Note: Descriptions are shown in the official language in which they were submitted.
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MACROLIDE FORMULATIONS FOR INHALATION AND METHODS OF
TREATMENT OF ENDOBRONCHIAL INFECTIONS
Field of the Invention
This invention concerns novel and improved macrolide formulations, such as
erythromycylamine formulations, for delivery by inhalation and to improved
methods
of treatment of susceptible acute or chronic endobronchial infections. In
particular,
the invention relates to formulations comprising at least one concentrated
macrolide
antibiotic in a physiologically acceptable liquid solution or dry powder form.
The
formulations are suitable for delivery of a macrolide antibiotic drug, such as
erythromycylamine, to the lung endobronchial airway space of a liquid aerosol
or dry
powder aerosol form, wherein a substantial portion of the aerosolized droplets
or
particles of the formulation have a mass median aerodynamic diameter between 1
to
5 Vim. Formulated and aerosol delivered efficacious amounts of the macrolides
are
effective for the treatment and/or prophylaacis of acute and chronic
endobronchial
infections, and pneumonia, particularly those caused by Streptococcus
pneumoniae,
Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis,
Legionella
pneumophila, Chlamydia pneunzoniae, and Mycoplasma pneumoniae. The novel
formulations have small volume yet deliver an effective dose of the macrolide
antibiotic to the site of infection. In yet other aspects, this invention
relates to new
and improved unit dose formulations of macrolide antibiotics for delivery by
aerosol
inhalation.
Background of the Invention
Streptococcus pneumonia and other typical and atypical pathogens infect the
endobronchi.al space in the lung of individuals who suffer from chronic
obstructive
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pulmonary disease (COPD) [S. Chodosh et al. Clinical Infectious Diseases 1998;
27:
730-738]. COPD is most commonly manifested as chronic bronchitis (CB) and
emphysema.
Chronic . bronchitis is a pulmonary disease that is characterized by the
inflammation and progressive destruction of lung tissue. The debilitation of
the lungs
in CB patients is associated with chronic cough, increased daily sputum
production,
and accumulation of purulent sputum produced as a result of chronic
endobronchial
infections caused by compromised pulmonary function. Acute exacerbation of
chronic bronchitis (AECB) is often characterized by increasing cough, purulent
sputum production, and clinical deterioration caused by Streptococcus
pneumonia, H.
influenzae, and Moraxella cata»halis. Pneumonia may also result from infection
by
these organisms either de novo or as a complication of COPD. Despite the
controversy over the appropriateness of antimicrobacterial therapy for the
treatment of
CB and in particular acute exacerbations of CB, Saint et al. (JAMA 1995; 273:
957-
960) demonstrated that oral antimicrobial therapy provided some clinical
benefit
when compared to no therapy. Furthermore, the dose of antimicrobial agent was
important with respect to time to relapse. Thus, higher doses of oral
antimicrobial
agents were associated with a higher median infection free-interval (S.
Chodosh et al.,
Clinical Infectious Diseases 1998; 27: 730-738).
Presently, oral administration of macrolides and fluoroquinolones active
against typical and atypical pathogens are treatments of choice for CB.
However, oral
administration of macrolide antibiotics has adverse side effects. The most
common
side effects associated with the treatment of oral/parental macrolide
antibiotics are
diarrhea/loose stools, nausea, abdominal pain and vomiting (R. N. Brogden D.
Peters,
Drugs, 1994; 48: 599-616 and H. D. Langtry, R. N. Brogden Drugs 1997; 53: 973-
1004 and references cited therein). In addition, pseudomembranous colitis is a
serious
side effect associated with oral antibiotic therapy including oral macrolide
therapy (S.
H. Ahmad et al. Indian J. Pediatr. 1993, 60: 591-594). Penetration of
macrolides into
lung tissue after oral administration varies according to dose and composition
(R. N.
Brogden D. Peters, Drugs, 1994; 48: 599-616 and H. D. Langtry, R. N. Brogden
Dmcgs 1997; 53: 973-1004 and references cited therein). Furthermore,
macrolides are
associated with alterations in the systemic concentrations of unrelated drugs,
such as
theophylline, due to interactions with the cytochrome-based metabolic system
of the
liver. Such drug-drug interactions often require dosage adjustment or
elimination of
one component from treatment regimes.
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Exythromycylamine is a 14-membered ring macrolide belonging to the
erythromycin family of antibiotics and possesses a similar in vitro antibiotic
spectrum
to erythromycin A, and like erythromycin A, is an effective treatment of
typical and
atypical pneumonias. Erythromycylamine has a C-9 amino function having the S-
configuration in place of the C-9 carbonyl group found in erythromycin A. One
significant limitation of erythromycylamine is its lack of oral absorption,
thus, in
order to achieve useful therapeutic concentrations a prodrug, dirithromycin,
was
developed. The prodrug of erythromycylamine is dirithromycin, which features a
bridged acetal function between the C-9 amino and C-11 hydroxy groups (see
Fig. I).
The cyclic acetal is rapidly hydrolysized in plasma by a nonenzymatic process
(half
life of approximately 30 minutes). Dirithromycin has been shown to
successfully
treat exacerbations that occur in patients with CB (M. Cazzola et al.,
Respinatofy
Medicine; 1998; 92: 895-901). A major advantage of erythromycylamine is its
long
half life (30-44 hours) (R. N. Brogden D. Peters, Df-ugs, 1994; 48: 599-616).
Unfortunately, oral bioavailability of dirithromycin is only 10-14% in humans
with
high elimination (62-81 %) into the feces mostly as erythromycylamine. Because
erythromycylamine is not absorbed and its prodrug, dirithromycin, is poorly
absorbed,
limited amounts of active drug substance are available systemically to treat
lung
infections caused by typical and atypical bacteria. While enough
erythromycylamine
concentrates at the site of infection to provide a therapeutic effect, the
concentration
of drug is limited. Higher oral doses or more frequent dosing of dirithromycin
increase_drug concentration at the site of action; however, increased adverse
events
are likely to occur and may increase patient hardship and compliance.
One of the first studies using aerosolized antibiotics for the treatment of
lung
infections was reported in Lancet, .22:1377-9 (1981). A controlled, double-
blind
study on twenty CF patients demonstrated that aerosol administration of
carbenicillin
and the aminoglycoside gentamicin can improve the health of CF patients. Since
that
time, scattered reports in the literature have examined aerosol delivery of
aminoglycosides in general and tobramycin in particular (see, for example,
U.S.
Patent No. 5,580,269). However, evaluation and comparison of these studies is
often
difficult because of the differences in antibiotic formulations, breathing
techniques,
nebulizers and compressors. Moreover, aerosol delivery is often difficult to
evaluate
because of differences in the formulations, aerosol delivery devices, dosages,
particle
sizes, regimens, and the like. When, for example, the mass median aerodynamic
diameter (MMAD) is greater than 5 Vim, the particles are typically deposited
in the
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upper airways, decreasing the amount of antibiotic delivered to the site of
infection in
the lower respiratory tract. An article published in Arch. Dis. Child., 68:788
(1993)
emphasized the need for standardized procedures and for improvement in aerosol
administration of drugs to CF patients.
Effective aerosol administration is currently compromised by the lack of
additive-free and physiologically compatible formulations and particularly by
the
inability of certain nebulizers to generate small and uniform particle sizes.
The size
range of aerosolized particles needed to deliver the drug to the endobronchial
space
and peripheral lung, the sites of the infection is preferably between about 1
and 5 pm.
Many nebulizers that aerosolize therapeutics, including aminoglycosides,
produce a
large number of aerosol particles having sizes less than 1 ~,m or greater than
5 Vim. In
order to be therapeutically effective, the majority of aerosolized antibiotic
particles
should not have a MMAD larger than 5 pm. When the aerosol contains a large
number of particles with a MMAD larger than 5 pm, the larger-sized particles
are
deposited in the upper airways, decreasing the amount of antibiotic delivered
to the
site of infection in the lower respiratory tract.
Currently, three types of available nebulizers, jet nebulizers, vibrating
porous
plate nebulizers and ultrasonic nebulizers, can produce and deliver aerosol
particles
with diameter sizes between 1 and 5 ~.m, a particle size that is preferable
for
treatment of bacterial infections of the lung. Therefore, it would be highly
advantageous to provide a macrolide formulation that could be efficiently
aerosolized
in a jet, vibrating porous plate, and ultrasonic nebulizer. In addition, newer
aerosol
generating technologies are now available, including mechanical extrusion and
both
passive and energized dry powder inhalers that are useful for the delivery of
therapeutic agents in dry powder form.
Another requirement for an acceptable formulation is adequate shelf life.
Generally, antibiotics, and particularly antibiotic solutions for intravenous
administration, contain phenol or other preservatives to maintain potency and
to
minimize the production of degradation products. However, phenol and other
preservatives, when aerosolized, may induce bronchospasm, an unwanted
occurrence
in patients with lung diseases such as chronic bronchitis.
Administration of macrolide antibiotics, such as erythromycylamine, for
inhalation in the form of a liquid or dry powder aerosol has the advantage of
overcoming poor oral bioavailability associated with the prodrug, while
providing
efficacious concentrations of the antibiotic to the lung that can not be
achieved by
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either the oral or intravenous route. An additional advantage of aerosol
delivery of
erythromycylamine is its inherent high affinity for lung tissue and
persistence in the
plasma compartment (long plasma/tissue half life). The combination of a high-
concentration aerosol delivery, long plasma/tissue half life and high lung
affinity
would allow for safer macrolide therapy, which is capable of eradicating or
substantially reducing endobronchial infections after a single aerosol dose.
It would be highly advantageous, therefore, to provide macrolide antibiotic
formulations, such as erythromycylamine formulations, containing no
preservatives,
at a pH adjusted to levels that slow or prevent degradation, and are tolerable
for a
patient, and that provide adequate shelf life suitable for commercial
distribution,
storage and use.
It is therefore an object of this invention to provide concentrated
formulations
of macrolide antibiotics, such as erythromycylamine, erythromycin A,
roxithromycin,
azithromycin and clarithromycin, that contain effective concentrations of the
macrolide antibiotic in a form that can be efficiently aerosolized by
nebulization, such
as by the use of jet, vibrating porous plate, or ultrasonic nebulizers, or dry
powder
inhalers, into aerosol particle sizes predominantly within a range from l and
5 Vim.
Summary of the Invention
In accordance with the present invention, it has now been discovered that
human and non-human animal subjects suffering from or at risk for
endobronchial
infection, such as an infection by bacterial Stf°eptococcus pneumoniae,
Haemophilus
influenzae, Staphylococcus aureus, Mof-axella catar~°halis and/or the
atypical
pathogens Legionella pneumonia, Chlamydia pneumoniae, and/or Mycoplasma
pneumoniae, can be effectively and efficiently treated by administering to the
subject
by inhalation an antibacterially effective amount of a macrolide antibiotic,
such as
erythromycylamine, erythromycin A, roxithromycin, azithromycin or
clarithromycin,
in a liquid solution or dry powder form suitable for aerosol generation.
Thus, one aspect of the current invention relates to concentrated formulations
suitable for efficacious delivery by inhalation of a macrolide antibiotic
drug, such as
erythromycylamine, erythromycin A, roxithromycin, azithromycin or
clarithromycin,
into the endobronchial space of a subject suffering from or at risk for a
bacterial
pulmonary infection.
Another aspect of the invention provides formulations suitable for efficacious
delivery of a macrolide antibiotic drug, such as erythromycylamine,
erythromycin A,
roxithromycin, azithromycin or clarithromycin, into the endobronchial space of
a
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subject suffering from bacterial Streptococcus pneumoniae, Haemophilus
influenzae,
Staphylococcus aureus, Moraxella catarf halis and/or the atypical pathogens
Legionella pneumonia, Chlamydia pneumoniae, and/or Mycoplasma pneumoniae
pulinonary infection.
Another aspect of the current invention provides formulations suitable for
efficacious delivery of a ' macrolide antibiotic drug, such as
erythromycylamine,
erythromycin A, roxithromycin, azithromycin or clarithromycin, into
endobronchial
space of a subject to prevent or substantially reduce the risk of pulmonary
infection in
at-risk patients caused by Stretococcus pneumoniae, Haemophilus influenzae,
Staphylococcus aureus, Moraxella catarrhalis and/or the atypical pathogens
Legionella pneumonia, Chlamydia pneumoniae, and/or Mycoplasma pneumoniae.
Still another aspect of the current invention provides liquid formulations
comprising the equivalent of 50 to 750 mg of a macrolide antibiotic drug, such
as
erythromycylamine, erythromycin A, roxithromycin, azithromycin or
clarithromycin,
in 0.5 to 5 ml of a physiologically acceptable carrier, such as saline diluted
into a
quarter normal saline strength wherein said formulation has a physiologically
tolerated osmolarity, salinity, and pH and is suitable for delivery to a
subject in
concentrated form by aerosol inhalation.
Still another aspect of the current invention provides dry powder formulations
comprising the equivalent of 25 to 250 mg of a macrolide antibiotic drug, such
as
erythromycylamine, erythromycin A, roxithromycin, azithromycin or
clarithromycin,
in a physiologically acceptable dry powder carrier for delivery to a subject
in
concentrated form by aerosol inhalation, wherein the dry powder formulations
comprise about 50 to 90% by weight of the macrolide antibiotic drug.
Still another aspect of the current invention provides methods for the
treatment
of pulmonary infections caused by susceptible bacteria by administering to a
subject
requiring such treatment by inhalation an aerosol formulation comprising an
antibacterially effective amount of a macrolide antibiotic drug, such as
erythromycylamine, erythromycin A, roxithromycin, azithromycin or
clarithromycin,
formulated in a physiologically compatible liquid solution or dry powder form,
wherein the mass median aerodynamic diameter (MMAD) of particles in the
aerosol
formulation is predominantly between l and 5 Vim.
In other aspects, the present invention provides unit dose formulations and
devices adapted for use in connection with a high efficiency inhalation
system, the
unit dose device comprising a container designed to hold and store the
relatively small
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volumes of the macrolide antibiotic formulations of the invention, and to
deliver the
formulations to an inhalation device for delivery to a subject in aerosol
form. In one
aspect, a unit dose device of the invention comprises a sealed container, such
as an
ampoule, containing less than about 2.0 ml of a liquid macrolide antibiotic
formulation comprising from about 50 to about 150 mg/ml of a macrolide
antibiotic
in a physiologically acceptable liquid carrier. Alternatively, the container
of the unit
dose device may contain less than about 1.5 ml, or less than about 1.0 ml, of
the liquid
macrolide antibiotic formulation, and the macrolide antibiotic formulation may
comprise from about 80 to about 180 mg/ml, or from about 90 to about 120
mg/ml, of
macrolide antibiotic. In another aspect, a unit dose device of the invention
comprises
a sealed container, such as an ampoule, containing a dry powder macrolide
antibiotic
formulation comprising from about 20 to about 250 mg of a macrolide antibiotic
in a
physiologically acceptable dry powder carrier. The sealed unit dose containers
of the
invention are preferably adapted to deliver the macrolide antibiotic
formulation to a
high efficiency inhalation device for aerosolization and inhalation by a
subject.
Brief Description of the Drawings
The foregoing aspects and many of the attendant advantages of this invention
will become more readily appreciated as the same becomes better understood by
reference to the following detailed description, when taken in conjunction
with the
accompanying drawings, wherein:
FIGURE 1 illustrates the chemical structure of erythromycylamine and
dirithromycin;
FIGURE 2 is a graphical representation of the stability of erythromycylamine
hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0,
and
7.0, at 4 degrees centigrade, as described in Example 4;
FIGURE 3 is a graphical representation of the stability of erythromycylamine
hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0,
and
7.0, at 25 degrees centigrade, as described in Example 4;
FIGURE 4 is a graphical representation of the stability of erythromycylamine
hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0,
and
7.0, at 40 degrees centigrade, as described in Example 4;
FIGURE 5 is a graphical representation of the stability of erythromycylamine
hydrochloride in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0,
and
7.0, at 60 degrees centigrade, as described in Example 4;
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_g_
FIGURE 6 is a graphical representation of the stability of erythromycylamine
sulfate in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and
7.0, at 60
degrees centigrade, as described in Example 4;
FIGURE 7 is a graphical representation of the stability of erythromycylamine
acetate in aqueous solution at 60, 100, and 150 mg/mL and pH 5.0, 6.0, and
7.0, at 60
degrees centigrade, as described in Example 4;
FIGURE 8 illustrates mean plasma concentrations of erythromycylamine
following a single 25 mg/kg intravenous dose, or single inhalation dose of 30
or 60
mg/ml solution for 30 minutes (0.7 or 1.77 mg/kg pulmonary dose) in rats
(n=3), as
described in Example 6;
FIGURE 9 illustrates mean lung concentrations of erythroniycylamine
following a single 25 mg/kg intravenous dose, or single inhalation dose of 30
or 60
mg/ml solution for 30 minutes (0.7 or 1.77 mg/kg pulmonary dose) in rats
(n=3), as
described in Example 6.
FIGURE 10 illustrates efficacy of erythromycylamine in the S, pneumoniae
pulmonary infection model after 30 minute inhalation administration daily for
three
(3) days to rats (n = 3) and comparing 5 mg/mL (0.13 mg/kg), 25 mg/mL (0.27
mg/kg), and 50 mg/mL (1.3 mg/kg) inhalation dose as described in Example 7;
and
FIGURE 11 illustrates efficacy of erythromycylamine in the S, pneumoniae
pulmonary infection model after 30 minute inhalation administration as a
single dose
to rats (n = 3) and comprising 1 mg/mL (0.03 mg/kg), 5 5 mg/mL (0.13 mg/kg),
mg/mL (0.27 mg/kg), and 50 mg/mL (1.3 mg/kg) inhalation dose as described in
Example 8.
FIGURE 12 illustrates the mean plasma and whole lung concentrations of
25 erythromycylamine following a single dose, 30 minute inhalation
administration of a
60 mg/mL sulfate solution in dogs, as described in Example 9.
FIGURE 13 illustrates the mean lung concentrations of erythromycylamine in
individual lung lobes following a single dose, 30 minute inhalation
administration of
a 60 mg/mL sulfate solution in dogs as described in Example 9.
Detailed Description of the Preferred Embodiment
Erythromycylamine and dirithromycin are macrolides having a chemical
structure depicted in FIG. 1. Dirithromycin, a prodrug of erythromycylamine,
is a
broad-spectrum macrolide antibiotic used for treatment of AECB and pneumonia.
Macrolide antibiotics useful in the present invention include, for example,
erythromycylamine, dirithromycin (a prodrug of erythromycylamine),
erythromycin
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A, clarithromycin (6-O-methyl erythromycin), azithromycin, and roxithromycin.
Other newer macrolides such as the ketolides (for example, ABT-773 (39t''
ICAAC
(1999), September 26-29, abstracts F-2133-2141, and HMR-3647 (Drugs ' of the
Future, 23, 591 (1998), 38~' ICAAC (1998), September 24-27, abstract A-49),
and
anhydrolides (see, J. Med. Chem., 1998, 41, 1651-1659 and 1660-1670) may also
be
used in the practice of the invention. In one aspect of the present invention,
the
macrolide antibiotic used in the aerosol formulations described herein is
erythromycylamine or dirithromycin. Erythromycylamine and dirithromycin have
the
chemical structures depicted in FIG. 1.
In accordance with the present invention, methods are provided for the
treatment of a subject in need of treatment, such as a subject suffering from
an
endobronchial infection, comprising administering to the subject by inhalation
an
antibacterially effective amount of a macrolide antibiotic formulation. This
aspect of
the invention is particularly suitable for formulation of concentrated
macrolides, such
as erythromycylamine, for aerosolization by small volume, breath actuated,
high
output rate and high efficiency inhalers to produce a m~crolide aerosol
particle size
between 1 and 5 ~,m desirable for efficacious delivery of the macrolide into
the
endobronchial space to treat susceptible microbial infections. . The
formulations
preferably contain minimal yet efficacious amounts of the macrolide formulated
in
small volumes of a physiologically acceptable solution. For example, an
aqueous
solution having a salinity adjusted to permit generation of macrolide aerosol
particles
that are well-tolerated by patients but prevent the development of secondary
undesirable side effects such as bronchospasm and cough. By way of example, a
quarter normal saline solution is useful for this purpose. By the more
efficient
administration of the macrolide formulation provided by the present invention,
substantially smaller volumes of macrolide than the conventional
administration
regime are administered in substantially shorter periods of time, thereby
reducing the
costs of administration and drug waste, and significantly enhancing the
likelihood of
patient compliance.
Thus, in accordance with one aspect of- the present invention, methods are
provided for the treatment of a subject in need of treatment, such as a
subject
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suffering from a susceptible endobronchial infection, comprising administering
to the
subject for inhalation a dose of a nebulized aerosol formulation comprising
from
about 50 to about 750 mg of a macrolide and a pharmaceutically acceptable
carrier.
In other aspects of the invention, the aerosol formulations administered in
the practice
of the invention may be liquid formulations comprising from about 50 to about
150
mg/ml of a macrolide antibiotic, preferably from about 70 to about 130 mg/ml
of a
macrolide antibiotic, and more preferably from about 90 to about 110 mg/ml of
a
macrolide antibiotic. Preferably, small volumes of aerosol formulation are
administered to the subject. Thus, in this aspect a dose of less than about
2.0 ml of a
nebulized liquid aerosol formulation is administered to the subject. In
another aspect,
a dose of less than about 1.5 ml of a nebulized aerosol formulation is
administered to
the subject. In yet another aspect, a dose of less than about 1.0 ml of a
nebulized
aerosol formulation is administered to the subject.
In other aspects, the macrolide compounds of the invention may be formulated
for aerosol delivery as a dry powder. As used herein, the term "powder" means
a
composition that consists of finely dispersed solid particles that are free
flowing and
capable of being readily dispersed in an inhalation device and subsequently
inhaled by
a subject so that the particles reach the lungs to permit penetration and
deposition in
the peripheral airways. Thus, powder formulations of the invention are said to
be
"respirable." Preferably the average powder particle size is less than about
10 pm in
diameter with a relatively uniform spheroidal shape. More preferably the
diameter is
less than about 7.5 ~,m and most preferably less than about 5.0 p.m. Usually
the
particle size distribution is between about 0.1 pm and about 5 p.m in
diameter,
particularly about 1 ~,m to about 5 p,m. Dry powder formulations of the
invention
have a moisture content such that the particles are readily dispersible in an
inhalation
device to form an aerosol. This moisture content will generally be below about
10%
by weight (% w) water, usually below about 5% w water and preferably less than
about 3% w water.
Dry powder formulations of the invention generally comprise a therapeutically
effective amount of a macrolide compound of the invention together with a
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pharmaceutically acceptable carrier. The dry powder formulations of the
invention
may comprise from about 25 to about 250 mg of a macrolide antibiotic,
preferably
from about 50 to about 200 mg of a macrolide antibiotic, and more preferably
from
about 75 to about 150 mg of a macrolide antibiotic. In this aspect of the
invention,
the dry powder formulations may comprise from about 50% to about 90% by weight
of the macrolide antibiotic, preferably from about 60% to about 88% by weight
of the
macrolide antibiotic, and more preferably from about 75% to about 85% by
weight of
the macrolide antibiotic.
Suitable pharmaceutically acceptable carriers include carriers that can be
taken
into the lungs of a patient with no significant adverse toxicological effects
on the
lungs, including, for example, stabilizers, bulking agents, buffers, salts and
the like.
A sufficient amount of the pharmaceutically acceptable carrier is employed to
obtain
desired stability, dispersibility, consistency and bulking characteristics to
ensure a
uniform pulmonary delivery of the composition to a subject in need thereof.
The
actual amount of pharmaceutically acceptable carrier employed may be from
about
0.05% w to about 99.95% w. More preferably, from about 5% w to about 95% w of
the pharmaceutically acceptable carrier will be used. Most preferably, from
about
10% w to about 90% w of the pharmaceutically acceptable carrier will be used.
Pharmaceutical excipients useful as carriers in this invention include
stabilizers such as human serum albumin (HSA), bulking agents such as
carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts
such as
sodium chloride; and the like. These carriers may be in a crystalline or
amorphous
form or may be a mixture of the two. Preferred bulking agents include
compatible
carbohydrates, polypeptides, amino acids or combinations thereof. Suitable
carbohydrates include monosaccharides such as galactose, D-mannose, sorbose,
and
the like; disaccharides, such as lactose, trehalose, and the like;
cyclodextrins, such as
2-hydroxypropyl-(3-cyclodextrin; and polysaccharides, such as raffinose,
maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol,
and the like.
A preferred group of carbohydrates includes lactose, threhalose, raffinose
maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino
acids
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include alanine and glycine, with glycine being preferred. Additives, which
may be
included as minor components of the dry powder formulations of the invention,
may
be included for conformational stability during spray drying and for improving
dispersibility of the powder. These additives include hydrophobic amino acids
such
tryptophan, tyrosine, leucine, phenylalanine, and the like. Suitable pH
adjusters or
buffers include organic salts prepared from organic acids and bases, such as
sodium
citrate, sodium ascorbate, and the like; sodium citrate is preferred.
In other aspects, the present invention relates to concentrated macrolide
formulations, such as a concentrated erythromycylamine formulation, suitable
for
efficacious delivery of the macrolide by aerosolization into endobronchial
space. The
invention is suitable for formulation of concentrated erythromycylamine for
aerosolization by jet, vibrating porous plate, ultrasonic or dry powder
nebulizers to
produce erythromycylamine aerosol particle size between l and 5 ~,m preferable
for
efficacious delivery of erythromycylamine into the endobronchial space to
treat
Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus
Moraxella catarrhalis and Legionella pneumonia, Chlamydia . pneumoniae, and
Mycoplasma pneumoniae infections. The formulations preferably contain minimal,
yet efficacious amounts of erythromycylamine formulated in a relatively small
volume of physiologically acceptable solution having a salinity, or a dry
powder,
adjusted to permit generation of an erythromycylamine aerosol that is well-
tolerated
by patients but preventing the development of secondary undesirable side
effects such
as bronchospasm and cough.
Primary requirements for any aerosolized formulation are its safety and
efficacy. Additional advantages are lower treatment cost, practicality of use,
long
shelf life, storage and optimization of nebulizer.
The aerosol formulation is nebulized predominantly into particle sizes which
can be delivered to the terminal and respiratory bronchioles where the
Streptococcus
pneumoniae, Haemoplailus influenzae, Staphylococcus aureus, and Moraxella
catarrhalis and the atypical bacteria Legionella pneumonia, Chlamydia
pneumoniae,
and Mycoplasma pfaeumoniae or other susceptible bacteria reside in patients
with
chronic bronchitis and pneumonia. Streptococcus pneumoniae, Haemophilus
influenzae, Staphylococcus aureus, Moraxella catarrhalis, Legionella
pneumonia,
Chlamydia pneumoniae, and Mycoplasma pneumoniae are present throughout the
airways including the bronchi, bronchioli and lung parenchema. However, they
are
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most predominant in terminal and respiratory bronchioles. During exacerbation
of
infection, bacteria can also be present in alveoli. Therefore, in one aspect,
the present
invention provides a formulation that is delivered throughout the
endobronchial tree
to the terminal bronchioles and eventually to the parenchymal tissue.
Aerosolized erythromycylamine formulation is formulated for efficacious
delivery of erythromycylamine. to the lung endobronchial space. A specific
jet,
vibrating porous plate or ultrasonic nebulizer is selected to allow the
formation of an
erythromycylamine aerosol particles with a mass median aerodynamic diameter
predominantly between 1 to 5 ~,m. The formulated and delivered amount of
erythromycylamine is efficacious for treatment and/or prophylaxis of
endobronchial
infections, particularly those caused by the bacteria Streptococcus
pneumoniae,
Haemophilus influenzae, Staphylococcus aureus, and Moraxella cataf~rhalis and
the
atypical pneumonias Legionella pneumonia, Chlamydia pneumoniae, and
Mycoplasma pneumoniae. The formulation has salinity adjusted to permit
generation
of erythromycylamine aerosol well tolerated by patients. Further, the
formulation has
suitable osmolarity. The formulation has a small aerosolizable volume and is
able to
deliver an effective dose of erythromycylamine to the site of the infection.
Additionally, the aerosolized formulation does not impair negatively the
function of
the airways by causing undesirable side effects.
The antibiotic formulation may be administered with the use of an inhalation
device having a relatively high rate of aerosol output, high emitted dose
efficiency,
and emission limited to periods of actual inhalation by the patient. Thus,
while
conventional air jet nebulizers exhibit a rate of aerosol output on the order
of 3 ~,l/sec,
inhalation devices useful for use in the practice of the present invention
will typically
exhibit a rate of aerosol output of not less that about 5 ~.1/sec, more
preferably not less
than about 6.5 ~1/sec, and most preferably not less than about 8 ~l/sec. In
addition,
while conventional air jet nebulizers have a relatively low emitted dose
efficiency and
typically release about 55% (or less) of the nominal dose as aerosol,
inhalation
devices useful for use in the practice of the present invention will typically
release at
least about 75%, more preferably at least about 80% and most preferably at
least
about 85% of the loaded dose as aerosol for inhalation by the subject. In
other
aspects, conventional air jet nebulizers typically continually release
aerosolized drug
throughout the delivery period, without regard to whether the subject is
inhaling,
exhaling or in a static portion of the breathing cycle, thereby wasting a
substantial
portion of the loaded drug dose. In contrast, preferred inhalation devices for
use in
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the present invention will be breath actuated, and restricted to delivery of
aerosolized
particles of the macrolide formulation to the period of actual inhalation by
the subject.
A representative inhalation device meeting the above criteria and suitable for
use in
the practice of the invention is the Aerodose inhaler, available from Aerogen,
Inc.,
Sunnyvale, California. The Aerodose~ inhaler generates an aerosol using a
porous
membrane driven by a piezoelectric oscillator. Aerosol delivery is breath
actuated,
and restricted to the inhalation phase of the breath cycle, i.e.,
aerosolization does not
occur during the exhalation phase of the breath cycle. The airflow path design
allows
normal inhale-exhale breathing, compared to breath-hold inhalers.
Additionally, the
Aerodose'T' inhaler is a hand-held, self contained, and easily transported
inhaler.
Although piezoelectric oscillator aerosol generators, such as the Aerodose'~''
inhaler,
are presently preferred for use in the practice of the invention, other
inhaler or
nebulizer devices may be employed that meet the above performance criteria and
are
capable of delivering the small dosage volumes of the invention with a
relative high
effective deposition rate in a comparatively short period of time.
In other aspects of the present invention, unit dose formulations and devices
are provided for administration of a macrolide antibiotic formulation to a
subject with
an inhaler, in accordance with the methods of the invention as described
supra.
Preferred unit dose devices comprise a container designed to hold and store
the
relatively small volumes of the macrolide antibiotic formulations of the
invention, and
to deliver the formulations to an inhalation device for delivery to a patient
in aerosol
form. In one aspect, unit dose containers of the invention comprise a plastic
ampoule
filled with a macrolide antibiotic formulation of the invention, and sealed
under sterile
conditions. Preferably, the unit dose ampoule is provided with a twist-off tab
or other
easy opening device for opening of the ampoule and delivery of the macrolide
antibiotic formulation to the inhalation device. Ampoules for containing drug
formulations are well known to those skilled in the art (see, for example,
U.S. Patent
Nos. 5,409,125, 5,379,898, 5,213,860, 5,046,627, 4,995,519, 4,979,630,
4,951,822,
4,502,616 and 3,993,223, the disclosures of which are incorporated herein by
this
reference). The unit dose containers of the invention may be designed to be
inserted
directly into an inhalation device of the invention for delivery of the
contained
macrolide antibiotic formulation to the inhalation device and ultimately to
the subject.
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In accordance with this aspect of the invention, a unit dose device is
provided
comprising a sealed container containing less than about 5.0 ml, preferably
less than
about 3.0 ml and most preferably less than about 2.0 ml of a liquid macrolide
antibiotic formulation comprising from about 50 to about 150 mg/ml of a
macrolide
antibiotic in a physiologically acceptable carrier, the sealed container being
adapted to
deliver the macrolide antibiotic formulation to an inhalation device for
aerosolization.
Suitable macrolide antibiotics for use in connection with this. aspect of the
invention
include those macrolide antibiotics described in detail, sup~~a. In a
presently preferred
embodiment, the macrolide antibiotic employed in the unit dose devices of the
IO invention is erythromycylamine. In other aspects of the invention, the unit
dose
devices of the invention may contain a liquid macrolide antibiotic formulation
comprising from about 70 to about 130 mg/ml of macrolide antibiotic. In yet
other
aspects of the invention, the unit dose devices of the invention may contain a
liquid
macrolide antibiotic formulation comprising from about 90 to about 110 mg/ml
of
macrolide antibiotic.
In preferred liquid unit dose formulations of the invention, the
physiologically
acceptable carrier may comprise a physiological saline solution such as a
solution of
one quarter strength of normal saline, having a salinity adjusted to permit
generation
of erythromycylamine aerosol well-tolerated by patients but to prevent
substantially
the development of secondary undesirable side effects such as bronchospasm and
cough.
In yet other aspects of the invention, dry powder formulations of the
invention
are placed within a suitable unit dose receptacle in an amount sufficient to
provide a
subject with a macrolide antibiotic compound of the invention for a unit
dosage
treatment by dry powder inhalation. Preferred 'dry powder unit dosage
receptacles fit
within a suitable inhalation device to allow for the aerosolization of the
macrolide-
based dry powder composition by dispersion into a gas stream to form an
aerosol and
then capturing the aerosol so produced in a chamber having a mouthpiece
attached for
subsequent inhalation by a subject in need of treatment. Such a dosage
receptacle
includes any container enclosing the formulations known in the art such as
gelatin or
plastic capsules with a removable portion that allows a stream of gas (e.g.,
air) to be
directed into the container to disperse the dry powder formulation. Such
containers
are exemplified by those shown in U.S. Patent Nos. 4,227,522, 4,192,309, and
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4,105,027. Suitable containers also include those used in conjunction with
Glaxo's
Ventolin Rotohaler brand powder inhaler or Fison's Spinhaler brand powder
inhaler.
Another suitable unit-dose container which provides a superior moisture
barrier is
formed from an aluminum foil plastic laminate. The macrolide powder is filled
by
weight or by volume into the depression in the formable foil and hermetically
sealed
with a covering foil-plastic laminate. Such a container for use with a powder
inhalation device is described in U.S. Pat. No. 4,778,054 and is used with
Glaxo's
Diskhaler® (LJ.S. Patent Nos. 4,627,432, 4,811,731; and 5,035,237). All of
these
references are incorporated herein by reference.
In accordance with this aspect of the invention, a unit dose device is
provided
comprising a sealed container containing a dry powder formulation comprising
from
about 25 to about 250 mg of a macrolide antibiotic, preferably from about 50
to about
200 mg of a macrolide antibiotic, and more preferably from about 75 to about
150 mg
of a macrolide antibiotic in a physiologically acceptable dry powder carrier,
the sealed
container being adapted to deliver the macrolide antibiotic formulation to an
inhalation device for aerosolization. In this aspect of the invention, the dry
powder
formulations may comprise from about 50% to about 90% by weight of the
macrolide
antibiotic, preferably from about 60% to about 88% by weight of the macrolide
antibiotic, and more preferably from about 75% to about 85% by weight of the
macrolide antibiotic.
Aerosol Erythromycylamine Formulation
In order to assess the stability of erythromycylamine in aqueous solutions
three salt forms of the antibiotic were prepared and submitted to varying
conditions of
temperature, time, concentration, and pH. Erythromycylamine concentrations
were
determined by HPLC methodology. The data from these stability studies are
shown
in Figures 2-7 and several important findings are revealed. First, the
stability of
erythromycylamine hydrochloride as expected, was directly proportional to
temperature of the solution (see Figures 2-5). Second, erythromycylamine
solutions
were more stable at neutral pH 7 than acidic pH 5 and 6 (Figure 5). This
result is
consistent with the known effects of pH on the degradation of macrolide
antibiotics.
One of the main degradation pathways is loss of the neutral sugar, cladinose
(see J.
Chrom. A, 812m 1998, 255-286). Third, solutions of erythromycylamine acetate
were more stable at pH 6 and 7 than the corresponding hydrochloride and
sulfate salts
at the same pH (compare Figure 7 to Figure 5 and 6).
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Liquid and dry powder formulations according to the invention contain from
about 50 to about 750 mg, preferably from about 75 to about 600 mg, and most
preferably from about 100 to about 500 mg of a macrolide antibiotic drug, such
as
erythromycylamine acetate, per dose. This corresponds to minimal yet
efficacious
amounts of erythromycylamine to suppress Streptococcus pneumoniae, Haemophilus
influenzae, Staphylococcus am eus, Moraxella catarrhalis, Legionella
pneumonia,
Chlamydia pneumoniae, and Mycoplasma pneumoniae infections in the
endobronchial space.
Presently preferred liquid aerosol erythromycylamine formulations according
to the invention comprise from about 90 to about 110 mg of erythromycylamine
sulfate per 1 mL of quarter normal saline. This corresponds to a
representative
efficacious amount of erythromycylamine to suppress bacterial infections of
AECB.
Both patients and aerosol generating devices are sensitive to the osmolarity,
pH, and ionic strength of the formulation. It has now been discovered that
this
problem is conveniently solved by formulating erythromycylamine solutions in
quarter normal saline, that is saline containing 0.225% of sodium chloride,
and that
quarter normal saline is a suitable vehicle for delivery of erythromycylamine
into the
endobronchial space.
Chronic bronchetic patients and other patients with chronic endobronchial
infections have a high incidence of bronchospastic or asthmatic airways. These
airways axe sensitive to hypotonic and hypertonic aerosols, to the
concentration of a
permeant ion, particularly a halide such as chloride, as well as to aerosols
that are
acidic or basic. The effects of irritating the airways can be clinically
manifested by
cough or bronchospasm. Both of these conditions can prevent efficient delivery
of
aerosolized erythromycylamine into the endobronchial space.
The erythromycylamine acetate, hydrochloride, and sulfate formulation
containing 60-100 mg of erythromycylamine per ml of quarter normal saline has
an
osmolarity in the range of 130-400 mOsm/kg. This is within the safe range of
aerosols administered to a chronic bronchitis patient (Table 1).
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TABLE 1
Osmolality of Erythromycylamine Solutions as a Function of Salt Form, pH, and
Concentration: Experimental and Theoretical Results
Conc. Experimental Theoretical
Salt pH (mg/ml) mOsm/k mOsm/k
Acetate 5.0 60 300 245
100 518 408
150 840 613
Acetate 6.0 60 365
100 639
150 701
Acetate 7.0 60 501
100 891
150 746
Hydrochloride5.0 60 227 245
100 382 408
150 601 613
Hydrochloride6.0 60 224
100 382
150 598
Hydrochloride7.0 60 226
100 386
150 594
Sulfate 5.0 60 130 163
100 239 272
150 396 409
Sulfate 6.0 60 132
100 238
150 395
Sulfate 7.0 60 132
100 241
150 391
The pH of the formulation is equally important for aerosol delivery. As noted
previously, when the aerosol is either acidic or basic, it can cause
bronchospasm and
cough. The safe range of pH is relative; some patients will tolerate a mildly
acidic
aerosol that in others will cause bronchospasm. Any aerosol with a pH of less
than
4.5 usually will induce bronchospasm in a susceptible individual; aerosols
with a pH
between 4.5 and 5.0 will occasionally cause this problem. An aerosol with a pH
between 5.0 and 7.0 is considered to be safe. Any aerosol having pH greater
than
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10.0 is to be avoided since irntation resulting in bronchospasm may occur. The
optimum pH for the aerosol formulation was determined to be between pH 5.0 and
7Ø
In one aspect, liquid formulations of the invention are preferably nebulized
predominantly into particle sizes allowing a delivery of the drug into the
terminal and
respiratory bronchioles and lower airways~where the bacteria reside. For
efficacious
delivery of erythromycylamine to the lung endobronchial airway space by
aerosol, the
formation of aerosol particles having a mass median aerodynamic diameter
predominantly between 1 to 5 ~m is necessary. The formulated and delivered
amount
of erythromycylamine for treatment and prophylaxis of endobronchial
infections,
particularly those caused by the bacteria Streptococcus pneumoniae,
Haemophilus
influenzae, Staphylococcus am°eus, Mof-axella eatars°halis,
Legio~zella pneumonia,
Chlamydia pneumoniae, and Mycoplasma pheumohiae, must effectively target the
endobronchial surface. Delivered doses of the formulations preferably have the
smallest practical aerosolizable volume able to deliver an effective dose of
erythromycylamine to the site of the infection. Preferred formulations
additionally
provide conditions that do not adversely affect the functionality of the
airways.
Consequently, preferred formulations contain a sufficient amount of the drug
formulated under conditions that allow its efficacious delivery, while
avoiding
undesirable reactions. The new formulations according to the invention meet
all these
requirements.
According to the invention, erythromycylamine is formulated in a dosage form
intended for inhalation therapy by patients with chronic bronchitis and
pneumonia.
Since the patients reside throughout the world, it is desirable that the
formulation has
reasonably long shelf life. Storage conditions and formulation stability thus
become
important.
As discussed above, the pH of the solution is important. A pH between 5.0
and 7.0, preferably about 6.0, is optimal from the storage and longer shelf
life point of
view.
The formulation is typically stored in a one- to two-milliliter low-density
polyethylene (LDPE) vials. The vials are aseptically filled using a blow-fill-
seal
process. The vials are sealed in foil overpouches.
Stability of the formulation with respect to oxidation is arxother very
important
issue. If the drug is degraded before aerosolization, a smaller amount of the
drug is
delivered to the lung, thus impairing'the treatment as well as provoking
conditions
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that could lead to the development of resistance to erythromycylamine, because
the
delivered dose would be too small. Moreover, erythromycylamine degradation
products may provoke bronchospasm and cough. To prevent oxidative degradation
of
erythromycylamine and in order to provide acceptable stability, a product with
low
oxygen content is produced by packaging the LDPE vials in oxygen-protective
packaging comprising foil overpouches, six vials per overpouch. Prior to vial
filling,
the solution in the mixing tank is nitrogen sparged and the annular overpouch
headspace is nitrogen purged. In this way, both hydrolysis and oxidation of
erythromycylamine is prevented.
II. Aerosolization Devices
Aerosolization devices, such as a jet, vibrating porous plate or ultrasonic
nebulizers, useful in the practice of the invention are generally able to
nebulize the
formulation of the invention into aerosol particles predominantly in the range
from 1
5 ~,m. Predominantly in this application means that at least 70% but
preferably more
than 90% of all generated aerosol particles are within 1-5 ~m range.
Nebulizers such as jet, ultrasonic, vibrating porous plate, and energized dry
powder inhalers, that can produce and deliver particles between the 1 and 5 ~m
particle size that is optimal for treatment of Streptococcus pheumohiae,
Haemophilus
influenzae, Staphylococcus auf°eus, Moraxella cataf~rhalis Legiohella
pheumonia,
Chlamydia pheumouiae, and Mycoplasma pneumouiae infections, are currently
available or can be produced using known methods and materials. A jet
nebulizer
works by air pressure to break a liquid solution into aerosol droplets.
Vibrating
porous plate nebulizers work by using a sonic vacuum produced by a rapidly
vibrating
porous plate to extrude a solvent droplet through a porous plate. An
ultrasonic
nebulizer works by a piezoelectric crystal that shears a liquid into small
aerosol
droplets. However, only some formulations of erythromycylamine can be
efficiently
nebulized by these three nebulizers, as these devices are sensitive to the pH
and ionic
strength of the formulation.
While a vaxiety of devices are available, only a limited number of these
nebulizers are suitable for the purposes of this invention. Preferred
nebulizers useful
in the present invention include, for example, AeroNeb~ and AeroDose~
vibrating
porous plate nebulizers (AeroGen, Inc., Sunnyvale, California), Sidestream~
nebulizers (Medic-Aid Ltd., West Sussex, England), Pari LC Plus~ and Pari LC
Star~ jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Virginia),
and
~ Aerosonic~ (DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden,
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Germany) and UltraAire~ (Omron Healthcare, Inc., Vernon Hills, Illinois)
ultrasonic
nebulizers.
III. Aerosol Pharmacokinetics
Solutions of erythromycylamine were administered to rats by the IV and
inhalation routes and drug concentrations in plasma and lung were measured.
Data
from these studies is shown in Figures 8 and 9. Two dose levels were selected
for the
inhalation delivery route, 1.7 and 0.7 mg/kg, and were compared to a single
intravenous dose (25 mg/kg).
Dose normalized AUC of erythromycylamine in the lung for IV (25 mg/kg),
inhalation (1.7 mg/kg), and (0.7 mg/kg) was 24.21, 1067.84 and 848.34
~g~h/gram,
respectively. Therefore, by administering erythromycylamine directly to the
lung via
inhalation route, lung drug levels achieved were approximately 40 times higher
on a
milligram basis than by the intravenous route of delivery. Thus, antibiotic
therapy by
inhalation should be more efficacious than treatment by oral or IV routes.
IV. Aerosol Efficacy
Erythromycylamine was very effective by both intravenous and aerosol
administration. At the lowest dose tested (10 mg/kg per day) intravenous
erythromycylamine reduced the lung burden of S. pheumohia to below the limits
of
detection (10 CFU/gram of lung) as shown in Example 7. Aerosol was also very
effective (see Figure 10) with only detectable recovery of S. pneumonia at 5
mg/ml
aerosol solution (calculated dose 0.13 mg/kg per day). In addition,
erythromyclamine
was very effective when administered as a single aerosol dose at
concentrations
greater than those required for single daily doses for 3 days. A single dose
of 0.13
mg/kg was less effective (less than 2 orders of magnitude reduction in
CFU/gram)
compared with 0.13 mg/kg for three consecutive days (5 orders of magnitude
reduction). However, a single dose of 0.67 mg/kg achieved almost complete
clearance of the organism from lung tissue, an efFect similar to the multiple
dose
efficacy indicating that at this concentration, the second and third doses
added little
value (see Figure 11).
The pharmacolcinetic evaluation of aerosolized erythromycylamine suggests,
and the efficacy data indicates, that equivalent lung concentrations to
multiple daily
IV, oral, or aerosol doses can be achieved by a single aerosol dose and that
the single
dose would be about 3-5 fold greater than required for similar effectiveness
as three
daily doses.
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Utili
One aspect of the utility of this invention is that small volume, high
concentration formulations of macrolide antibiotics, such as
erythromycylamine, can
be used with suitable nebulizers to deliver an efficacious dose of
erythromycylamine
to the endobronchial space in people with chronic bronchitis, bronchiectasis,
and
pneumonia caused by macrolide susceptible bacteria or other infections. The
formulation is safe and very cost effective. Furthermore, the formulations may
be
kept in a nitrogen environment, with pH controhed for tolerance, to provide
adequate
shelf life for commercial distribution.
EXAMPLE 1
General Procedure For The Preparation Of Erythromycylamine Salts:
Synthesis Of Erythromycyclamine Acetate
To a solution of 10.0 g (13.6 mmol) of erythromyclamine in 100mL of MeOH
cooled in an ice bath was added dropwise 1.56 mL (27.2 mmol, 2.0 eq) of
glacial
acetic acid. The solution was warmed to ambient over a period of 30 min, then
the
solvent removed under reduced pressure. EtzO (50 mL) was added and the slurry
concentrated. This was repeated to provide 11.52 g (96.9%) of erythromyclamine
acetate monohydrate as a white powder; IR (I~Br, cm 1) 1718, 1560, 1406, 1168,
1080, 1055, 1012; 'H NMR (400 MHz, CD30D) 8 0.89 (t, 3H, J = 7.2 Hz), 1.06-
1.32
(m, 27H), 1.35-1.47 (m, 4H), 1.52-1.66 (m, 3H), 1.85-2.02 (m, 8H), 2.03-2.26
(m,
2H), 2.45-2.49 (m, 1H), 2.66-2.77 (m, SH), 2.91-3.09 (m, 3H), 3.21-3.40 (m,
6H),
3.58 (d, 1H, J = 7.0 Hz), 3.67 (s, 1H), 3.78-3.83 (m, 2H), 4.10-4.13 (m, 1H),
4.59 (d,
1H, J = 7.0 Hz), 4.88-5.01 (m, 12H); MS mlz 735.6 (M+-2AcOH-2Hz0); KF 2.33
H20.
Anal. Calcd for C4,H$oN201~: C, 56.40; H, 9.24; N, 3.21. Found: C, 56.38; H,
9.21; N, 3.16.
EXAMPLE 2
Synthesis Of Erythromycylamine Sulfate
To a solution of 10.0 g (13.6 mmol) of erythromyclamine in 100mL of MeOH
cooled in an ice bath was added dropwise 0.73 mL (13.6 mmol, 1.0 eq) of
concentrated sulfuric acid. The solution was warmed to ambient over a period
of 30
min, then the solvent removed under reduced pressure. Et20 (50 mL) was added
and
the slurry concentrated. This was repeated to provide 11.13 g (96.1 %) of
erythromyclamine sulfate monohydrate as a white powder; IR (KBr, cTri') 1718,
1384, 1168, 1122, 1078, 1012; 1H NMR (400 MHz, CD30D) 8 0.89 (t, 3H, J = 7.2
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Hz), 1.08-1.32 (m, 27H), 1.45-1.63 (m, 7H), 1.89-2.04 (m, 2H), 2.23-2.31 (m,
2H),
2.44-2.47 (m, 1H), 2.84-2.89 (5H), 2.99-3.07 (m, 3H), 3.30-3.49 (m, 6H), 3.58
(d,
1H, J = 7.0 Hz), 3.69 (s, 1H), 3.78-3.86 (m, 2H), 4.09-4.11 (m, 1H), 4.60 (d,
1H, J =
6.8 Hz), 4.87-4.99 (m, 12H); MS mlz 735.7 (M+-HZSO4 2H20); KF 2.93 % HaO.
Anal. Calcd for C3~H~4NZO16S: C, 52.22; H, 8.76; N, 3.29. Found: C, 52.55;
H, 8.91; N, 3.27.
EXAMPLE 3
Synthesis Of Erythromycylamine Hydrochloride
To a solution of 10.0 g (13.6 mmol) of erythromyclamine in 100mL of MeOH
cooled in an ice bath was added dropwise 2.34 mL (27.2 inmol, 2.0 eq) of 37%
hydrochloric acid. The solution was warmed to ambient over a period of 30 min,
then
the solvent removed under reduced pressure. Et20 (50 mL) was added and the
slurry
concentrated. This was repeated to provide 11.24 g (97.9%) of erythromyclamine
hydrochloride dihydrate as a white powder; IR (KBr, crri') 1718, 1466, 1383,
1170,
1078, 1055, 1011; 1H NMR (400 MHz, CD30D) 8 0.87-0.91 (m, 3H), 1.10-1.31 (m,
27H), 1.43-1.65 (m, 7H), 1.89-2.01 (m, 2H), 2.25-2.27 (m, 2H), 2.45-2.48 (m,
1H),
2.82-3.10 (m, 8H), 3.34-3.42 (m, 8H), 3.57-3.58 (m, 1H), 3.67 (s, 1H), 3.80-
3.82 (m,
2H), 4.08-4.11 (m, 1H), 4.61-5.00 (m, 13H); MS m/z 735.6 (M+-2HCl-2H20); I~F
4.38 % HZO.
Anal. Calcd for C3~H~6C12Nz0,z: C, 52.65; H, 9.08; N, 3.32. Found: C, 52.21;
H, 9.18; N, 3.20.
EXAMPLE 4
Aqueous Formulation And Stability Of Erythromycylamine Salts
Preparation of Solutions Erythromyclamine (9.0g, 12.2 mM) free base was
added to a tared 100 mL Erlenmeyer flask . De-ionized water (25 mL) was added
to
the flask with agitation by magnetic stirrer. 1N sulfuric acid (24.5 mL, 2
equivalents)
was gradually added while stirring. When the solution was clear, it was
removed
from the stir plate and re-weighed. Deionized water was added dropwise to
obtain a
final.solution weight of 62.9g. The solution was divided into three 20 mL
portions,
and the pH was adjusted to the desired value (5.0, 6.0, or 7.0) by dropwise
addition of
1N sodium hydroxide or sulfuric acid while monitoring with a pH meter. The
above
procedure was used to prepare solutions at 100 mg/mL and 60 mg/mL by adjusting
the weight of erythromyclamine (6.0g and 3.6g) and the volume of 1N sulfuric
acid
(16.3 and 9.8 mL).
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Solutions of the acetate and hydrochloride salts of erythromyclamine at 150,
100
and 60 mg/mL were prepared as described above, except that 1N Acetic acid and
1N
Hydrochloric acid (2 equivalents) were added to prepare the salts and adjust
the pH.
Aliquots of each salt form at each concentration and each pH were stored at 4,
40,
and 60 °C, and at ambient temperature.
Stability Determination All solutions were analyzed immediately after
preparation (t = 0) and at 24 hours, 48 hours, eight days, 15 days and 22 days
following preparation, excepting that samples that appeared substantially
degraded at
eight days were omitted from subsequent analyses.
Refrigerated and heated samples were equilibrated to ambient temperature for
at least one hour prior to sample preparation. Final dilution volume for all
samples
was 10 mL. The diluent for all samples consists of an 80:20 (v/v) mixture of
50 mM
phosphate buffer at pH 6.5 and acetonitrile.
An appropriate amount of sample (40 microliters for a 150 mg/mL solution,
50 microliters for a 100 mg/mL, or 100 microliters for a 60 mg/mL solution)
was
transferred to a 20 mL scintillation vial. 10 mL of the diluent were added to
the vial
and mixed thoroughly.
Standard Preparation Standards were prepared in duplicate and used for a
maximum of three days. Ery-amine free base (30 mg) was transferred to a tared
50
mL volumetric flask and exact weight was recorded. Sample diluent (45 mL) is
added and sonicated briefly to dissolve. The standard was cooled and diluted
to
volume with diluent.
Sample and Standard Analysis Samples and standards were analyzed by
reversed-phase high performance liquid chromatography. A 250 x 4.6 mm
Phenomenex Luna CN column with 5 micron particle size was used to perform the
separation. All analyses were performed on an Agilent Technologies HP 1100
chromatography system, and the data were acquired and stored using an Agilent
Technologies ChemStation data system. Analytical parameters were as shown
below
in Table 2.
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Table 2
Flow Rate 1.0 mL/min
Column Temperature30 C
Injection Volume 20 ~L
Detector UV absorbance at
200 nm
Run Time 10 min
Mobile Phase A 50 mM Phosphate pH
2.1
Mobile Phase B Acetonitrile
Composition 80/20 A/B
EXAMPLE 5
Osmolality Of Erythromyclamine Salt Solutions
Three portions of erythromyclamine HCl salt (0.6g, 1.0g and 1.5g) were
weighed into separate 10 mL volumetric flasks. Easypure UV water (8 mL) was
added to each flask and sonicated until completely dissolved, then diluted to
volume.
This procedure was repeated for the Erythromyclamine sulfate and acetate. The
pH
and osmolality of each solution were measured, and the measured osmolality
compared to the theoretical values.
The salts prepared in Example 4 (4C) were
allowed to equilibrate to room
temperature, and the osmolality was measured.
The results are shown in Table 3:
Table 3
Erythromyclamine Salt Osmolality Study
Erythromyclamine HCl Salt Lot # TEM-702-171 843.91 g/m
dihydrate mw=
Theoretical Actual
Target Actual Osmolarity Osmolarity
Wei ht Wei ht mOsm mOsm ~H Comments
0.600 0.59957 279 213 7.57 wh.cloudy
1.00 1.00443 474 357 7.59 wh.cloudy
1.50 1.50258 733 534 7.60 wh.cloudy
Erythromyclamine Sulfate Salt Lot # TEM-702-169
monohydrate mw=883.14 g/m
0.600 0.60368 146 137 77.62 wh.cloudy
1.00 1.00430 258 227 77.63 wh.cloudy
1.50 1.49941 408 340 77.61 wh.cloudy
Erythromyclamine Acetate Salt Lot # TEM-702-167
monohydrate mw= 873.08 g/m
0.600 0.60189 g 195 207 6.62 s1. cloudy
1.00 1.00713 g 345 346 6.67 s1. cloudy
1.50 1.50178 g 552 516 6.62 s1. cloudy
(((wt. in grams/molecular wt )*number of species)/O.O1L)*(1000
mOsm/lOsm)= X
mOsm
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EXAMPLE 6
Aerosol Delivery Of Erythromycylamine To Rats:
Characterization Of Aerosol Pharmacokinetics
IV Pharmacokinetics: Erythromycylamine (2S0 mg) was dissolved in a S mL
S of DI water, and 12 mL of concentrated sulfuric acid was added. A solution
of dilute
sulfuric acid (1:10 v/v) was added gradually to the solution to dissolve the
drug
completely. The solution of dilute sulfuric acid was added gradually to bring
the pH
of solution to 6.8-7.2. By adding DI water, the total volume of solution was
brought
up to 8 mL. A 200 ~L solution of erythromycylamine sulfate (2S mg/kg) was
~ delivered to male Sprague-Dawley rats (Simonsen Laboratories, 1180 C Day
Road,
Gilroy, CA 95020) by intravenous administration via the lateral tail vein.
Animals
were anesthetized with 1-4% isoflurane and lung and blood samples were
collected
from 3 rats at 0.083, 0.25, O.S 1, 2, 4, 6, 8 and 24 hours post dosing. The
blood
samples were collected via cardiac puncture using heparin as an anticoagulant.
Lungs
1 S were removed surgically following blood sampling, and the bronchi and
trachea were
removed and discarded. The remaining lung tissue was processed as described
below.
Both the lung and blood samples were immediately placed on ice, and the blood
samples were centrifuged immediately following collection to harvest plasma
samples. Both lung and plasma samples were stored at -80°C until
assayed.
20' Erythromycylamine concentrations in plasma and the lung (per gram of lung
tissue) were determined using a validated LC-MS method. Plasma samples (100
~,g)
were spiked with oleandomycin (internal standard, 1 ~,g/mL) before extraction.
Plasma samples (100 ~,L) were deproteinated with 3.3% trichloroacetic acid
(TCA).
Samples were centrifuged (10,000 rpm, 10 min.) and the supernatant was
transferred
2S to HPLC centrifilter for centrifiltration (10,000 rpm, 10 min). The mobile
phase
consisted of 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) solution at a
flow rate
of O.S ml/min for 3 minutes, followed by 0.1% acetic acid-acetonitrile (60:40,
v/v) at
a flow rate of 0.8 ml/min for 3 minutes. A stainless steel analytical column
(Zorbax
SB-C18, 2.1 mm ID x 150.0 mm, S ~.m with a Phenomenex cartridge guard column)
30 was used as the stationary phase. The column temperature was SO° C.
Quantification
of the erythromycylamine was performed using a HP 1100 LC/MSD API-
Electrospray System. Data acquisition was set in the selective ion monitoring
mode.
'The method was linear (r>0.9990) in the concentration range of 0.01 to SO
~,g/ml.
The absolute recovery was 95.0 ~ 2.19%.
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Lung samples were homogenized with DI water. Oleandomycin was added to
the samples as an internal standard. The homogenate was deproteinated with 0.9
M
TCA. Samples were centrifuged at 10,000 rpm for 10 minutes and the supernatant
was transferred to HPLC centrifilters for centrifiltration. The mobile phase
consisted
of 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) at a flow rate of 0.5
ml/min for 3
minutes, followed by 0.1 % acetic acid-acetonitrile (60:40, v/v) at a flow
rate of 0.8
ml/min for 3 minutes. A stainless steel analytical column (Zorbax SB-C18, 2.1
mm
ID x 150.0 mm, 5 ~m with a Phenomenex cartridge guard column) was used as the
stationary phase. The column temperature was 50° C. Quantification of
the
erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray
System. Data acquisition was set in the selective ion monitoring mode. The
linearity
(r>0.9990) of the assay ranged from 0.1 to 200 pg/g. The extraction efficiency
was
93.8 ~ 2.54%.
Pharmacokinetic parameters, area under the curve (AUC) and mean residence.
time (MRT), were estimated based on the statistical moment theory using
WinNonlinTM Professional Version 2.0 software (Pharsight Corporation). The
peak
concentration (C~) was not estimated but observed.
Inhalation Pharmacokinetics: For a 60 mg/mL solution, 3.191 g (4.08 mmol)
of erythromycylamine (94% purity) was added to 43 mL of DI water and 4.27 mL
(4.27 mmol) of 1 M sulfuric acid in a 50 ml volumetric flask. The solution was
then
adjusted to pH 6.5 with the addition of another 53 ~,L (0.053 mmol) of 1 M
sulfuric
acid. The volume was brought up to 50 mL with additional DI water. The 30
mg/mL
solution was made by diluting the 60 mg/mL solution in 1/2 normal saline. The
. osmolality of the resulting solutions were 148 mOsm as determined using The
AdvancedTM Micro-Osmometer Model 3300 (Advanced Instruments, Inc., Norwood,
Mass.)
Rats were exposed once to either 30 or 60 mg/mL solution of
erythromycylamine sulfate via inhalation in a 32-port nose-only rodent
exposure
system (Battelle, Richland, WA) for 30 minutes. The Battelle system nose-only
rodent exposure system is based on the Cannon Flow-Past Nose only system (Am.
Ind
Hyg Assoc J 1983 Dec;44(12)923-8) and is made up of four stackable stain-less
steel
tiers with a total of 32 ports. The system includes inlet and exhaust flow
monitoring
and control, aerosol data was collected using the NORES version 1.1.4 software
provided by Battelle. Erythromycylamine solutions were aerosolized using the
PARI
LC STARTM nebulizer. Mean aerosol concentrations were determined by
gravimetric
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analysis of filter samples taken at 10 and 20 minutes following the start of
exposure.
The mean aerosol concentrations were 0.540.06 and 1.360.30 mg/L, respectively,
for 30 and 60 mg/mL solutions.
Lung and blood samples were collected from 3 rats at 0.083, 0.25, 0.5 l, 2, 4,
8 and 24 hours post dosing as described above. The sample collection and
handling
procedures for the inhalation study were same as for the intravenous study.
Bioanalytical assay procedures for the inhalation study were the same as for
the intravenous study. The calculated deposited dose in the lung (pulmonary
dose)
was approximately 0.70 or 1.77 mg/kg following an inhalation dose of 30 or 60
mg/mL erythromycylamine solution for 30 minutes, respectively. The pulmonary
dose in the lung was calculated as follows:
LDD = MAC x MV x DE x FLD = MBW
Where,
LDD = Lung Deposited Dose
MAC = Mean Aerosol Concentration = 0.54 and 1.36 mg/L for 30 and 60
mg/mL solutions, respectively.
MV = Minute Volume = 0.1 L/min.
DE = Duration of Exposure = 30 minutes
FLD = Fraction of Lung Deposit = 0.1
MBW = Mean Body Weight = 0.23 kg
Pharmacokinetic parameters in the lung following intravenous and inhalation
administration of erythromycylamine are summarized below in Table 4:
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Table 4
Pharmacokinetic parameters of erythromycylamine in the lung following an
intravenous or two inhalation doses in the rat (N=3).
Drug Administration
Routa and
Dose
Inhalation Inhalation
30 mg/mL, 60 mg/mL,
PharmacokineticIntravenous (Pulmonary (Pulmonary
Dose:
Parameter 25 m Dose: 0.7 mg/kg)1.77 mg/kg)
C",a,~(~,g/gram)68.99 89.33 155.24
AUC (~gh/gram)'605.19 854.27 1357.35
AUC (~gh/gram)Z24.21 1220.39 766.86.
MRT(h)3 10.8 10.5 11.2
1. Area under the curve estimated 0-24 hours postdose.
2. Area under the curve dose-normalized to 1 mg/kg.
3. Mean residence time estimated 0-24 hours postdose.
n.e.: Not estimated.
EXAMPLE 7
Aerosol And IV Efficacy Of Erythromycylamine
In The S. Pneumonia Rat Lung Model Of~Infection
Methods Male Sprague-Dawley rats were infected by intratracheal
administration with 50-100 microliters of S. pheumoniae A66 (Strain # PGO
4716)
prepared in agar beads. The inoculum was prepared by suspending a broth
culture of
PGO 4716 in molten agar, suspending the agar suspension in sterile mineral oil
with
mixing to generate small beads of agar containing the bacteria. The beads are
recovered by centrifugation, resuspended in sterile saline, and administered
to each
animal through a tracheal incision by injection directly into the.lung.
Erythromycylamine solutions are prepared in sterile saline. Antibiotic was
administered either by intravenous injection into the tail vein or by aerosol
exposure.
The aerosol exposure was accomplished by nose-only exposure using the In-Tox
Aerosol Exposure System (model No. 04-1100). This system is a closed aerosol
delivery system designed to expose rodents that are confined in plastic tubes,
open to
the system at one end (nose port) and sealed at the other to maintain system
integrity.
The aerosol is generated by a Pari LC Star TM air jet nebulizer at a flow of
approximately 6.5 liters per minute. Vacuum is set at 9 liters per minute such
that the
total flow through the system with diluter air is 7.5 liters per minute.
Treatment is initiated 24 hours after infection and continued once per day,
for
3 days. Aerosol was administered for 30 minutes each day. On day four after
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infection and 12 hours after the last dose, animals are sacrificed and lungs
surgically
removed. After removal, lungs are homogenized, diluted and quantitatively
plated
onto blood agar. Plates are incubated for 24 hours'and colonies of S.
pneumoniae
counted to determine bacterial load. The results are shown in Table 5:
Table 5
Efficacy of Erythromycylamine vs.
S. pneumoniae in the Rat Pneumonia
Model
Dose CFU/gram
Route (mg/kg per day) Recovered
IV 0 8.5x10'
BQL*
BQL*
40 BQL*
Aerosol 0 4.1 x 10'
0.13 3.5 x 102
0.67 BQL*
1.33 BQL*
*BQL=Below Quantitation Limit
EXAMPLE 8
Aerosol Efficacy Of Ervthromvcvlamine
10 _In The S. Pneumonia Rat Pulmonary Model Of Infection
After a Single Dose Treatment of Erythromycylamine
Male Sprague-Dawley rats were infected and exposed to aerosol treatment as
described in Example 7. The single treatment was initiated 24 hours after
infection
with aerosol administered at the doses indicated for 30 minutes. No further
treatment
15 was undertaken and animals were observed until surgery. On day four after
infection
(day 3 after dosing), the animals were sacrificed and their lungs were
surgically
removed. After removal, the lungs are homogenized, diluted and quantitatively
plated
onto blood agar. The plates are incubated for 24 hours and colonies of S.
pneumoniae
are counted to determine bacteria load. The results after a single dose
administration
20 are shown in Figure 11. Further results are shown in Table 6:
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Table 6
Efficacy of Erythromycylamine vs. S. pneumoniae in the Rat Pneumonia Model
Number Dose CFU/gram
of Doses (mg/kg/day) Recovered
3 0 4.1 x 10~
3 0.13 3.5 x 10~
3 ' 0.67 BQL
3 1.33 BQL
BQL = below quantitation limit
EXAMPLE 9
Aerosol Delivery Of Erythromycylamine To Dogs
Characterization Of Aerosol Pharmacokinetics
Inhalation Pharmacokinetics: For a 60 mg/mL solution, 3.191 g (4.08 mmol)
of erythromycylamine (94% purity) was added to 43 mL of DI water and 4.27 mL
(4.27 mmol) of 1 M sulfuric acid in a 50 ml volumetric flask. The solution was
then
adjusted to pH 6.5 with the addition of another 53 ~,L (0.053 mmol) of 1 M
sulfuric
acid. The volume was brought up to 50 mL with additional DI water. Dogs were
exposed once to either 60 mg/mL solution of erythromycylamine sulfate via an
inhalation mask exposure system (Inveresk Research, Scotland, UK) for 30
minutes.
The dogs were removed from their pen in the dog holding area and transferred
to the dosing laboratory. During dosing the animals were either restrained by
an
animal attendant or in a sling/harness system. Inhalation dosing was
undertaken using
a closed facemask connected to a nebulizer that was suitably characterized
prior to
commencement of dosing. The dosing apparatus incorporates a , facemask and
mouthpiece attached to flexible tubing, which was connected to the nebulizer
device.
The mouthpiece was located inside the animal's mouth, on top of the tongue,
and the
facemask sealed around the dog's snout by means of a rubber sleeve. An exhaust
valve from the mask was connected to an extract system. When the dosing
apparatus
is fully assembled and fitted to the dog, inspiration is shown by movement of
the
aerosol through the flexible tubing to the dog.
Lung samples were collected from 2 dogs at 2, 24, 48, 72, 96 and 120 hours
post-dosing. Lungs were removed surgically from the dogs, and each lobe (right
caudal, left caudal, right cranial, left cranial, right middle and accessory).
was
separated for assay. Plasma samples were collected from all surviving animals
at 2,
24, 48, 72, 96 and 120 hours post.
Erythromycylamine concentrations in plasma and the lung (per gram of lung
tissue) were determined using a LC-MS method. Plasma samples (100 ~,g) were
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spiked with oleandomycin (internal standard, 1 ~,g/mL) before extraction.
Plasma
samples (100 ~,L) were deproteinated with 3.3% trichloroacetic acid (TCA).
Samples
were centrifuged (10,000 rpm, 10 min.) and the supernatant was transferred to
HPLC
centrifilter for centrifiltration (10,000 rpm, 10 min). The mobile phase
consisted of
0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) solution at a flow rate of
0.5
ml/min for 3 minutes, followed by 0.1 % acetic acid-acetonitrile (60:40, v/v)
at a flow
rate of 0.8 ml/min for 3 minutes. A stainless steel analytical column (Zorbax
SB-C18,
2.1 mm ID x 150.0 mm, 5 ~,m with a Phenomenex cartridge guard column) was used
as the stationary phase. The column temperature was 50°C.
Quantification of
erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray
System. Data acquisition was set in the selective ion monitoring mode. The
method
was linear (r>0.9990) in the concentration range of 0.01 to 50 ~g/ml. The
absolute
recovery was greater than 90%.
Lung samples were homogenized with DI water. Oleandomycin was added to
the samples as an internal standard. The homogenate was deproteinated with 0.9
M
TCA. Samples were centrifuged at 10,000 rpm for 10 minutes and the supernatant
was transferred to HPLC centrifilters for centrifiltration. The mobile phase
consisted
of 0.1% acetic acid-acetonitrile (70:30, v/v, pH=3.2) at a flow rate of 0.5
ml/min for 3
minutes, followed by 0.1 % acetic acid-acetonitrile (60:40, v/v) at a flow
rate of 0.8
ml/min for 3 minutes. A stainless steel analytical column (Zorbax SB-C18, 2.1
mm
ID x 150.0 mm, 5 ~,m with a Phenomenex cartridge guard column) was used as the
stationary phase. The column temperature was 50° C. Quantification of
the
erythromycylamine was performed using a HP 1100 LC/MSD API-Electrospray
System. Data acquisition was set in the selective ion monitoring mode. The
linearity
(r>0.99) of the assay ranged from 2 to 100 ~g/g for lung. The extraction
efficiency
was greater than 90%.
Pharmacokinetic parameters, area under the curve (AUC) and mean residence
time (MRT) and half life (T1,2) were estimated based on the statistical moment
theory
using WinNonlinTM Professional Version 3.1 software (Pharsight Corporation).
The
peak concentration (C",~) was not estimated but observed.
Pharmacokinetic parameters in the lung and plasma inhalation following
administration of erythromycylamine in the dog are summarized in Table 7
below,
and in Figures 13 and 14:
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Table 7
Pharmacokinetic Parameters of Erythromycylamine in the Lung and Plasma
following
a 30-minutes Inhalation Adxilinistration of 60 mg/mL Solution in the Dog
(N=2).
Pharmacokinetic
Parameter
(unit)
Matrix Ca,~ AUC(0-120h)
(wgl~'~) (!~g~~'~)1 tv2 ~) MRT (h)i
Whole Lung 69 2085 27 29
Plasma 1.0 29 n.e. 37
Right Caudal77 2078 26 26
Left Caudal68 2000 26 28
Right Cranial87 2517 24 27
Left Cranial54 1857 29 31
Right Middle48 1979 30 34
Accessory 68 2256 31 31
1. Mean residence time
n.e.: Not estimated.
EXAMPLE 10
Liquid Aerosol Delivery Of Erythromycylamine
A solution of erythromycylamine sulfate (100 mglmL) in quarter normal
saline at pH 7.0 is prepared in accordance with the general procedure of the.
foregoing
examples. A 1.0 mL dose of the solution is administered by aerosol inhalation
in less
than 10 minutes to a human subject suffering from acute exacerbation of
chronic
bronchitis (AECB) using an AeroGen AerodoseTM inhaler. A reduction in the
bacteria
associated with AECB and symptoms of AECB is observed.
EXAMPLE 10
D Powder Aerosol Delive Of E om c famine
A dry powder formulation of erythromycylamine sulfate (100 mg) and a dry
powder carrier (equal parts of lactose, 2-hydroxypropyl-(3-cyclodextrin,
mannitol and
aspartame; , total weight 25mg) is prepared. The formulation is administered
by
aerosol inhalation in less than 2 minutes to a human subject suffering from
acute
exacerbation of chronic bronchitis (AECB) using a Glaxo Ventolin Rotohale'T"
inhaler.
A reduction in the bacteria associated with AECB and symptoms of AECB is
observed.
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While the preferred embodiment of the invention. has been illustrated and
described, it will be appreciated that various changes can be made therein
without
departing from the spirit and scope of the invention.
