulation
preparation process (Example 9 Method A and Example 9 Method B) and its content
was estimated. The polycarbonate filters (Example 9 Method A and Example 9
Method B) were washed with 5 mL of 20 % DMSO in methanol. This sample was
injected in HPLC as 'membrane residue'.
The RB flask used to prepare the formulation was washed with 10 mL of 20 %
DMSO in methanol. This sample was injected in HPLC as 'flask residue'.
The reference standard was injected six times on the HPLC and peak areas were
noted. The relative standard deviation of six injections of the standard was below 2.0
%. The samples were injected in duplicate on the HPLC and mean of the peak areas
were considered for calculation.
Results obtained are given in Table 4.
Table 4
Conclusion:
Entrapment efficiency of the formulation is comparatively better when prepared with
Method A of Example 9 than when prepared with Method B of Example 9.
Example 11
Determination of particle size of microparticles of formulation of Example 9 Method A
Procedure:
The hydrodynamic diameter and size distribution of microparticles of formulation of
Example 9 Method A was determined using Photon Correlation Spectroscopy
(DELSA Nano, Beckmann Coulter, USA). 1 mL of formulation was diluted to 10 mL
with SLF. 1 mL of this solution was used for determining particle size.
Result:
The mean particle size of microparticles of formulation of Example 9 Method A is 2
mi to 3 mi . For each individual population, the dispersion of the particle size around
the mean is about 0.45 - 0.47.
Table 5 provides the three cut-off diameters. Di 0 , D5o and D90 indicate the average
diameter of the 10 %, 50 % and 90 % of the particle population respectively.
Table 5
Conclusion:
The solvent evaporation method (Example 9 Method A) for compound of formula I
lipid based microparticles has led to generation of micron size particles in the range
of 1 m to 10 mi (as depicted in Table 5), with more than 99 % particles having
particle size less than 10 mi (depicted in Table 6).
Table 6
Example 12
Determination of pH of formulation of Example 9 Method A
Procedure:
pH meter (Eutech, USA) was standardized electronically against standard pH buffers
of pH 4, 7 and 9. pH of the formulation of Example 9 Method A was determined by
using the same pH meter.
Result:
pH of the formulation of Example 9 Method A is 6.1 4.
Conclusion:
The pH of the formulation of Example 9 Method A is physiologically compatible with
administration of the drug (compound of formula I) for a short period of up to 30
minutes of one time nebulization or intratracheal installation.
Example 13
Determination of osmolality of formulation of Example 9 Method A
Materials:
Reference solution : Wheecon Instruments Private Limited
Osmometer : Wheecon Instruments Private Limited
Procedure:
Osmolality of the formulation of Example 9 Method A was determined by using
Osmometer. The Osmometer was standardized using reference solution of 290
mOsmol/kg.
Result:
Osmolality of the formulation of Example 9 Method A is 340 mOsmol/kg.
Conclusion:
The physiological osmolality of body fluids is 300 mOsmol/kg. Any significant change
in this osmolality has pathological implications. Administration of large volumes of
hyper tonic solutions (significantly higher osmolality) or hypotonic solutions
(significantly lower osmolality) causes either excessive loss/gain of fluid leading to
pathological conditions of pulmonary and tissue edema or dehydration. Introduction
of small amounts of fluids, in aerosol form at normal osmolality concentrations
precludes the development of aforementioned pathologies. The formulation of
Example 9 Method A has osmolality of 340 mOsmol/kg and hence is within the safe
limits.
Example 14
Evaluation of excipient compatibility of compound of formula I in the formulation of
Example 9 Method A
Materials:
Hermetic sealing pans with O-rings : Perkin Elmer India Limited
DSC : Perkin Elmer, USA
Procedure:
50 m of formulation of Example 9 Method A was pipetted in DSC pan. The sample
and empty reference pan (reference standard) was hermetically sealed and placed in
Hyper DSC and the temperature program was run as follows:
Temperature program : 0 C to 50 C
Heating rate : 5 C/minute
Purge gas : Nitrogen
Flow rate : 30 imL/minute
Result:
Liposomes prepared without compound of formula I exhibited an endotherm with an
onset at 4 1. 1 °C. This endotherm may be attributed to the transition from ripped gel to
liquid crystalline phase. The microparticle formulation exhibited an endotherm at
approximately 42.5 °C. This shift in onset temperatures may be due to physical
interaction of compound of formula I with DPPC liposomes. The shift in the main
transition marks the transition of the phospholipid bilayer from a highly hindered wellorganized
hydrocarbon chain packed state to a state in which some acyl chains
show presence of kinks.
Conclusion:
The results exhibit drug-excipient compatibility relationship between the compound
of formula I and DPPC. There is absence of significant shift in the endotherm onset
temperature as well as absence of peak abolition.
Example 15
Estimation of solvent content in the formulation of Example 9 Method A
Materials:
Methanol (HPLC grade) : RFCL Limited, India
Chloroform (HPLC grade) : RFCL Limited, India
DMSO (HPLC grade) : RFCL Limited, India
Glass GC-HS vials : Perkin Elmer India Limited
Glassware : Merck Limited, India
Procedure:
The experiment was done to study residual solvent content in formulation of
Example 9 Method A using GC-HS. This experiment evaluates the feasibility of using
the solvent evaporation method (Example 9 Method A) to be used for large-scale
commercial production of formulation of Example 9 Method A.
Blank Preparation: 5 mL of DMSO was added to a 20 mL GC-HS glass vial. The vial
was crimped with a teflon stopper and aluminum cap. Two such vials were prepared
for blank injection.
Standard Preparation: 10 mg of chloroform and 100 mg of methanol were accurately
weighed in a 100 mL standard volumetric flask. The solvents were dissolved and
volume was made up to 100 mL with DMSO. 5 mL of stock solution was pipetted out
in a 20 ml_ GC-HS glass vial. The vial was crimped with a teflon stopper and
aluminum cap. Six such vials were prepared for standard injection.
Sample preparation: 5 ml_ of DMSO was added to a 20 ml_ GC-HS glass vial. The
vial was placed on an electronic balance and the weight was tared. 500 m I_ of
formulation of Example 9 Method A was added to the glass vial and the weight was
accurately noted. The vial was crimped with a teflon stopper and aluminum cap.
Three such vials were prepared for sample injection.
Table 7 gives chromatographic conditions and method for estimation of solvent
content in the formulation of Example 9 Method A.
Table 7
Instrument Gas Chromatograph :
Make: Perkin Elmer
Model No: Clarus 500
Head space Sampler:
Make: Perkin Elmer
Model No: Turbo Matrix 40
Column DB-1 (30 meter X 0.53 mm, 5 m)
Make: Agilent
Oven Temperature Initial temp. 40 °C (hold for 7 minutes ) to
Programming: 200 @ 20 O/min. (5 minutes hold)
Injection Temperature 220 °C
Detector Flame Ionization Detector
Detector Temperature 250 °C
Detector Range 1
Detector Attenuation: 5
Carrier Gas Helium
Carrier flow 2.3 psi
Split ratio 1 : 10
Run Time 20 minutes
Retention Time Chloroform ~ 3 minutes
Methanol ~ 11 minutes
DMSO ~ 14 minutes
Thermo stating Temperature 90 °C
Needle Temperature 95 °C
Transfer line temperature 100 €
Pressurization time 1.0 minutes
Thermo stating time 15 minutes
GC Cycle Time 30 minutes
Injection Time 0.07 minutes
Withdrawal Time 0.20 minutes
Result:
Table 8 gives a break up of effect of evaporation time on solvent content and
entrapment of formulation of Example 9 Method A.
Table 8
Observation:
It has been observed that evaporation time of 2 hours gives a lower solvent content
and desired entrapment and mass balance qualities of the formulation of Example 9
Method A. Also the residual solvent content as detected by GC-HS is below the
permissible limit for each individual solvent (permitted daily exposure limit for
chloroform is 60 ppm and for methanol is 3000 ppm - ( ICH Quality Guidelines,
Impurities: Guidelines for Residual Solvents, Q3C(R5), Feb 201 1) for the API solvent
content and permissible daily exposure limit for the pharmaceutical product.
Conclusion:
The solvent evaporation method (Example 9 Method A) can be considered as a
suitable method to develop this formulation.
Example 16
Nebulization of formulation of Example 9 Method A
Materials:
Twin Stage Impinger Unit Copley Scientific, U.K.
Nebulizer DeVILBISS®, Sunrise Medical, USA
Methanol RFCL Limited, India
DMSO RFCL Limited, India
HPLC Agilent, USA
Electronic Balance Denver Instruments, USA
Procedure:
TSI Unit was used for this study and was assembled as per the instruction manual.
TSI is an in vitro glass model of the human pulmonary tract and is used to quantify in
vitro the pulmonary drug deposition potential.
The vacuum pump of the TSI was switched on and the flow meter was used to
accurately check the flow of the system. The flow valve on the vacuum pump was
adjusted to ensure exact flow specification (28.3 L/min). After calibration of the
airflow, the whole unit was disassembled. 7 mL of methanol was added to the upper
impinger and 20 mL methanol was added to the lower impinger. The TSI unit was
assembled again as per the instruction manual.
5mL of the formulation of Example 9 Method A was added to the medication cup of
the nebulizer. Mouthpiece of the nebulizer was attached to the mouth of the TSI. It
was ensured that all parts were fit to avoid any vacuum loss.
The vacuum pump of the TSI was started to ensure uniform airflow. After 30 seconds
the nebulizer was started and start time was noted. After exactly 5 minutes, the
nebulizer was stopped. The pump was allowed to run for another 30 seconds after
which it was switched off. The upper impingement chamber mimics the throat and
upper airways and the lower impingement chamber mimics the alveoli.
Methanol was used as a rinsing solvent and the contents of the upper and lower
impingement chamber were collected in appropriate standard volumetric flasks. 20
% DMSO was added to ensure dissolution of compound of formula I. The final
volumes of the volumetric flasks were made up by methanol. The above experiment
was repeated with six batches of formulation and two batches of unformulated
compound of formula I.
The samples were evaluated at several concentrations starting from:
0.5 mg of compound of formula I in 5 ml formulation of Example 9 Method A -
Sample 1;
1 mg of compound of formula I in 5 ml formulation of Example 9 Method A - Sample
2;
2.5 mg of compound of formula I in 5 ml formulation of Example 9 Method A -
Sample 3;
5 mg of compound of formula I in 5 ml formulation of Example 9 Method A - Sample
4; and
100 mg/mL of unformulated compound of formula I (only compound of formula I) -
Sample 5.
This study was done to understand effect of concentration on in vitro drug
deposition. The study was further continued to estimate the amount of drug
(compound of formula I) lost to the process of nebulization and hence left behind in
the nebulizer cup.
Compound of formula I deposited in the upper and lower impingement chamber of
the TSI was analyzed by an HPLC with gradient pump and autosampler. The
chromatographic conditions used in HPLC analysis are provided in Table 9.
Table 9
Column Lichrosphere® 100, RP-1 8e, 150 x 4.6 mm, 5 mi
Mobile Phase A) 0.01 M ammonium acetate + 0.5 %
triethylamine in 1000 mL water; pH adjusted
to 6.5 with glacial acetic acid
B) acetonitrile
Composition A : B :: 50 : 50 (Isocratic)
Run Time 5 minutes
Column Temperature 25 °C
Injection Volume 20 m
Retention Time ~ 3.5 minutes
Solvent 20 % DMSO in methanol
100 m /i . of compound of formula I (2.5 mg of compound of formula I was dissolved
in 20 % DMSO in methanol) was used as reference standard.
The reference standard was injected six times on the HPLC and peak areas were
noted. The relative standard deviation of six injections of the standard was below 2.0
%. The samples were injected in duplicate on the HPLC and mean of the peak areas
were considered for calculation.
Result:
The % of compound of formula I deposited by the various samples (samples 1 to 6)
in the upper and lower impinger of the TSI is provided in Table 10.
Table 10
(B) Amount ^g) of compound of formula I lost (during nebulization process) in the
nebulization cup is provided in Table 11.
Table 11
Observations:
The formulation when nebulized at a concentration of 1 mg/mL and 2.5 mg/mL
deposits about 16 % and 10 % of the initial amount of compound of formula I
introduced into the nebulizer cup. The minimum inhibitory concentration of
compound of formula I is in the range of 0.1 25 g/mL to 5 g/mL (PCT application
publication WO201 1027290). The amount of compound of formula I deposited is
sufficient and above the minimum inhibitory concentration in the in vitro model. The
results suggest that 1 mg/mL and 2.5 mg/mL compound of formula I formulation
concentrations could be the concentrations which need to be evaluated in the in vivo
studies. At concentrations lower than 1 mg/mL, the percentage of compound of
formula I deposited is higher but the quantity is not significant while at concentration
of 5 mg/mL, significant amount of compound of formula I is retained back in the
nebulizer cup leading to wastage of compound of formula I.
Conclusion:
The results clearly establish that the formulation of Example 9 Method A is capable
of depositing significant amount of compound of formula I in the TSI in vitro lung
model while the unformulated compound of formula I is not deposited in the TSI on
nebulization. Thus the formulation of Example 9 Method A can be used for
inhalation-based delivery of compound of formula I.
BIOLOGICAL EVALUATION OF THE MICROPARTICLE FORMULATION
In vitro assays
Example 17
Microbial assay
The assay was carried out based on the reference; Nathan P et al, 1978, Laboratory
Methods for Selection of Topical antimicrobial Agents to treat infected Burn Wounds,
Burns 4: 177-1 87.
(A) Bacterial test models used in the assay:
Staphylococcus aureus 209P (MSSA)
Staphylococcus aureus ATCC 33591 (MRSA)
Enterococcus faecium R-2 (VRE)
(B) Inoculum preparation
Culture from cryovials were streaked on TSA slant and incubated at 37 °C for 18 to
24 hours. Using growth on the slant saline suspension was prepared and the optical
density adjusted to 0.3 units at 560 nm (~1 08 CFU/ml).
(C) Sample preparation
Samples tested in the assay are:
(i) Sample 1: Unfiltered suspension of the formulation of Example 9 Method B.
(ii) Sample 2: Filtered suspension of the formulation of Example 9 Method B
(after filtration through 0.22 mi filter).
(iii) Sample 3: Sample prepared by methanol disruption method: Sample 2 was
mixed with equal volumes of methanol and incubated for one
hour. Dilutions of this solution were prepared in methanol and
used for evaluating the efficacy of the released compound of
formula I.
(iv) Sample 4: Unformulated compound of formula I (compound of formula I
dissolved in methanol chloroform in 1:80 ratio).
The concentrations of samples 1, 2, 3 and 4 evaluated in the assay were: 100, 50,
25, 12.5, 6.25, 3.1 25, 1.56, 0.78, 0.39 and 0.1 95 mg/mL.
(D) Assay procedure:
40 m I_ of inoculum suspension of the test culture obtained from (B) was put into each
of 40 ml_ melted TSA butt (maintained at 38°C to 39 °C) in 100 ml_ capacity sterile
conical flasks and swirled for uniform mixing. The seeded butts were poured into
petri plates ( 150 mm outer diameter) allowing them to set for about 30 minutes. The
plates were kept at 4°C to 8°C for complete setting. Required numbers of wells (of
diameter 6 mm) were punched out from the set medium. 50 m I_ of sample 1, sample
2 and sample 3 were added in corresponding wells in the plates. The plates were
pre-incubated at low temperature (2°C-8°C) for about 30 minutes to allow diffusion.
The plates were then incubated at 37 °C for 18 to 24 hours. Vancomycin at 20 mg/mL
was used as standard antibiotic. The results of activity of various samples were
interpreted as size of zone of inhibitions in mm.
Result:
Sample 3 showed clear zones from 100 mg/mL to 0.78 mg/mL while sample 4
showed clear zones from 100 m/mL to 0.39 mg/mL.
The activity (with respect to zone size) of sample 2 is better than sample 1 as
sample 2 showed clear zone of inhibition at 12^g/ml while sample 1 showed clear
zones at 25mg/ml .
In case of sample 3, the zone size of compound of formula I is less than the
unformulated compound of formula I (sample 4). This may be due to the slow
release of compound of formula I from the lipid formulation.
Conclusion:
The activity of compound of formula I released from the formulation of Example 9
Method B (sample 3) is comparable to the unformulated compound of formula I
(sample 4) and hence can be carried forward for in vivo testing and evaluation.
Example 18
In vitro cytocompatibility assay
The assay was carried out based on the reference Journal of Biomedical Materials
Research, 2009, 89A, 281 -292.
This assay was performed to evaluate the cytocompatibility of the formulation of
Example 9 Method A with the relevant cell lines and standard cells which are
specified by the regulatory agencies.
Cell lines evaluated:
MRC5 (Lung fibroblast cell line)
A549 (Type I I alveolar cell line isolated from of malignant tumour)
L929 (Mouse fibroblast cell line, is an ASTM standard)
Experimental details:
Cell density: 10,000/well for MRC 5 and L292, and 3000/well for A549
Time points: 24 hours and 48 hours.
Concentrations evaluated:
1, 0.7, 0.3, 0.1 mg/mL for microparticles of formulation of Example 9 Method B -
Sample 1
0.3, 0.1 mg/mL for microparticles without compound of formula I, DPPC - Sample 2
0.1 , 0.01 mg/mL for only compound of formula I - Sample 3
Procedure:
The toxicological evaluation was done using The CellTiter 96 Aqueous One Solution
Assay by Promega (Cat.no: G3582). The assay is a standard colorimetric method for
determining the number of viable cells in proliferation or cytotoxicity assays. The
MTS used in the assay is bioreduced by cells into a colored formazan product that is
soluble in tissue culture medium. The quantity of formazan product as measured by
the amount of 490 nm absorbance is directly proportional to the number of living
cells in culture.
Briefly, the cell lines were plated in triplet with 96-well plate and allowed to adhere
and proliferate over a period of 24 hours. 24 hours after cell adhesion, the
compounds were added. 48 hours post cell plating, the assay was terminated by
replacing the medium with 100 m I_ fresh medium, and addition of 20 m I_ of CellTiter
96 Aqueous One Solution (Promega, Cat.no: G3582). A set up in triplet of "no-cell"
control containing 100 m I_ of culture medium and 20 m I_ of "One Solution" was also
maintained as control in the assay. The plate was incubated for 30 minutes to 4
hours for the color to develop. The ELISA 96-well plate was then subjected to
absorbance recordings at 490 nm (450-540 nm) with a 96-well plate reader. The
average 490 nm absorbance from the "no-cell" control was subtracted from all other
absorbance values and the corrected absorbance was utilized for further
calculations. (Background absorbance from "no-cell" control is typically 0.2 to 0.3
absorbance units after 4 hours of incubation).
Result:
Viability of all the cell lines evaluated in presence of the Sample 1 was more than
98 %. The morphology of the cell lines was unaltered and monolayer confluence was
present. This shows that at the evaluated concentrations the sample 1 did not exhibit
any toxicity in the cell-based assay. Compound of formula I in its unformulated form
however exhibited cytotoxicity in all the different cell lines evaluated. Hence the
formulation has an additional advantage of limiting the toxicity manifested by
compound of formula I. The results are summarized in Table 12.
Table 12
Conclusion:
Sample 3 has the potential to result or express in vivo adverse effects and toxicity.
However sample 1 that includes the use of DPPC, the toxic potential of compound of
formula I is masked and biological efficacy is observed in absence of toxicity.
Example 19
In vitro macrophage uptake assay
The assay was carried out based on the reference Respiratory Research, 2009, 10,
44.
Isolation and culture of rat alveolar macrophages:
Bronchoalveolar lavage washings from the rats were taken after appropriate ethical
permission. Sterile warmed saline was introduced into the lung and then removed by
suction. Bronchoalveolar lavage fluid was transported on icepacks (usually 100 to
200 mL of bronchoalveolar lavage fluid per donor is desired). The fluid was
subjected to centrifugation at 250 g for 10 minutes and the cells were collected.
Pooled the cell pellets by resuspending the cells in a total of 10 mL of HBSS. 100 L
aliquot was removed and stained with Wright-Giemsa stain to do a differential cell
count. The cell count was adjusted and plated the cells in macrophage culture
medium (HAM F 12 medium, Amimed, Switzerland). 0.5 mL of the cell suspension
was added to each of the 6 well tissue culture plates with 0.5 mL of the macrophage
culture medium. The cells were incubated for 24 hours at 37 °C and 5 % CO2. Cell
adherence and cell growth was evaluated and treated with the formulation of
Example 9 Method A tagged with sulforhodamine dye. Post treatment the cells were
evaluated for fluorescence. This fluorescence results for selective uptake of the
formulation by the alveolar macrophages.
Result:
The microparticles of formulation of Example 9 Method A were selectively taken up
by the alveolar macrophages starting from 1-hour post treatment. The dye shows
gradual saturation in intensity at 3 hours. This is indicative of active uptake and a
saturation point of the uptake. The mycobacteria reside and survive in the alveolar
macrophages, hence it is very critical to ensure that the drug reaches the
macrophages. By the designed microparticle formulation of Example 9 Method A it is
evident that not only is compound of formula I reaching the macrophages but is
being actively taken up by them, which is desired for the therapy to be successful in
vivo.
The Formulation of Example 9 Method A is actively taken up (gradual increase in the
compound concentration) and metabolized by the macrophages (decrease in the
compound concentration) as evident from the values in table 12. The free drug
(compound of formula I) does not show active uptake (evident from the saturated
concentrations of the free drug).
Table 1
Conclusion:
Compound of formula I when developed as a lipid based microparticle formulation is
actively taken up by the alveolar macrophages, which are the target for treatment.
The therapy has the potential to actively reach the target and relevant cells.
In vivo assay
Animals used in the experiments were housed and cared for, in accordance with the
Guidelines in force published by CPCSEA, Tamil Nadu, India. Procedures using
laboratory animals were approved by the IAEC of Piramal Life Sciences Limited,
Goregaon, Mumbai, India.
Example 20
Lung deposition studies
The assay was done as reported in The AAPS Journal, 2005, 7 ( 1 ) , E20-E41 .
A pilot in vivo lung deposition study was carried out as per the reference mentioned.
Guinea pigs were divided in three groups. Group 1 received formulation of Example
9 Method A, group 2 received unformulated compound of formula I and group 3 was
untreated (na'ive group). 10 mg/kg of the formulation was aerosolized and the
animals were allowed to breathe passively. Following administration of the
formulation of Example 9 Method A the animals were sacrificed at 30 minutes time
point. The lungs were collected and analyzed by HPLC. HPLC conditions are shown
in Table 13.
HPLC System Waters Alliance HPLC
Column BDS Hypersil, C 18 (250 x 4.6 mm)
5 m
Mobile phase A- Acetonitrile,
B- 0.5 % Formic Acid pH 3.5 with
TEA
Flow 1 imL/min
Gradient program 0.01/20, 10/80, 13/80, 15/20,
(Time/%A) 17/20
Injection Volume 50 L
Column Temperature 25 C
Wavelength 240 nm
Retention Times compound of formula I - 10.21 min
Results obtained are summarized in Table 14.
Table 14
Observations:
On nebulization, the unformulated compound of formula I is unable to reach the
lungs. The formulation of Example 9 Method A was able to reach the lungs on
passive respiration. Further exposure of the animals will lead to increased levels of
compound of formula I in the lungs.
Conclusion:
The formulation of Example 9 Method A is able to reach the lungs.
Example 2 1
Determination of pulmonary bioavailability of the formulation of Example 9 Method A.
The assay was done as reported in The AAPS Journal, 2005, 7 ( 1 ) , E20-E41 .
A nose-only exposure in vivo lung deposition study was carried out as per the
reference mentioned. Guinea pigs were divided in two groups. Group 1 received
unformulated compound of formula I (3 mg/kg) and group 2 received formulation of
Example 9 Method A (3 mg/kg). The unformulated compound of formula I and
formulation of Example 9 Method A were aerosolized and the nose-only exposure
was carried out using jet nebulizer wherein the nebulization period was one hour.
Following aerosol administration, the animals were sacrificed at 1 hour, 2 hours, 6
hours, 12 hours and 24 hours post aerosol exposure and the lungs were collected
and analyzed to quantitate the amount of compound of formula I (ng/g) deposited in
the lung by LC-MS. LC-MS conditions are specified below:
Table 15
Chromatographic Conditions:
Source/Gas parameters:
Parameter Value
Collision Gas (CAD) 7.00
Curtain Gas (CUR) 25.00
Ion source Gas 1 (GS1 ) 50.00
Ion source Gas 2 (GS2) 55.00
Ion Spray Voltage (ISV) 5500.00
Capillary Temperature 500.00
Table 17
MS-MS Parameters:
Observations
On nebulization, the unformulated compound of formula I was unable to reach the
lungs. The formulation of Example 9 Method A was able to reach the lungs and was
retained for 24 hours.
Conclusion
The formulation of Example 9 Method A was able to overcome the poor
bioavailability profile of unformulated compound of formula I. Further the lung
retention profile of formulation of Example 9 Method A can also make it suitable for
once a day inhalation.
Example 22
Evaluation of cumulative accumulation of formulation of Example 9 Method A after
once a day dosing for 5 days for nebulization exposure period of 1 hour/day
The assay was done as reported in The AAPS Journal, 2005, 7 ( 1 ) , E20-E41 .
A nose-only exposure in vivo lung deposition study was carried out as per the
reference mentioned. Guinea pigs were divided in two groups. Group 1 received
unformulated compound of formula I (3 mg/kg) and group 2 received formulation of
Example 9 Method A (3 mg/kg). The unformulated compound of formula I and
formulation of Example 9 Method A were aerosolized and the nose-only exposure
was carried out using jet nebulizer wherein the nebulization period was one hour
everyday for five consecutive days and the animals were sacrificed on 6th day. The
lungs were collected and analyzed to quantitate the amount of compound of formula
I (ng/g) deposited in the lung by LC-MS. LC-MS conditions are as provided in Tables
15, 16 and 17 of Example 2 1.
Results obtained are summarized in Table 19.
Observations:
On nebulization, the unformulated compound of formula I was unable to reach the
lungs. The formulation of Example 9 Method A was able to reach the lungs and
retain for 24 hours and also exhibit compound of formula I after chronic daily
exposure for 5 days.
Conclusion:
The capability of the lungs to retain the compound of formula I as formulation
(formulation of Example 9 Method A) for five days can be used as five days of
nebulization as a therapeutic regimen.
Example 23
To evaluate dose scheduling of the formulation of Example 9 Method A
The assay was done as reported in The AAPS Journal, 2005, 7 ( 1 ) , E20-E41 .
A nose-only exposure in vivo lung deposition study was carried out as per the
reference mentioned. Guinea pigs were divided in three groups. Group 1 received
unformulated compound of formula I (3 mg/kg); group 2 received formulation of
Example 9 Method A (3 mg/kg) once a day for one day and group 3 received
formulation of Example 9 Method A (3 mg/kg) twice a day for one day. The
unformulated compound of formula I and formulation of Example 9 Method A were
aerosolized and the nose-only exposure was carried out using jet nebulizer. The
animals were sacrificed and the lungs were collected and analyzed to quantitate the
amount of compound of formula I (ng/g) deposited in the lung by LC-MS. LC-MS
conditions are as provided in Tables 15, 16 and 17 of Example 2 1.
Results obtained are summarized in Table 20.
Observations:
On nebulization, the unformulated compound of formula I was unable to reach the
lungs. The formulation of Example 9 Method A was able to reach the lungs and the
amount of compound of formula I absorbed by the lungs increased as the dose was
doubled in a day.
1. A microparticle formulation comprising compound of formula I;
and a biodegradable lipid for drug delivery wherein the ratio of drug (compound of
formula I) to lipid is 1: 1 5 to 1:25; wherein said formulation is a biodegradable and
inhalable formulation.
2. The microparticle formulation as claimed in claim 1, wherein compound of formula
I constitutes 1 % to 5 % (w/w) of the formulation.
3. The microparticle formulation as claimed in claim 1 or 2, wherein the
biodegradable lipid is dipalmitoylphosphatidylcholine (DPPC).
4. The microparticle formulation as claimed in any one of the preceding claims 1 to 3,
wherein the particle size of the microparticles ranges between 0.5 and 10 microns.
5. The microparticle formulation as claimed in claim 4, wherein at least 90 % of the
microparticles are of particle size less than 10 microns.
6. The microparticle formulation as claimed in any one of the preceding claims 1 to 5,
wherein the formulation is an aqueous liposomal dispersion.
7. The microparticle formulation as claimed in any one of the preceding claims 1 to 5,
wherein pH of the formulation ranges from 6 to 7.
8. The microparticle formulation as claimed in any one of the preceding claims 1 to 5,
wherein the phase transition temperature of the formulation ranges from 4 1 C to
43 C.
9. A process for the preparation of a microparticle formulation comprising the
compound of formula I as defined in claim 1 and dipalmitoylphosphatidylcholine
(DPPC) wherein the ratio of drug (compound of formula I) to DPPC is 1: 1 to 1:20,
wherein said process comprises the steps of:
(a) dissolving compound of formula I and DPPC ( 1 : 1 to 1:20 ratio) in
3 ml_ to 15 ml_ chloroform to obtain a solution;
(b) adding 20 ml_ to 45 ml_ of methanol to the solution of step (a)
and mixing well to ensure homogeneity;
(c) adding 20 ml_ to 50 ml_ of simulated lung fluid (SLF) to the
solution of step (b);
(d) evaporating the solvents;
(e) making up the volume obtained in step (d) to 30 ml_ with SLF
and centrifuging at 15000 G TO 35000 G at 4 C for ten minutes
to obtain a pellet;
(f) resuspending the pellet obtained in step (e) in SLF to obtain a
suspension of concentration 0.5 img/mL to 10 img/mL; and
(g) filtering the suspension obtained in step (f) through 0.5 mi - 5
mi polycarbonate filter to obtain uniform particle size of the
microparticles formed.
10. The process as claimed in claim 9, wherein the particle size of the microparticles
ranges between 0.5 and 10 microns.
11. The process as claimed in claim 10, wherein at least 90 % of the microparticles
are of size less than 10 microns.
12. A process for the preparation of the microparticle formulation comprising the
compound of formula I as defined in claim 1 and dipalmitoylphosphatidylcholine
(DPPC) wherein the ratio of drug (compound of formula I) to DPPC is 1: 1 to 1:20,
wherein said process comprises the steps of:
(i) adding 20 ml_ to 45 ml_ of simulated lung fluid (SLF) to a mixture
of compound of formula I and dipalmitoylphosphatidylcholine
(DPPC) ( 1 : 1 to 1:20 ratio);
(ii) subjecting the mixture of step (i) to 100 rpm to 200 rpm rotation
at 45 C for one hour to obtain a suspension;
(iii) centrifuging the suspension obtained in step (ii) at 15000 G -
35000 G at 4 C for ten minutes to obtain a pellet;
(iv) resuspending the pellet obtained in step (iii) in SLF to obtain a
suspension of concentration 0.5 mg/mL to 10 mg/mL; and
(v) filtering the suspension obtained in step (iv) through 0.5 mi - 5
mi polycarbonate filter to obtain uniform particle size of the
microparticles formed.
13. A method for the treatment of pulmonary tuberculosis, multi drug resistant
tuberculosis, methicillin resistant Staphylococcus aureus pneumonias or methicillin
sensitive Staphylococcus aureus pneumonias, comprising administering by
inhalation to a mammal in need thereof the microparticle formulation as claimed in
claim 1.
14. The method as claimed in claim 13, wherein said method targets alveolar
macrophages.
15. A method of delivering microparticle formulation as claimed in claim 1 to a
mammal in need thereof, wherein the formulation is administered to the mammal by
inhalation or intratracheal instillation for pulmonary delivery.
16. The method as claimed in claim 15, wherein the formulation is administered by
inhalation.
17. The method as claimed in claim 16, wherein the administration of formulation by
inhalation is done by nebulization in which the drug (compound of formula I) is
entrapped in microparticles.
18. The method as claimed in claim 17, wherein the retention of the entrapped
compound of formula I range from 30 % to 70 %.
19. The method as claimed in claim 16, wherein the drug (compound of formula I),
contained in the formulation administered by inhalation is retained in the lungs over a
period of 24 hours.
20. The method as claimed in claim 16, wherein the dosage for inhalation ranges
between 0.05 and 10 mg/kg body weight/day.
2 1. The method as claimed in claim 13, wherein said method is non-invasive.
22. Use of the microparticle formulation comprising compound of formula I and a
biodegradable lipid for drug delivery wherein the ratio of drug (compound of formula
I) to lipid is 1: 15 to 1:25 for the treatment of pulmonary tuberculosis, multi drug
resistant tuberculosis, methicillin resistant Staphylococcus aureus pneumonias or
methicillin sensitive Staphylococcus aureus pneumonias.
23. Use of the microparticle formulation comprising compound of formula I and a
biodegradable lipid for drug delivery wherein the ratio of drug (compound of formula
I) to lipid is 1: 1 5 to 1:25 for the manufacture of a medicament for the treatment of
pulmonary tuberculosis, multi drug resistant tuberculosis, methicillin resistant
Staphylococcus aureus pneumonias and methicillin sensitive Staphylococcus aureus
pneumonias.
24. A microparticle formulation comprising compound of formula I and a
biodegradable lipid for drug delivery wherein the ratio of drug (compound of formula
I) to lipid is 1: 1 5 to 1:25 for treatment of pulmonary tuberculosis, multi drug resistant
tuberculosis, methicillin resistant Staphylococcus aureus pneumonias or methicillin
sensitive Staphylococcus aureus pneumonias.