Note: Descriptions are shown in the official language in which they were submitted.
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PARTICULATE DELIVERY SYSTEMS AND METHODS OF USE
Field of the Invention:
The present invention generally relates to compositions and methods for the
administration of particulates
comprising at least one bioactive agent which, in selected embodiments, may
comprise an immunoactive agent. in this
respect, the invention provides for both topical and systemic delivery of the
bioactive agent using, for example, the
respiratory, gastrointestinal or urogenital tracts. In particularly preferred
embodiments, the disclosed compositions
will be used in conjunction with inhalation devices such as metered dose
inhalers, dry powder inhalers, atomizers or
nebulizers for targeted delivery to mucosal surfaces.
Background of the Invention:
Vertebrates possess the ability to mount an immune response as a defense
against pathogens from the
environment as well as against aberrant cells, such as tumor cells, which
develop internally. This can take the form of
innate or passive immunity, which is mediated by neutrophils and cells of the
monocytelmacrophage lineage, or the form of
acquired or active immunity mediated by lymphocytes against a specific
antigenic sequence. Active immune responses can
themselves be further subdivided into two arms, the humoral response which
entails the production of specific antibodies
which serve to neutralize antigens exposed to the systemic circulation and aid
in their uptake by professional phagocytic
cells, and the cellular arm which is required for recognition of infected or
aberrant cells within the body.
In both cases the specific response is triggered by the intracellular
processing of antigen. When the antigen is
processed through the cytoplasmic route, the resultant peptides are bound to
nascent MHC class I molecules which
facilitates appropriate presentation to effector T-cells. MHC class I
presentation favors recognition by cytotoxic T
lymphocytes. In contrast, intracellular processing via the endocytic route
results in presentation on MHC class II molecules
which favors T helper responses involved in stimulation of the humoral arm.
The goal of vaccination is to prime both
responses and generate memory T cells, such that the immune system is primed
to react to a pathogenic infection. Such a
response is promoted by the co-administration of signals that promote
costimulatory molecule expression, so called
"adjuvants." Engagement of both the humoral and cellular immune responses
leads to broad based immunity and is the
preferred goal for intracellular pathogens. The absence of appropriate
costimulatory molecule expression can lead to a
state of T cell unresponsiveness.
In this regard, modulation of an immune response can take one of two
directions; either to elicit an immune
response directed against a foreign pathogenic agent or antigen thereof, or to
suppress an inappropriate reaction mounted
against a self-epitope that leads to chronic inflammation. Such chronic
reactions against self-epitopes are associated with
various autoimmune diseases such as diabetes, typically type I, multiple
sclerosis, rheumatoid arthritis or lupus
erythrematosis. In either case, the active agent frequently takes the form of
a relatively complex peptide, protein, RNA or
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DNA-based entity or other macromolecular structure rather than small chemical
entities typical of classical pharmaceutical
agents. These complex bioactive agents generally exhibit poor bioavailability
when administered orally, and therefore have
traditionally been administered by invasive parenteral injection. Recently
however, it has been suggested that relatively
large biomolecules may be delivered via mucosal routes, e.g. by inhalation.
Delivery of these agents into systemic
circulation through inhalation is particularly attractive since administration
via the respiratory mucosa bypasses the
digestive enzymes of the GI tract. Furthermore, it offers the potential for
increased bioavailability for peptides and proteins
because of the large surface area available for exchange with systemic
circulation. While the molecular weight cut-off for
oral bioavailability is generally regarded to be in the range of 500 Daltons,
peptide hormones or analogues of larger
molecular weight (e.g., 1.8 kD desmopressin, 5.8 kD insulin, 9.5 kD
parathyroid hormone), have been shown to be
absorbed across the nasal or pulmonary mucosa intact into the systemic
circulation.
Besides allowing for the effective delivery of protein, peptide, viral and DNA
formulations without degradation,
targeted delivery to the mucosal surface itself may offer a benefit if it
elicits a local immune response within the MALT
(mucosa-associated lymphoid tissue) lymphoid system. Mucosal vaccination is of
particular interest for vaccines designed
against pathogens whose port of entry is typically at one of the mucosal
surfaces interfacing the body with the external
environment. The MALT lymphoid system resides within the lamina propria of the
mucosa. When foreign antigen is
presented to local dendritic cells, there is a local amplification and
maturation of B-cell precursors, which produce IgA and
IgM antibodies in addition to the IgG antibodies typically induced by systemic
delivery of antigen. The former are secreted
through specialized transport receptors by a process known as transcytosis
across the mucosal surface into the lumen.
There, they provide a first line of defense against invading pathogens at the
mucosal surface. Recent evidence indicates
that, in addition to binding pathogenic antigens, the resultant formation of
immune complexes may in and of itself inhibit
viral transmission occurring via the transcytotic route. By priming this first
line immune response to antigens derived from
pathogens, mucosal bnmunization should greatly enhance the efficiency with
which the organism first intercepts an
invading pathogen.
Several previous attempts have been made to exploit this uptake mechanism and
provide for the effective
delivery of peptides or proteins. For example, U.S. Pat. No. 5,756,104
describes the use of liposome formulations for
intranasal vaccine formulations. These formulations appear to comprise aqueous
carriers having liposomes and free
antigenic material dispersed therein. While the compositions were found to
elicit an immune response, they appear to be
extremely labile and susceptible to degradation over time. In a practical
sense this is a substantial drawback.
Attempts to overcome such limitations and further increase delivery efficiency
have resulted in the development
of dry powders for the administration of relatively large biomolecules.
Unfortunately, conventional powdered preparations
(i.e. micronizedl often fail to provide accurate, reproducible dosing over
extended periods. In part, this is because the
powders tend to aggregate due to hydrophobic or electrostatic interactions
between the fine particles. Such cohesion may
be partially overcome through the use of larger carrier particles (i.e.
lactose) to inhibit aggregation. However, these larger
particles and associated drug often fail to reach the targeted cells resulting
in uneven delivery profiles. Further, crude
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mixtures comprising carrier molecules provide little, if any, protection for
the incorporated biomolecule. Accordingly, as
with the aqueous compositions described above, such preparations are subject
to degradation and loss of activity over
time.
More recently, improved formulation methods have been undertaken in order to
overcome the limitations
associated with conventional prior art powders and aqueous preparations. In
this regard, U.S. patent applications serial
nos. 091218,209 and 091219,736, incorporated herein by reference, describe
methods and processes for generating
preparations comprising bioactive agents in microparticulate form. The
resultant powders, which preferably exhibit a
hollow, porous morphology, are suitable for use in inhalation devices such as
dry powder inhalers (DPIs) or, when
suspended in a nonaqueous liquid (i.e, a hydrofluoroalkane or fluorocarbon),
metered dose inhalers (MDIs) and nebulizers.
Moreover, the mild conditions used during the formulation process support
retention of biological activity making the
preparations particularly compatible for use with proteins and peptides as
well as more complex macromolecular
structures such as viruses. Additionally, since the resultant powders have
very low residual water content, which can
be further maintained by formulation in short~chain fluorocarbons or
fluorochemicals such as propellants ar the longer
chain fluorochemicals such as perfluorooctyl bromide (PFOB), these
formulations provide a stable means for storage of
labile bioactive agents.
Besides enhanced stability, the preferred hallow, porous morphology of the
microparticulates provides
aerodynamic characteristics that are particularly compatible with inhalation
therapies. Further, the particulate
characteristics allows for the formation of exceptionally stable dispersions
and makes them especially compatible with
hydrofluoroalkane propellants such as HFA-134a as well as other fluorocarbon
liquid vehicles like PFOB. Thus, whether
used in a dry form or as a nonaqueous dispersion, the microparticulates
provide for good dose reproducibility, excellent
plume characteristics (a measure of the uniformity of a propellant or dry
powder spray) and a high percentage of the dose
delivered as the respirable fraction (as opposed to deposition in the device
or throat). These properties suggest that the
disclosed microparticles offer substantial theoretical advantages as far as
delivery deep into the lung. Such deep
deposition is preferred where delivery into the systemic circulation is
desired since uptake of large macromolecules like
proteins and peptides is optimal at the level of the alveoli.
While the use of such microparticulate preparations is a substantial
improvement over conventional prior art
delivery methods, there still remains a need to provide for the targeted
delivery of bioactive, immunomodulating or
immunoactive agents that results in an enhanced physiological response.
Accordingly, it is an object of the present invention to provide compositions,
systems and methods that provide
for the generation of an enhanced immune response.
It is another object to provide for the effective delivery of immunoactive
agents, including vaccines and
immunomodulating agents, to the mucosal surfaces of a patient in need thereof.
It is yet a further object of the present invention to provide vaccine or
other bioactive formulations that do not
require refrigeration or freezing to maintain activity.
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It is still a further object of the present invention to provide for the
establishment of passive and active immunity
via inhalation therapies.
It is yet another object of the present invention to provide for stable
preparations of immunoactive agents that
may be used to confer immunity or down regulate the immune system of a patient
in need thereof.
Summary of the Invention:
These and other objects are provided for by the invention disclosed and
claimed herein. To that end, the methods
and associated compositions of the present invention allow, in a broad aspect,
for the improved delivery of bioactive
agents to selected target sites in a powdered or particulate form. More
particularly, it has been surprisingly been found
that the disclosed methods and compositions may be used to enhance or increase
the activity of an incorporated bioactive
agent, which preferably comprises an immunoactive agent, following
administration. In this regard, the vaccines of the
instant invention appear to exhibit an "adjuvant effect" that may provoke an
enhanced immune response an order of
magnitude or more greater than that provoked by a comparable prior art vaccine
formulation. Besides this unexpected
improvement in potency, relatively gentle formulation techniques may be
combined with particulate morphology and
composition to protect and enhance the activity of any incorporated agents.
This allows for the formation of relatively
efficacious preparations that retain their biological activity without the
need for refrigeration or freezing. Further, unlike
prior art powders or dispersions for drug delivery, the present invention
preferably employs novel techniques to reduce
attractive forces between the particles, resulting in improved flowability and
dispersibilty. When these powders are
incorporated in a nanaqueous suspension medium (e.g. a liquid fluorochemical)
these same characteristics provide for
reduced flocculation, sedimentation or creaming that may further reduce the
rate of agent degradation. Finally,
administration of the disclosed particulates or dispersions to selected target
sites such as mucosal surfaces may further
serve to optimize or enhance bioactivity. As such, the dispersions or powders
of the present invention may be used to
effectively deliver bioactive agents in conjunction with metered dose
inhalers, dry powder inhalers, atomizers, aeroselizers,
nasal pumps, spray bottles, nebulizers or liquid dose instillation (LDI)
techniques.
A particularly beneficial feature of the disclosed particulate formulation
technology is that a wide range of
bioactive structures can be incorporated in the stabilized dispersions or
powders irrespective of their hydrophobicity or
hydrophilicity. In preferred embodiments, the bioactive powders will be
produced using relatively mild spray drying
methodology. Due to such compatible particulate formulation techniques,
larger, more labile biomolecules such as
peptides, proteins or genetic material may readily be incorporated in the
disclosed compositions without adverse effects or
undue loss of activity. These same formulation techniques and resulting
particulates further provide for the incorporation
and delivery of relatively high doses (ca. 10 mg) of bioactive agents using
conventional administration techniques and
systems. Thus, whether administered in the form of a dry powder or stabilized
dispersion, the novel particulate fabrication
techniques and enhanced response afforded by the disclosed preparations lead
to the effective delivery of bioactive agents
to targeted sites such as the mucosa.
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In connection with the present invention, the term "bioactive agent" refers to
any active peptide or protein, such
as a hormone, cytokine or chemokine or an immunoactive agent. That is, while
the disclosed compositions and methods
are compatible with almost any bioactive agent, they have been discovered to
be surprisingly effective for the delivery
or administration of immunoactive agents designed to modulate immune responses
such as, for example, eliciting an
immune response to a foreign antigen or pathogen or down regulating an active
immune reaction. Accordingly, as used
herein, the terms "immunoactive agents," or "immunologically active agents,"
will comprise any molecule that may be used
to elicit a physiological or immune response or modulate pre-existing
responses in a subject. Such immunoactive agents or
biologics may comprise peptides, polypeptides, proteins, carbohydrates,
genetic material including DNA, RNA and
antisense constructs, as well as microbes including viruses, phages and
bacteria.
In addition, molecules that may function as cofactors, potentiators or
penetration enhancers can be readily
co-formulated in the particulates described herein. Those skilled in the art
will appreciate that any compound which
acts to improve the uptake, presentation er bioavailability may function as a
potentiator or penetration enhancer in
accordance with the teachings herein. For instance, compounds that can alter
or increase the membrane permeability
of a cell may function as potentiators or penetration enhancers. Exemplary
potentiators or penetration enhancers may
include chelating agents (e.g. EDTA, citric acid), detergents or surfactants
(e.g. 9-iauryl ether), fatty acids (e.g. oleic
acid) and bile salts (e.g. sodium glycocholatel. Particularly preferred
penetration enhancers comprise relatively short
chain phospholipids having chain lengths of less than about 10 carbons. As
with the bioactive agents, and as will be
discussed in more detail below, the selected potentiators or penetration
enhancers may be incorporated in, or
associated with, particulates in varying concentrations.
With regard to the particulates, microparticulates or perforated
microstructures of the present invention, those
skilled in the art wilt appreciate that they may be formed of any
biocompatible material providing the desired physical
characteristics or morphology. In this respect, perforated microstructures
will preferably comprise pores, voids, defects or
other interstitial spaces that act to reduce attractive forces by minimizing
surface interactions and decreasing shear
forces. This morphology acts to reduce aggregation and improve dispersability.
Yet, given these constraints, it will be
appreciated that any biocompatible material or configuration may be used to
form the microstructure matrix. As to the
selected materials, it is desirable that the microstructure incorporates at
least one surfactant which, in preferred
embodiments, will act as a penetration enhancer. Preferably, this surfactant
will comprise a phospholipid or other
surfactant or amphiphile approved for pharmaceutical use. Similarly, it is
preferred that the microstructures incorporate at
least one bioactive agent or biologic. As to the configuration, selected
embodiments of the invention comprise spray dried,
hollow microspheres having a relatively thin porous wall defining a large
internal void, although, other void containing or
perforated structures are contemplated as well.
It has unexpectedly been found that the use of hollow andlor porous perforated
microstructures may
substantially reduce attractive molecular forces, such as van der Waals
forces, which dominate prior art powdered
preparations and dispersions. In this respect, the powdered compositions
typically have relatively low bulk densities that
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contribute to the flowability of the preparations while providing the desired
characteristics for inhalation therapies. More
particularly, the use of relatively low density perforated (or porous)
microstructures or microparticulates significantly
reduces attractive forces between the particles thereby lowering the shear
forces required to achieve fiowability of the
resulting powders. The relatively low density of the perforated
microstructures also provides for superior aerodynamic
performance when used in inhalation therapy. In dispersions, the physical
characteristics of these powders provide for the
formation of stable preparations. Moreover, by selecting dispersion components
in accordance with the teachings herein,
interparticie attractive forces may further be reduced to provide formulations
or preparations having enhanced stability.
While preferred embodiments of the invention comprise perforated
microstructures or porous particulates,
relatively nonporous or solid particulates may also be used to prepare powders
or dispersions that are compatible with the
teachings herein. That is, powders or dispersions comprising suspensions of
relatively nonporous or solid particulates are
also contemplated as being within the scope of the present invention. In this
respect, such relatively nonporous
particulates may comprise micronized particles, milled particles or
nanocrystals. Accordingly, as used herein the term
"particulate" shall be interpreted broadly and held to comprise particles of
any porosity and or density, including both
perforated microstructures and relatively nonporous particles.
As previously alluded to, the disclosed powders may be dispersed in an
appropriate nonaqueous suspension
medium to provide stabilized dispersions comprising a selected bioactive
agent. Such dispersions are particularly
useful in metered dose inhalers, atomizers nasal pumps, spray bottles and
nebulizars. Other embodiments of the
invention comprise stabilized dispersions that may be administered directly to
the lung or nasal cavity using direct
instillation techniques. In any case, particularly preferred suspension
mediums comprise fluorochemicals (i.e.
perfluorocarbons or fluorocarbons) that are liquid at room temperature or
fluorinated propellants (i.e.
hydrofluoroalkanes or chlorofluorocarbons). Because of their beneficial
wetting characteristics, some fluorochemicals
may be able to provide for the dispersion of particles deeper into the lung or
other mucosal surface, thereby improving
systemic delivery. Moreover, such suspension media tend to be anhydrous
thereby retarding hydrolytic degradation of
the incorporated bioactive agents. Finally, fluorochemicals are generally
bacteriostatic thus decreasing the potential
for microbial growth and associated proteolytic decay in compatible
preparations.
With regard to the delivery of the disclosed powders or stabilized
dispersions, another aspect of the present
invention is directed to inhalation systems for the administration of one or
more bioactive agents or biologics to a
patient. As alluded to above, exemplary inhalation devices compatible with the
present invention may comprise an
atomizer, a nasal pump, a sprayer or spray bottle, a dry powder inhaler, a
metered dose inhaler or a nebulizer. In preferred
embodiments, these inhalation systems will deliver the bioactive agent to the
desired physiological site (e.g. a mucosal
surfaces as an aerosol. For the purposes of the instant application the term
"aerosolized" shall be held to mean a
gaseous suspension of fine solid or liquid particles unless otherwise dictated
by contextual restraints. That is, an
aerosol or aerosolized medicament may be generated, for example, by a dry
powder inhaler, a metered dose inhaler, an
atomizer, a spray bottle or a nebulizer. Of course, as explained in more
detail below, the compositions of the present
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invention may also be delivered directly (e.g, by conventional injection or
needleless injection) or using such techniques
as liquid dose instillation. In especially preferred embodiments the
compositions of the present invention are contacted
with a mucosal surface /e.g. via inhalation) to elicit both mucosal and
systemic immunity.
While the powders or stabilized dispersions of the present invention are
particularly suitable for
administration of bioactive agents to mucosal surfaces, it will be appreciated
that they may also be used for the
localized or systemic administration of compounds to any location of the body.
Accordingly, it should be emphasized
that, in preferred embodiments, the formulations may be administered using a
number of different routes including, but
not limited to, the gastrointestinal tract, the respiratory tract, topically,
intramuscularly, parenterally, intradermally,
transdermally, intraperitoneally, nasally, vaginally, rectally, aurally,
buccally, orally or ocularly. In this respect those
skilled in the art will appreciate that the selected route of administration
wilt largely be determined by the choice of
bioactive agent and the desired response of the subject.
Other objects, features and advantages of the present invention will be
apparent to those skilled in the art from a
consideration of the following detailed description of preferred exemplary
embodiments thereof.
Brief Description of the Drawines:
Fig. 1 is a graphical representation of levels of functional HA peptide f
residues 110-120 of the hemagglutinin
of the influenza virus) following formulation in microstructures according to
the present invention;
Fig. 2 illustrates the fact that antigens formulated in microstructures do not
require intracellular processing
to activate T cells;
Fig. 3 graphically compares the plasma concentration of HA peptide delivered
using nasally administered
microparticulates and intravenous injection;
Fig. 4 depicts calibration curves for human IgG formulated in
microparticulates as described in the instant
application along with selected controls;
Figs. 5A and 5B graphically illustrate release kinetics for IgG formulated
microparticulates and HA peptide
formulated microparticulates respectively;
Figs. 6A and 6B show the persistence of IgG in the plasma following
intratracheal and nasal administration
using formulated microparticuiates;
Figs. 7A and 7B show, respectively, systemic and localized antibody responses
to IgG administered
intratracheally as formulated microparticulates in accordance with the present
invention;
Fig. 8 graphically illustrates levels of cytokines indicative of a T cell
response following intratracheal
administration of IgG microparticulates to mice;
Fig. 9 depicts murine antibody response to IgG microparticulates administered
intranasally;
Figs. 10A and 10B present murine antibody titers at 7 and 14 days respectively
following intraperitoneal
administration of IgG microparticulates;
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Figs. 11 A, 11 B and 11 C respectively illustrate T cell responses to
microparticulate formulated virus, viral
control and infectious titer of both formulated and unformulated virus;
Fig. 12 depicts murine antibody responses to microparticulate formulated live
and killed influenza virus at 7
and 14 days following intranasal administration;
Figs. 13A, 13B and 13C show, respectively, murine levels of factors indicative
of a T cell response following
intranasal inoculation of viral microparticulates or live virus or killed
virus along with control antigens;
Figs. 14A and 14B respectively illustrate viral shedding and body weight
variation in mice intranasally
inoculated with microparticulates comprising both live and killed virus; and
Fig. 15 presents results of an in vitro Andersen cascade impactor study
showing efficient delivery of
formulated microspheres comprising bovine gamma globulin from a metered dose
inhaler.
Detailed Description of Preferred Embodiments:
A. Introduction
While the present invention may be embodied in many different forms, disclosed
herein are specific
illustrative embodiments thereof that exemplify the principles of the
invention. It should be emphasized that the
present invention is not limited to the specific embodiments as illustrated.
As discussed above, the present invention provides methods, systems and
compositions comprising powders
or microparticulates that may advantageously be used for the delivery of
bioactive agents. Preferably the bioactive
agent will comprise active peptides or proteins or an immunoactive agent. In
the context of the present invention,
immunoactive agents may comprise any molecule that may be used to elicit an
immune response or modulate pre-existing
responses such as vaccines, immunoglobulins or autoantigens. Those skilled in
the art will appreciate that the disclosed
powders may advantageously be used to deliver bioactive agents in a dry state
(e.g. with a DPI or gas driven powder
injector) or in the form of a stabilized dispersion (e.g. with an atomizer,
spray bottle, MDI, LDI, needleless injector,
syringe, nasal pump or nebulizerl. In particularly preferred embodiments, the
powders or microparticulates will
comprise perforated microstructures which, as disclosed herein, comprise a
structural matrix that exhibits, defines or
comprises voids, pores, defects, hollows, spaces, interstitial spaces,
apertures, perforations or holes. These perforated
microstructure powders have aerodynamic characteristics that make them
particularly useful for inhalation therapy and
exhibit morphologies that allow for the formation of stabilized dispersions in
propellants or nonaqueous delivery vehicles.
More generally, the relatively mild conditions employed during the formation
of the disclosed bioactive powders and
advantages associated with compatible delivery methods allow for the efficient
administration of comparatively fragile
biologic agents.
While not wishing to be bound by any particular theory, it is believed that
the relatively gentle methods used
to form, store and administer the disclosed compositions provide for the
effective retention of biological activity in
generally unstable agents. In this respect, preferred formulations do not
require refrigeration to maintain their activity.
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Moreover, selection of appropriate compounds for use in the disclosed powders
and delivery to selected physiological
sites (e.g. mucosal surfaces) may promote the uptake of the incorporated agent
or agents as well as enhancing the
activity thereof. In addition, the compositions andJor delivery techniques of
the present invention appear to generate
an unexpected "adjuvant effect" that may provide for an enhanced immune
response or bioactivity following
S administration of the selected agent. More specifically, as will be
discussed below and seen in the Examples, the
present invention may be used to elicit an immune response comparable to that
achieved by administering an antigen in
complete Freund's adjuvant (i.e. an order of magnitude or more higher than
conventional pharmaceutical formulations).
Accordingly, the present invention provides for the effective delivery of
active peptides, proteins, genetic material, or
pathogenic particles (either five or inactivated) to induce active localized
or systemic immunization or to achieve
passive immunization, immune modulation, hormonal regulation or gene therapy.
B. Bioactive Agents
In a broad aspect, the powdered or microparticulate compositions of the
present invention, including dispersions
incorporating such powders, will preferably comprise at least one bioactive
agent. As used herein, the term "bioactive
agent" shall be held to comprise any active peptide or protein or any
immunoactive agent. With respect to the latter,
particularly preferred embodiments of the present invention will comprise an
immunoactive agent designed capable of
modulating an immune response. In accordance with the teachings herein,
modulation of a subject's immune response
shall comprise eliciting a response against a potential pathogenic infection
or foreign antigen, stimulating an existing
immune response, inducing localized or systemic passive immunity or
suppressing an autoimmune response or allergenic
response. For the purposes of the instant application the terms "bioactive
agent" or immunoactive agent" shall be broadly
construed to comprise any molecule or organism, or analog, homologue or
derivative thereof, that provides a desired
physiological or immune response in a subject. It will be appreciated that the
term "bioactive agent" shall be held
inclusive of the term "immunoactive agent" and its equivalents unless
otherwise dictated by contextual restraints.
Exemplary bioactive agents that may be used in conjunction with the invention
comprise peptides, polypeptides,
proteins, fusion or chimeric proteins, immunoglobulins, genetic material
including DNA, RNA, recombinant and
antisense constructs, microbes including viruses, phages, bacterial
carbohydrates and bacteria as well as smaller
molecules that may function as potentiators, cofactors ar penetration
enhancers. The bioactive compositions according
to the present invention find use as vaccines, immunomodulators, effectors or
replicons for gene therapy applications.
It will be appreciated that the powders or microparticulate compositions of
the present invention may exclusively
comprise one or more bioactive agents) (i.e. up to 100% wlw). However, in
selected embodiments the perforated
microstructures may incorporate much less bioactive agent depending on the
activity thereof. Accordingly, for highly
active materials the particulates, microparticulates or perforated
microstructures may incorporate as little as 0.001 % by
weight although a concentration of greater than about 0.1 % wIw is preferred.
Other embodiments of the invention may
comprise greater than about 5%,10%,15%, 20%, 25%, 30% or even 40% wfw active
or bioactive agent or biologic. Still
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more preferably the disclosed powders may comprise greater than about 50%,
60%, 70%, 75%, 60% or even 90% wIw of
a bioactive agent. The precise amount of bioactive agent incorporated in the
powders or perforated microstructures of the
present invention is dependent upon the agent of choice, the required dose,
method of administration and the form of the
agent actually used. Those skilled in the art will appreciate that such
determinations may be made by using well-known
pharmacological techniques in combination with the teachings of the present
invention.
With regard to pharmaceutical preparations, any bioactive agent that may be
formulated in the disclosed
powders or perforated microstructures for the purpose of eliciting a
physiological response, including an immune
response, is expressly held to be within the scope of the present invention.
In accordance with the teachings herein
the selected bioactive agents) may be associated with, or incorporated in, the
powders or perforated microstructures
in any form that provides the desired efficacy and is compatible with the
chosen production techniques. As used
herein, the terms "associate" or "associating" mean that the particulate,
microparticulate, structural matrix or perforated
microstructure may comprise, incorporate, adsorb, absorb, be coated with or be
formed by the bioactive agent. Where
appropriate, the agent may be used in the form of salts (e.g. alkali metal or
amine salts or as acid addition salts) ar as
esters or as solvates (hydrates). In this regard the form of the bioactive
agent may be selected to optimize the activity
andlor stability of the compound andlor to minimize the solubility of the
agent in the suspension medium andlor to
minimize particle aggregation.
At least to some extent, the advantages provided by the instant invention
reside in the unique formulation,
storage and delivery aspects afforded by the disclosed powders and
dispersions. In this respect, and as will be
discussed in mote detail below, the conditions under which the disclosed
powders or perforated microstructures may
be formed are relatively mild. That is, particulates comprising bioactive
agents may be formed according to the
present invention without subjecting the active compound or agent to extreme
physical or chemical conditions. This is
of extreme importance with regard to relatively large macromolecules or agents
such as proteins, genetic material or
attenuated viruses that may easily be degraded or inactivated. Moreover,
selected embodiments of the present
invention further serve to maintain the biological activity of incorporated
agents by forming relatively stable
dispersions comprising nonaqueous suspension media. These dispersions of
active powder in suspension medium
(preferably a liquid fluorochemical or fluorochemical propellant) tend to be
both bacteriostatic and anhydrous, thereby
inhibiting hydrolysis or proteolytic decay of the incorporated agent. It will
be appreciated that such compositions have
been found to maintain comparatively high levels of activity over prolonged
storage periods. Finally, it has been found
surprisingly that both the composition of the disclosed powders and delivery
techniques thereof can be adjusted to
potentiate or enhance the activity of the associated bioactive agent. Taken
together, these advantages of the present
invention provide for the efficient delivery and efficacy of highly active
agents to the selected physiological site.
As indicated above the compositions, methods and systems of the instant
invention are useful for the
delivery of bioactive agents such as peptides, polypeptides, bacterial
carbohydrates, viruses and genetic material. In
this respect, the disclosed invention is particularly useful for the
administration of vaccines for active immunization
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(e.g. mucosai and systemic vaccination), immunoglobulins for passive
immunization, immunomodulators for the
treatment of autoimmune diseases, active peptides or proteins, and effectors
and expression vectors for gene therapy
or vaccination. As will be explained in more detail below, powders comprising
the selected agent may be formed
through a variety of different means. Preferably, the powders or
microparticulates will be in the form of perforated
microstructures and will comprise additional components to enhance the
stability andlor efficacy of the incorporated
bioactive agent. Optionally, the powders may be formulated in a suspension
medium to provide stabilized dispersions.
Particularly preferred classes of bioactive agents will be discussed in more
detail immediately below.
B(i). Antigens and Vaccines (for Active Immunization)
In accordance with the teachings herein, particularly preferred bioactive
agents will comprise vaccines. As
discussed throughout the instant specification and accompanying examples,
compatible vaccines may comprise inactivated
or killed microbes (e.g. viruses), live attenuated microbes, phages, subunit
vaccines such as proteins, peptides or
carbohydrates (e.g. bacterial carbohydrates), genetic material including
replicons, viral vectors, and plasmids and
recombinant molecules such as fusion proteins or chimeric antibodies.
Regardless of which type of agent or biologic is
selected, the resulting powdered compositions may be used to immunize a
subject against one or more target antigens.
Further, the adjuvant effect or enhanced immunity associated with the
disclosed invention provides for particularly
effective immunization.
As defined herein a °target antigen" refers to an antigen, typically a
portion of a protein or a peptide, toward
which it is desirable to induce an immune response. Such an antigen may be
comprised in a pathogen, such as a viral,
bacterial, protozoan, fungal, yeast, or parasitic antigen, or may be comprised
in a cell, such as a cancer cell. Tumor
antigens and viral antigens are especially preferred target antigens. In the
case of genetic vaccines, one or more target
antigens will be expressed by the host following transfection or
transformation of autologous cells with the
administered genetic material. Conversely, in protein or peptide based
vaccines, including those comprising chimeric or
fusion proteins or killed or attenuated microbes, the target antigen or
antigens will be presented directly to the immune
system. In either case, presentation of the target antigens using the powders
or dispersions of the instant invention
will provoke the desired immune response. Interestingly, it has been found
that when live viruses, or combinations of
live and killed viruses have been used as vaccines in accordance with the
teachings herein, a particularly vigorous
immune response is generated by the subject.
Those skilled in the art will appreciate that, in general, an effective anti-
viral immune response comprises
both a cell-mediated response, typically involving ThIICTL cell response and a
B cell-mediated humoral response.
Whereas a purified protein or killed microbe usually elicit B, Th2 without CTL
responses, certain formulations of
subunit or killed vaccines, as well as live vaccines can elicit B, Th1
associated with CTL responses. Preferably, the
vaccine compositions of the present invention will induce a broad range of
immune responses upon administration,
including B, Th1 and CTL responses. However, infection with live virus during
vaccination can lead to unacceptable
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side effects. Therefore the goal of a successful vaccination strategy is to
engage both the cellular and humoral
branches of immunity without incurring undue adverse effects. As will be
disclosed below, the compositions of the
present invention may be used to induce both types of response upon
administration.
In this respect, compatible vaccines may include any molecule, organism or
compound that results in the
generation of B cell response, a T cell response or a combination thereof to
the target antigen. As such, the agent
actually presented to the host immune system (whether directly or following
transformation of host cells) may be an
analog, homologue or derivative of the naturally occurring target antigen or
molecule comprising the target antigen.
Moreover, the immunization may be local or systemic in nature depending on the
type of target antigen presented and
the form of delivery. For example, in particularly preferred embodiments the
immunogenic response will be largely
mucosal in nature (e.g. within the mucosa-associated lymphoid tissue [MALT]
lymphoid system). As previously discussed,
when foreign antigen is presented by local dendritic cells, there is a local
amplification and maturation of T~cells and B-
cells, which produce IgA and IgM antibodies in addition to the IgG antibodies
typically induced by systemic delivery of
antigen. Such localized immunization, particularly in the nasal passages and
sinuses, has been found to be particularly
effective in preventing infection by airborne pathogens such as influenza
virus and respiratory syncytial virus.
More generally, the vaccine compositions of the present invention may comprise
one or more target antigens
from a number of pathogens. For example, but not by way of limitation, the
target antigen may be comprised in an
influenza virus, a cytomegalovirus, a herpes virus (including HSV-1 and HSV-
11), a vaccinia virus, a hepatitis virus
(including but not limited to hepatitis A, B, C, or D), a varicella virus, a
rotavirus, a papilloma virus, a measles virus, an
Epstein Barr virus, a coxsackie virus, a polio virus, an enterovirus, an
adenovirus, a retrovirus (including, Gut not limited
to, HIV~1 or HIV-21, a respiratory syncytial virus, a rubella virus, a
Streptococcus bacterium (such as Streptococcus
pneumoniaeJ, a Staphylococcus bacterium (such as Staphylococcus aureus), a
Hemophilus bacterium Isuch as
Hemophilus unfluenzael, a Listeria bacterium (such as Listeria monocytogenes),
a Klebsiella bacterium, a Gram~negative
bacillus bacterium, an Escherichia bacterium (such as Escherichia coh), a
Salmonella bacterium Isuch as Salmonella
typhimurium), a Vibrio bacterium isuch as Vibrio cho%rae), a Yersinia
bacterium (such as Yeisinia pestis or Yeisinia
enterocoliticusl, an Enterococcus bacterium, a Neisseria bacterium(such as
Nelsseria meningitidis), a Corynebacterium
bacterium (such as Corynebacterium diphtheriae), a Clostridium bacterium (such
as Clostridium tetam), a Mycoplasma
(such as Mycoplasma tuberculosis), a Candida yeast, an Aspergillus fungus, a
Mucor fungus, a toxoplasma, an amoeba,
a malarial parasite, a trypanosomal parasite, a leishmanial parasite, a
helminth, etc. Specific nonlimiting examples of
such target antigens include hemagglutinin, nucleoprotein, M protein, F
protein, HBS protein, gp120 protein of HIV, nef
protein of HIV, and listerialysine.
Regardless of what type of antigen is selected to be the target antigen it
will comprise at least one relevant
epitope. The term "relevant epitope", as used herein, refers to an epitope
comprised in the target antigen which is
accessible to the immune system. For example, a relevant epitope may be
processed after penetration of a microbe
into a cell or recognized by antibodies on the surface of the microbe or
microbial proteins. Preferably, an immune
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response directed toward the epitope confers a beneficial effect; for example,
where the target antigen is a viral
protein, an immune response toward a relevant epitope of the target antigen at
least partially neutralizes the infectivity
or pathogenicity of the virus. Those skilled in the art will appreciate that
the relevant epitopes may be B-cell or T-cell
epitopes.
The term "B cell epitope", as used herein, refers to a peptide, including a
peptide sequence contained within a
larger protein, which can elicit antibody production by B cells.
For example, and not by way of limitation, the hypervariable region 3 loop
("V3 loop") of the envelope protein
of human immunodeficiency virus ("HIV") type 1 is known to be a B cell
epitope. Other examples of known B cell
epitopes which may be used according to the invention, include, but are not
limited to, epitopes associated with
influenza virus strains, such as site B of influenza HA 1 hemagglutinin, which
has been shown to be an
immunodominant B cell epitope (Li et al., 1992, J. Virol. 66:399-4041; an
epitope of F protein of measles virus
(residues 404-414, Parlidos et al., 1992, Eur. J. Immunol. 22:2675-26801; an
epitope of hepatitis virus pre-S1 region,
from residues 132-145 (Leclerc, 1991, J. Immunol. 147:3545-3552); and an
epitope of foot and mouth disease VP1
protein, (residues 141-160. Clarke et al., 1987, Nature 330381-384)- Still
further B cell epitopes which may be used
are known or may be identified by methods known in the art, as set forth in
Caton et al.,1982, Cell 31:417-427.
In additional embodiments of the invention, the peptides may comprise T cell
epitopes. The term "T cell
epitope", as used herein, refers to a peptide, including a peptide sequence
within a larger protein, which when
associated with MHC self antigens and recognized by a T cell, functionally
activates the T cell. In this regard the
present invention provides for the Th epitopes which, in the context of MHC
class II self antigens, may be recognized
by a helper T cell and thereby promote the facilitation of B cell antibody
production via the Th cell.
For example, and not by way limitation, influenza A hemaggiutinin (HA) protein
of PR8 strain, bears, at amino
acid residues 110-120, a Th epitope. Other examples of known T cell epitopes
include. but are not limited to, two
promiscuous epitopes of tetanus toxoid (Ho et al., 1990, Eur J.Immunal. 20:477-
4831; an epitope of cytochrome c,
(residues 88-1031; an epitope of Mycrobacteria heatshock protein, (residues
350-369, Vordermir et al., Eur. J.
Immunol. 24:2061-20671; an epitope of hen egg white lysozyme, (residues 48-61,
Neilsonet al., 1992, Proc. Natl.
Acad. Sci. U.S.A. 89:7380-7383); an epitope of StreptococcusA M protein,
(residues 308-319, Rossiter et al., 1994,
Eur. J. Immunol. 24:1244-12471; and an epitope of Staphylococcus nuclease
protein, (residues B1-100, de Magistris,
1992, Dell 68:1-201. Still further Th epitopes which may be used in
conjunction with the instant invention are known
or may be readily identified by methods known in the art.
As a further example, a relevant epitope may be a CTL epitope, which, in the
context of MHC class I self
antigens, may be recognized by a cytotoxic T cell and thereby promote CTL-
mediated lysis of cells comprising the
target antigen. Nonlimiting examples of such epitopes include epitopes of
influenza virus nucleoproteins corresponding
to amino acid residues 147-161 and 365-379, respectively (Taylor et al., 1989
immunogenetics 26:267; Townsend
et al., 1983, Nature 348:674); LSMV peptide, /amino acid residues 33-41;
Zinkernagel et al., 1974, Nature 248:701-
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702): and ovalbumin peptide, corresponding to amino acid residues 257264
(Cerbone et al., 1983, J. Exp. Med
163:603-612).
With regard to genetic vaccines, one or more target antigens will be expressed
by the host following
transfection or transformation of autologous cells with the administered
genetic material. The expressed antigenls)
then elicit the desired immune response in the subject. Those skilled in the
art will appreciate that genetic material
may be associated with the powder in the form of naked molecules (e.g. DNA or
RNA) or in a viral vector form. In
either case, nucleic acids compatible with the invention will preferably
encode one or more relevant epitopes, and may
optionally further comprise elements that regulate the expression andlor
stability andlor immunogenicity of the relevant
epitope.
For example, elements that regulate the expression of the epitope encoded
within the genetic construct
include, but are not limited to, a promoterlenhancer element, a
transcriptional initiation site, a polyadenylation site, a
transcriptional termination site, a ribosome binding site, a translational
start codon, a translational stop codon, a signal
peptide, etc. Specific examples include, but are not limited to, a promoter
and intran A sequence of the initial early
gene of cytomegalovirus (CMU or SU40 virus ("SU40"1; Montgomery et al., 1993,
DNA and Cell Biology 12:777
783). Alternatively, more than one epitope may be expressed within the same
open reading frame. Examples of
genetic vaccines which may be used according to the invention, and methods for
their production, are set forth in
International Application Publication No. WO 94121797, by Merck & Co. and
Vical, Inc., International Application
Publication No. WO 97121687, by Mt. Sinai, United States Patent Nos. 5,589,466
and 5,580,859 and in International
Application Publication No. WO 90f 11092, by Uical, Inc., the contents of
which are incorporated by reference in their
entireties.
To provide enhanced stability andlor immunogenicity of the relevant epitope,
it may be desirable to present
the epitope in the context of a larger peptide or protein. For example, the
relevant epitope may be expressed in the
variable region of a chimeric antibody or as a portion of a fusion protein. In
other preferred embodiments, it may be
advantageous to administer a fulhlength protein (e.g. a viral coat protein)
comprising one or more relevant epitopes.
Alternatively, it may be desirable to administer powders or perforated
microstructures comprising combinations or
cocktails of immunogenic peptides or proteins. In this regard it will be
appreciated that the relevant epitapes may be
derived from the same or different pathogens. With respect to the latter,
opportunistic pathogens may be targeted
along with the primary disease causing agent. In addition to the broad target
range, the disclosed compositions may
comprise various epitope combinations. For example, the compositions of the
present invention may comprise nucleic
acids or peptides or proteins comprising mixtures of B cell epitopes, mixtures
of T cell epitopes, or combinations of B
and T cell epitopes.
Mare particularly, the administration of compositions that comprise or express
more than one relevant
epitope may exhibit an unexpected synergistic effect. It will be appreciated
that such combination vaccines may prove
to be much more efficient at conferring the desired immunity with respect to
the selected pathogents) than
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compositions comprising a single nucleic acid species encoding a single
relevant epitope. Those skilled in the art will
further appreciate that such synergism could allow for an effective
immunoprophylactic or immunotherapeutic
response to be generated with lower dosing and less frequent administration
than single-epitope vaccines. Moreover,
the use of such multi-epitope vaccine compositions may provide more
comprehensive protection as the induced multi-
site immunity would tend to be more resistant to natural phenotypic variation
within a species or rapid mutation of a
target antigen by the selected pathogen. Of course, effective immunity may
also be imparted by vaccines encoding a
single B or T cell epitope and such compositions are clearly contemplated as
being within the scope of the present
invention.
In addition to the antigens themselves, the current invention permits
manipulation of the excipient
components of the particle shell itself to enhance or modify the
immunogenicity of the formulated antigen. For
example, efficient antigen capture by dendritic cells has been shown to be
facilitated when the antigen uptake is
facilitated by the mannose receptor and hence improves targeting to the
lysosomal compartment (Salusto et al, J.
Expt Med. 182:389-400, 1995). Therefore, incorporation of a low percent of
mannans, or other polysaccharides that
bind to receptors on cells, into the particufates would be predicted to
enhance the immunogenicity. Furthermore, as
1 S will be discussed in more detail below, the use of cofactor or cytokines
to promote APC responses might also serve to
enhance or suppress an immune response as required. The current invention
permits for co-formulation of antigen with
cofactors that might augment stimulation local immune responses within the
mucosa or other targeted sites of delivery
(e.g. transdermal) directed to local dendritic cell or other APC. By
facilitating APC activation and enhancing antigen
uptake and presentation within a local environment, such combination
formulations provided by the current invention
could lead to increased efficiency of the resultant immune response.
More generally, the methods and compositions of the present invention provide
for an enhanced immune
response when used to immunize or vaccinate a subject. This "adjuvant effect"
provided by the disclosed particulates
may be used to elicit an immune response comparable to that elicited by an
antigen administered with an adjuvant Ii.e.
alum or complete Freund's adjuvantl. Unlike the present invention, those
skilled in the art will appreciate that such
traditional adjuvants are typically associated with undesirable side effects
and, in many cases, are not available for
use in humans. Conversely, the present invention can afford an enhanced immune
response (i.e. an immune response
greater than that generated by a comparable antigen presented using art
recognized techniques such as CTL levels for
antibody titers), without the administration of potentially toxic adjuvants.
While not wishing to be bound by any
particular theory, it is believed that the observed enhancement is, at least
in part, a result of the particulate
configuration or morphology, antigen release profile and possible antigen
aggregation within the particulate. In any
event, the effect allows the generation of a clinically useful immune response
with lower levels of antigen andlor fewer
inoculations.
By this adjuvant effect, the immune response provided by the compositions and
methods of the instant
invention is enhanced relative to prior art inoculation techniques. In
particular, the immune response elicited by the
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compositions of present invention will generally be greater than the immune
response provoked by intravenous or
intraperitoneal administration of the same antigen solubilized or suspended in
an aqueous carrier. Of course, the
magnitude of the elicited immune response may be measured using any one of a
variety of techniques well known to
those in the art including compatible methods set forth in the Examples below.
Using such comparisons, the
S preparations of the present invention will preferably provoke an immune
response that is 25%, 50%, 75% or 100%
greater than that provoked by administration of the same antigen using the
prior art methods discussed above. More
preferably, the present invention will provoke a response that is 2, 3, 4 or 5
times greater than the baseline response
obtained using the antigen in an aqueous carrier. In even more preferred
embodiments, the disclosed preparations and
methods will elicit an immune response that is 6, 7, 6, 9 or even 10 times
greater than the baseline response. Still
IO other embodiments may produce responses that are 20, 30, 40, 50 times or
even two orders of magnitude greater
than baseline. Those skilled in the art will appreciate that these novel, and
heretofore unexpected properties, of the
disclosed particulates make them extremely effective in generating the desired
immune response in a subject.
Besides the aforementioned adjuvant effect, other mechanisms may also
contribute to an enhanced immune
response in accordance with the teachings herein. For example, it has
surprisingly been found that combinations of
15 live and killed virus provoke a much stronger response than that provided
by the killed virus alone. More particularly, in
preferred embodiments the powders may be formulated using a live attenuated
virus which is, to some extent, killed or
inactivated during the particulate fabrication. As will be demonstrated in
conjunction with the Examples below, this
mixture of live and killed virus appears to elicit a surprisingly strong, or
enhanced, immune response. Moreover, in
keeping with the teachings herein, the selected virus or virus mixture may
comprise a naturally occurring inactivated or
20 attenuated virus or may be engineered to express one or more foreign
antigens. An alternative method of formulating
live virus provided for by the present invention involves the formulation of
viral receptors within the particle matrix
followed by binding the selected virus to the particles after fabrication
(i.e. after spray dryingl. There are a wide
variety of cellular viral receptors that have now been well defined, for
example, the prolactin receptor which can
function as a retrovirus receptor, CCRS, the cellular receptor for HIV, the
Polio virus receptor, the IgG Fc region which
25 binds HSV1 and receptors that bind influenza virus.
Regardless of the antigen selected or the form of the antigen (virus, peptide,
genetic material, etc.), those
skilled in the art will further appreciate that effective immunization of a
subject may include more than one inoculation.
As used herein, the terms "immunize" or "immunization" or related terms refer
herein to conferring the ability to mount
a substantial immune response (consisting of antibodies or cellular immunity
such as effector CTL) against a target
30 antigen or epitope. These terms do not require that completely protective
immunity be created, but rather that a
protective immune response be produced which is substantially greater than
baseline. For example, a mammalian may
be considered to be immunized against a target antigen if the cellular andlor
humoral immune response to the target
antigen is enhanced following the application of methods of the invention.
Assays demonstrating the enhancement of
both B cell or T cell responses are well known and could easily be performed
by those skilled in the art. Preferably,
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immunization results in significant resistance to the disease caused or
triggered by pathogens expressing target
antigens.
Similarly, the term "inoculating", as used herein, refers to administering or
introducing a composition
comprising at least one vaccine comprising a relevant epitope, or capable of
generating or expressing a relevant
epitope. according to the instant disclosure. While an effective immune
response may be induced with a single
inoculation, effective immunization of a subject may comprise multiple
inoculations or a subsequent booster or
boosters. As such, the methods of the present invention may comprise one, two,
three, four or even five inoculations
in order to achieve the desired immunoprophylactic effect. Moreover, as
previously alluded to the administered vaccine
will preferably contact andlor be absorbed by a mucosal surface. In
particularly preferred embodiments, the mucosal
surface will be associated with oral or nasal passages or cavities or a
pulmonary air passage. Those skilled in the art
will further appreciate that the vaccine compositions of the present invention
(i.e. powders or dispersions) may be used
to inoculate neonates (0-6 mol, infants (6 mo-2 yr), children (2 yr-13 yr) or
adults (73 yr +).
B(ii). Immunoglobulins (Passive Immunotherapy)
While the methods and compositions of the present invention provide effective
means for inducing localized
and systemic active immunity, they may also be used for the induction of
localized or systemic passive immunity. In
particular, the disclosed powders and microparticulates may be used to
administer immunoglobulins, or fragments or
portions thereof, to provide rapid prophylaxis or therapy with regard to
infection or disease. The administered
immunoglobulins, which may be monoclonal or polyclonal, will recognize at
least one antigen on the target pathogen.
Preferably, the recognized antigen or antigens will comprise one or more
relatively conserved epitopes. For the
purposes of the present invention, the administered compositions may comprise
neutralizing, therapeutic or
prophylactic antibodies or combinations thereof. In particularly preferred
embodiments, the administered compositions
will comprise one or more species of monoclonal antibodies or immunoreactive
fragments.
Following administration, the active immunoglobulin or immunoglobulins can
either function at the site of
delivery or be taken up into the systemic circulation. Antibodies retained at
the site of administration could rapidly bind
to any target infectious agent (e.g. an airborne virus) coming in contact with
the treated site (i.e. a mucosal surface)
and prevent subsequent infection or clear the microbes. Alternatively, the
relatively high levels of circulating
antibodies provided by preferred embodiments of the instant invention will
allow rapid clearance of the target pathogen
from the bloodstream thereby preventing, or at least ameliorating, symptoms
associated with infection. Of course it
will be appreciated that, unlike active immunization which can last for the
lifetime of the subject, passive immunization
is relatively transitory, lasting as long as the delivered immunoglobulin dose
remains in the circulation.
As alluded to above any immunoglobulin, or immunoreactive fragment thereof,
that recognizes an antigen or
antigens on a target pathogen, may be used to confer the desired immunity on a
subject. The ability to provide bath
monoclonal and polyclonal antibodies to particular pathogens andfor antigens
and/or epitopes is well known in the art.
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With respect to the form of antibody actually administered, it will be
appreciated that bath native and engineered
antibodies are compatible with the teachings herein, as are different classes
of antibodies including IgA, IgD, IgE, IgG
and IgM. Similarly, any immunoreactive fragment or domain of an
immunoglobulin, including F(ab')Z, Fab, or Fv can be
used to provide the desired protection. Regarding engineered antibodies,
humanized constructs (i.e. chimeric
antibodies) are particularly preferred. While such immunoglobulins typically
contain the antigen binding complementarity
determining regions (CDRs) of murine antibodies, the remainder of the molecule
is comprises human antibody sequences
which are not recognized as foreign. See, for example, Jones et al., Nature,
321:522-525 (1986) which is incorporated
herein by reference. As human polyclonal IgG is not typically recognized as
foreign by the subject, these antibodies da not
tend to produce undesirable side effects if infrequently administered and are
not rapidly eliminated by the body.
Passive immunity is particularly effective in preventing or reducing the
chances of infection by readily
transmitted pathogens, particularly those that are air ar water borne. As
such, powders and dispersions of the present
invention comprising the appropriate immunoglobulins are especially effective
against respiratory viruses and
pathogens such as influenza or respiratory syncytial virus. For example, a
stabilized dispersion comprising
immunoglobulin laden perforated microstructures in a liquid fluorocarbon
medium could be administered to the nasal
passages via an atomizer or spray bottle. The composition, which could easily
be administered as needed, would
provide both localized and systemic passive immunity with respect to a target
pathogen such as a cold virus
(Orthomyxovirus, Paramyxovirus, Rhinovirus). Similarly, readily administered
compositions could be provided in
accordance with the present invention to provide protection against water
borne agents such as Vibrio choleiae.
Passive immunity as disclosed herein could also be used to provide at least
some protection with respect to various
organisms including, but not limited to, Rabies virus, Hepatitis (A, B C)
viruses, HIU and Clast~idium tetanii. Other
infectious agents for which passive immunity may be imparted by the disclosed
compositions may easily be identified
by those skilled in the art.
B(iii). Tumor Antigens
In alternative embodiments, the target antigen may be a tumor antigen. Those
skilled in the art will
appreciate that tumor antigens are often peptide fragments derived from cell
proteins that either are restricted to the
type of tissue from which the tumor is derived or are mutated during the
course of malignant transformation. Other
tumor antigens are often aberrantly expressed by the tumor cell andlor
represent "neo" antigens resulting from errors
in transcription, RNA processing due to mutations that are idiosyncratic to
the tumor cells. Alternatively, changes in
post-translational modifications of a normal protein (e.g. glycosylation) may
aid in revealing hitherto hidden (cryptic)
epitopes not normally recognized by the immune system (e.g., as is the case
with the mucin, MUC1). B cell epitopes
associated with tumor antigens are expressed at the surface of tumor cells and
are recognized by specific antibodies.
In contrast, T cell epitopes are of two types: CTL epitopes that are MHC class
I-restricted peptides derived from tumor
associated antigens and Th epitopes that are MHC class II-restricted peptides
derived from tumor antigens. Whereas
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Th epitopes are mostly presented by antigen presenting cells (APC) to CD4' T
cells, CTL epitopes are presented by
APC as well as tumor cells and are recognized by tumor-specific CD8" T cells.
Exemplary tumor antigens include, but
are not limited to, carcinoembryonic antigen ("CEA"1, melanoma associated
antigens, alpha fetoprotein, papilloma virus
antigens, Epstein Barr antigens, MUC 1, p53, etc. Several other tumor antigens
are reportedly recognized by
S autologous cytotoxic T lymphocytes as set forth in Boon, T., et al. J. Exp.
Med., 183:725-729, 1996; Disis, M.L., et
al. Curr. Opin. Immunol. 8:637-642, 1996; Bobbins, P.F., et al. Curr. Opin.
Immunol. 8:628-636, 1996, Salgaller et
al., J. Surg. Oncol. 68:122-138, 1998, each of which is incorporated herein
their entirety.
Bliv). Immune Modulation
Autoimmune diseases are mediated by autoreactive T and B cells as well as
other immune cell subtypes that
may exert regulatory or effector roles. It is thought that T cells recognizing
organ-specific self epitopes are a key element
in the pathogenesis of autoimmune diseases such as diabetes type I, multiple
sclerosis (MS) or rheumatoid arthritis (RAI.
CD4+ and in certain cases, CD8+ T cells recognizing antigens presented in
certain locations of the body may infiltrate the
tissue and trigger destruction of various cell types and persisting
inflammation. Whereas CD4+ Th1 cells that produce IL-
2, lFN-, TNF- and LT- are considered pathogenic, CD4+ Th2 cells that produce
IL-4, IL-10, IL-5, IL-13 and IL-9 are
considered non-pathogenic relative to autoimmunity and in certain
circumstances may suppress disease. Furthermore, Th3
cells induced by mucosaf exposure to antigens, to secrete TGF- and IL-10, are
thought to be crucial mediators of mucosal-
induced tolerance.
As a strategy to prevent or suppress the autoimmune diseases, autoreactive T
cells provide a good therapeutic
target. There are several means of inactivating the pathogenic autoreactive T
cells (general designation of "tolerance",
which is not necessarily restricted to "deletion"), responsible for the
autoimmune disease: (1) to directly turn-off ar
energize the pathogenic cells by providing long-time exposure to high levels
of antigen; (2) to energize or switch the
function of pathogenic T cells by exposing them to antigens in context of non-
professional APC or certain modulating
factors; and (3) to induce antigen-specific Th suppressor cells of Th21Th3
phenotype that migrate to the site of disease
and inhibits the function of pathogenic T cells.
Surprisingly, it has been found that tolerance may be induced in accordance
with the present invention through
the use of inhalation therapies. The advantage of the respiratory tract as the
target site for immune tolerance induction is
two-fold: first, it is a non-invasive route that allows local and systemic
delivery of complex antigens; and secondly,
mucosal immunity is likely to comprise Th21Th3 suppressor cells to the
administered antigens. Such antigens may be
whole self antigens (recombinant or purified), antigen fragments (obtained by
molecular biology or biochemical techniques
well known in the art) or peptides limited to epitopes. In other embodiments
they may be incorporated as virus
components, phages, chimeric antibodies, fusion proteins, replicons, bacteria
or delivered via nucleic acid-based or viral
vectors. They may be incorporated in self molecules like immunoglobulins or
any natural or synthetic ligand for receptors
on body cells. They may be administered as isolated, individual components or
in mixtures. Examples for diabetes type I
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include but are not limited to such peptides and antigens as: GAD65 (glutamic
acid decarboxylase 65 - Baekkeskov et al.,
Nature 1990, 347:151), insulin (Palmer et al., Science 1983, 222:1337),
ICA51211A-2 (islet cell antigen 512; Robin et
al., J. Immunol. 1994, 152:31831. In the case of MS, such proteins and
peptides are: MBP (myelin basic protein,
Steinman et al-, 1995, Mol. Med. Today, 1:79; Warren et al., 1995, Proc. Natl.
Acad. Sci. USA , 92:11061 ). PLP.
transaldolase, 2',3' cyclic nucleotide 3' phosphodiesterases (CNP), MOG and
MAG (Steinman L., 1995, Nature, 375:739).
Besides autoimmune diseases, it will be appreciated that the compositions and
methods of the present invention may also
be used to down regulate immune responses provoked by allergens.
B/v). Active Peptides and Proteins
Certain peptides and proteins are known to have to ability to modulate, up-
regulate or down-regulate immune
responses to foreign or self antigens. Such peptides or proteins may act by
engaging endogenous receptors leading to
activation or inhibition of certain processes, or by interfering with the
ligand-receptor binding of endogenous elements.
Examples of such proteins or peptides are cytokines that exert immune
modulatory function leading to suppression of
autoimmunity: interferon- , IL-4, IL-10, IL-13, IL-9, native or in the form of
fragments attached, incorporated or complexed
with other molecules. Other cytokines may act as immune activators, leading to
increased immunity against microbes or
tumor cells: IL-12, IL-2, interferon-, interferon-, TNF-, TNF-, lymphotoxins,
and GM-CSF. For example, co-administration
of GC-MSF, IFN-a, IL-2, IL-12 or TNF-a has been demonstrated to enhance an
immune response and antigen
presentation. However, systemic delivery of such agents in many cases has led
to unacceptable side effects, leading
to a concerted effort directed at targeted delivery of these pluripotent
factors. The current invention advantageously
permits for co-formulation of a selected antigen or antigens with cofactors
that might augment stimulation local
immune responses within the mucosa or other targeted sites of delivery (e.g.
transdermal or intradermal) directed to
local dendritic cell or other APC presentation. By facilitating APC activation
and enhancing antigen uptake and
presentation within a local environment such combination formulations of the
current invention could provide for
increased efficiency of the resultant immune response.
Other active proteins or peptides that may be used in accordance with the
present invention comprise
chemokines in native form or as fragments, constructs or complexes with other
molecules which may increase, modulate
or inhibit the recruitment of lymphocytes. Far example, whereas eotaximl,
eotaxin-2, TARC, MIP-3b, SLC are thought to
mediate the recruitment of Th2 cells, MIG, IP-10, MIP-1 , MIP-1 and RANTES are
thought to mediate the recruitment of
Tht cells fSallusto et al., 1998, J. Exp. Med., 187:875; Ward et al., 1998,
Irrununity, 9:1). Similarly, cytokine or
chemakine receptors in native form, or as fragments, recombinant constructs or
complexes with other molecules may
inhibit the recruitment or activation of certain lymphocytes. Examples of
cytokine and chemokine receptors that are likely
to inhibit ongoing Th1 responses comprise the IL-12 receptor, IFN- receptor,
IL-2 receptor, TNF- receptor, CXCR3 or
CCR5. Examples of cytokine and chemokine receptors that are likely to inhibit
ongoing Th2 responses are the IL-4 receptor,
IL-13 receptor, IL-9 receptor, IL-10 receptor, CCR3, CCR4 ar CCR7. Of course,
it will be appreciated that compatible
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compounds are not limited to cytokines, chemokines or their receptors, but may
include other ligands or receptors (in native
form, fragments, constructs or complexes with other molecules) like integrins
and homing receptors. In preferred
embodiments all these categories of compounds may be formulated and
administered either locally or systemically via the
respiratory tract in order to enhance, suppress, or modulate an immune
response.
It will further be appreciated that the perforated microstructures according
to the invention may, if desired,
contain a combination of two or more active ingredients. The agents may be
provided in combination in a single
species of perforated microstructure or individually in separate species of
perforated microstructures. For example,
two or more active or bioactive agents may be incorporated in a single feed
stock preparation and spray dried to
provide a single microstructure species comprising a plurality of bioactive
agents. Conversely, the individual agents
could be added to separate stocks and spray dried separately to provide a
plurality of microstructure species with
different compositions. These individual species could be added to the
suspension medium or dry powder dispensing
compartment in any desired proportion and placed in the aerosol delivery
system as described below.
Based on the foregoing, it will be appreciated by those skilled in the art
that a wide variety of bioactive agents
may be incorporated in the disclosed powders. Accordingly, the list of
preferred bioactive agents above is exemplary only
and not intended to be limiting. It will also be appreciated by those skilled
in the art that the proper amount of bioactive
agent and the timing of the dosages may be determined for the formulations in
accordance with already existing
information and without undue experimentation.
C. Powder Composition
As may be seen from the discussion above, the present invention may be used to
effectively deliver a wide
variety of bioactive agents. While the particulates may be formed exclusively
by the bioactive agent, they will preferably
comprise one or more additional materials which, in selected embodiments, may
comprise absorption enhancers,
potentiators, excipients or structural components. More generally, the
particulates (i.e. the structural matrix may be
formed of or comprise any material which possesses physical and chemical
characteristics that are compatible with any
incorporated active agents. While a wide variety of materials may be used to
form the powders, in particularly preferred
pharmaceutical embodiments the particulate is associated with, or comprises, a
surfactant such as phospholipid or
fluorinated surfactant. Although not required, the incorporation of a
compatible surfactant can improve powder
ffowability, increase aerosol efficiency, improve dispersion stability, and
facilitate preparation of a suspension. Moreover,
selected surfactants may also function as absorption enhancers thereby
increasing uptake and improving bioactivity of the
selected agent. Of course it will be appreciated that the powders of the
present invention may also be formed using
nothing more than traditional non-surfactant excipients and one or more
incorporated bioactive agents.
As indicated, the disclosed powders may optionally be associated with, or
comprise, one or more surfactants. In
accordance with the teachings herein, these compounds may serve to stabilize
any incorporated bioactive agent, assist in
stabilizing particulates suspended in a nonaqueous media or potentiate the
uptake of an agent at the target site. Besides
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those surfactants associated with the disclosed particulates, miscible
surfactants may optionally be combined in the case
where the microparticles are formulated in a suspension medium liquid phase.
It will be appreciated by those skilled in the
art that the use of surfactants, while not necessary to practice the instant
invention, may further increase dispersion
stability, powder flowability, simplify formulation procedures or increase
efficiency of delivery. Of course combinations of
surfactants, including the use of one or more in the liquid phase and one or
more associated with the perforated
microstructures are contemplated as being within the scope of the invention.
By "associated with or comprise" it is meant
that the particulate or perforated microstructure may incorporate, adsorb,
absorb, be coated with or be formed by the
surfactant-
In a broad sense, surfactants suitable for use in the present invention
include any compound or composition that
aids in the formation of perforated microparticles or provides enhanced
suspension stability, improved powder dispersibility
or decreased particle aggregation. The surfactant may comprise a single
compound or any combination of compounds,
such as in the case of ca-surfactants. Particularly preferred surfactants are
nonfluorinated and selected from the group
consisting of saturated and unsaturated lipids, nonionic detergents, nonionic
block copolymers, ionic surfactants and
combinations thereof. In those embodiments comprising stabilized dispersions,
such nonfluorinated surfactants will
preferably be relatively insoluble in the suspension medium. It should be
emphasized that, in addition to the
aforementioned surfactants. suitable fluorinated surfactants are compatible
with the teachings herein and may be used
to provide the desired preparations.
Lipids, including phospholipids, from both natural and synthetic sources are
particularly compatible with the
present invention and may be used in varying concentrations to form the
particulates or structural matrix. Generally
compatible lipids comprise those that have a gel to liquid crystal phase
transition greater than about 40°C. Preferably
the incorporated lipids are relatively long chain (i.e. C,s-CZZ) saturated
lipids and more preferably comprise
phospholipids. Exemplary phospholipids useful in the disclosed stabilized
preparations comprise,
dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,
diarachidoylphosphatidylcholine
dibehenoylphosphatidylcholine, short-chain phosphatidylcholines, long-chain
saturated phosphatidylethanolamines,
long-chain saturated phosphatidylserines, long-chain saturated
phosphatidylglycerols, long-chain saturated
phosphatidylinositols, glycolipids, ganglioside GM1, sphingomyelin,
phosphatidic acid, cardiolipin; lipids bearing polymer
chains such as polyethylene glycol, chitin, hyaluronic acid, or
polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-,
and polysaccharides: fatty acids such as palmitic acid, stearic acid, and
oleic acid; cholesterol, cholesterol esters, and
cholesterol hemisuccinate.
Compatible nonionic detergents comprise: sorbitan esters including sorbitan
trioleate (Span~ 851, sorbitan
sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20)
sorbitan monolaurate, and
polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether,
stearyl polyoxyethylene (2) ether, lauryl
polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable
nonionic detergents can be easily
identified using McCutcheon's fmulsifiers and Detergents (McPublishing Co.,
Glen Rock, New Jersey) which is
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incorporated herein in its entirety. Preferred block copolymers include
diblock and triblock copolymers of
polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic~ F-
681, polaxamer 407 (Pluronic~ F-127),
and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty
acid soaps may also be utilized. In
preferred embodiments the microstructures may comprise oleic acid or its
alkali salt. Due to their excellent
biocompatibility characteristics, phospholipids and combinations of
phospholipids and poloxamers are particularly
suitable for use in the pharmaceutical embodiments disclosed herein.
In addition to the aforementioned surfactants, cationic surfactants or lipids
are preferred especially in the
case of delivery or RNA or DNA. Examples of suitable cationic lipids include:
DOTMA, N-[1-(2,3-dioleyloxy)propyl]-
N,N,N-trimethylammonium chloride; DOTAP, 1,2-dioleyloxy-3-
(trimethylammoniolpropane; and DOTB, 1,2-dioleyl-3-14'-
trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as
polyiysine, and polyarginine are also
contemplated.
Besides those surfactants enumerated above, it will further be appreciated
that a wide range of surfactants
may optionally be used in conjunction with the present invention. Moreover,
the optimum surfactant or combination
thereof for a given application can readily be determined by empirical studies
that do not require undue
experimentation. Finally, as discussed in more detail below, surfactants
comprising the particulate or structural matrix
may also be useful in the formation of precursor oil-in-water emulsions (i.e.
spray drying feed stock) used during
processing to form the perforated microstructures.
Unlike prior art formulations, it has surprisingly been found that the
incorporation of relatively high levels of
surfactants or biocompatible wall forming material (e.g., phospholipids) may
be used to improve powder dispersibility,
increase suspension stability and decrease powder aggregation of the disclosed
applications. That is, on a weight to
weight basis, the particulate or structural matrix of the perforated
microstructures may comprise relatively high levels of
surfactant. In this regard, the particulates will preferably comprise greater
than about 1 %, 5%, 10%, 15%, 18°Yo, or even
20% wlw surfactant. More preferably, the microparticulates or microstructures
will comprise greater than about 25%,
30%, 35%, 40%, 45%, or 50% wlw surfactant. Still other exemplary embodiments
will comprise particulates wherein the
surfactant or surfactants are present at greater than about 55%, 60%, 659'0,
70%, 75%, 8090, 85%, 90% ar even 95%
wlw. In selected embodiments the powders will comprise essentially 100% wlw of
a surfactant such as a phospholipid.
Those skilled in the art will appreciate that, in such cases, the balance of
the particulate or structural matrix (where
applicable) will likely comprise a bioactive agent, excipients or other
additives.
As will be discussed below, surfactants may be incorporated in any type of
particulate. That is, while the
aforementioned surfactant levels are preferably employed in perforated
microstructures, they may be used to provide
powders or stabilized dispersions comprising relatively nonporous, or
substantially solid, particulates. While selected
embodiments of the present invention will comprise perforated microstructures
associated with high levels of surfactant,
compatible powders may be formed using relatively low porosity particulates of
equivalent surfactant concentrations.
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Preferably, such particulates will comprise relatively high levels of
surfactant on the order of greater than about 5% wlw.
In this respect, such embod'unents are specifically contemplated as being
within the scope of the present invention.
In other preferred embodiments of the invention, the particulates optionally
comprise synthetic or natural
polymers or combinations thereof. In this respect useful polymers comprise
polylactides, polylactide-co-glycolides,
cyclodextrins, polyacrylates, methylcellulose, carboxymethylcellulose,
polyvinyl alcohols, polyanhydrides, polylactams,
polyvinyl pyrrolidones, monosaccharides, disaccharides or polysaccharides
(dextrans, starches, chitin, chitosan, etc.),
hyaluronic acid, proteins, (albumin, collagen, gelatin, etc.). Examples of
polymeric resins that might prove useful for the
preparation of microparticles include: styrene-butadiene, styrene-isoprene,
styrene-acrylonitrile, ethylene-vinyl acetate,
ethylene-acrylate, ethylene-acrylic acid, ethylene-methylacrylatate, ethylene-
ethyl acrylate, vinyl-methyl methacrylate,
acrylic acid-methyl methacrylate, and vinyl chloride-vinyl acetate. Those
skilled in the art will appreciate that, by
selecting the appropriate polymers, the delivery efficiency of the
particulates andlor the stability of the dispersions may be
tailored to optimize the effectiveness of the active ar bioactive agent.
Besides the aforementioned polymer materials and surfactants, various
excipients may be incorporated in, or
added to, the particulates to provide structure and, in preferred embodiments
form perforated microstructures (i.e.
1 S microspheres such as latex particles). In this regard it will be
appreciated that the rigidifying components can be
removed using a post-production technique such as selective solvent
extraction. Compatible excipients may include,
but are not limited to, carbohydrates including monosaccharides, disaccharides
and polysaccharides. For example,
monosaccharides such as dextrose (anhydrous and monohydrate), galactose,
mannitol, D-mannose, sorbitol, sorbose
and the like; disaccharides such as lactose, maltose, sucrose, trehalose, and
the like; trisaccharides such as raffinose
and the like; and other carbohydrates such as starches (hydroxyethylstarchl,
cyclodextrins and maltodextrins. Amino
acids are also suitable excipients with glycine preferred. Mixtures of
carbohydrates and amino acids are further held
to 6e within the scope of the present invention. The inclusion of both
inorganic (e.g. sodium chloride, calcium chloride,
etc.), organic salts (e.g. sodium citrate, sodium ascorbate, magnesium
gluconate, sodium gluconate, tromethamine
hydrochloride, etc.) and buffers is also contemplated. The inclusion of salts
and organic solids such as ammonium
carbonate, ammonium acetate, ammonium chloride or camphor are also
contemplated.
Along with the compounds discussed above, it may be desirable to add other
excipients to a microsphere
formulation to improve particle rigidity, production yield, delivery
efficiency and deposition, shelf-life and patient
acceptance. Such optional excipients include, but are not limited to: coloring
agents, taste masking agents, buffers,
hygroscopic agents, antioxidants, and chemical stabilizers. Moreover, as
discussed above, the particulates may
comprise compounds that can potentiate, induce or modulate the uptake of the
associated bioactive agent. Further,
the particulates of the invention may comprise targeting molecules such as
antibodies, cofactors, receptors, ligands
and substrates that preferentially direct the particulates, or allow them to
bind, to molecules associated with cells at
the target site. For example, particulates could be formed comprising an
antibody targeting a mucosal cell receptor
and an immunoactive compound. Such targeting molecules would likely increase
the concentration of bioactive
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particulates at the target mucosal site and further enhance any localized
immune response. It will be appreciated that
ligands directed to receptors preferentially expressed on the surface of
mucosal or other target cells could also be used
to increase the binding of particulates at the desired site.
Yet other preferred embodiments include perforated microstructures that may
comprise, or may be coated with,
charged species that prolong residence time at the paint of contact or enhance
penetration through mucosae. For example,
anionic charges are known to favor mucoadhesion while cationic charges may be
used to associate the formed
microparticulate with negatively charged bioactive agents such as genetic
material. The charges may be imparted through
the association or incorporation of polyanionic or polycationic materials such
as polyacrylic acids, polylysine, polylactic
acid and chitosan.
D. Powder Morphology
Those skilled in the art will appreciate that powders or particulates of
various compositions, configurations
and morphologies may be used in accordance with the present invention as long
as they provide desired stability and
delivery characteristics. In this respect, it may be advantageous to use
relatively dense, solid particulates or powders
for same applications (e.g. for intradermal administration of a stabilized
dispersion via a air gun or needleless injector)
while in other embodiments (e.g. DPI administration) a relatively porous,
aerodynamically light perforated
microstructure may be preferred. Accordingly, while the present invention may
be discussed below in terms of
preferred embodiments, it must be emphasized that it is not limited to any
particular particle composition,
configuration or morphology. Rather, selection of particulate characteristics
(charge, density, composition, etc.) is
largely based on the form of administration, targeted delivery site and choice
of bioactive agent.
While various particulate configurations, including micronized and milled
particulates, may be used in
accordance with the teachings herein, the present invention provides unique
methods and compositions to reduce
cohesive forces between dry particles, thereby minimizing particulate
aggregation that can result in improved delivery
efficiency. As such, selected disclosed preparations provide a highly
flowable, dry powders that can be efficiently
aerosolized, uniformly delivered and penetrate deeply in the lung ar nasal
passages. Moreover, selected powder
configurations and morphologies have been found to provide relatively stable
dispersions when combined with a
nonaqueous suspension medium. In either case, the disclosed particulates may
be fabricated so as to result in
surprisingly low throat deposition upon administration.
As previously discussed, particularly preferred embodiments of the present
invention incorporate powders or
particulates in the form of porous or perforated microstructures comprising a
structural matrix. It will be appreciated that,
as used herein, the terms "structural matrix" or "microstructure matrix" are
equivalent and shall be held to mean any solid
material forming perforated microstructures which define a plurality of voids,
apertures, hollows, defects, pores, holes,
fissures, etc. that provide the desired characteristics. In selected
embodiments, the perforated microstructures defined by
the structural matrix comprise a spray dried hollow porous microsphere
incorporating at least one surfactant. ~t will
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further be appreciated that, by altering the matrix components, the density of
the structural matrix may be adjusted so as
to further increase dispersion stability or delivery efficiency.
The absolute shape (as opposed to the morphology) of the particuiates or
perforated microstructures is generally
not critical and any overall configuration that provides the desired
characteristics is contemplated as being within the
scope of the invention. Accordingly, preferred embodiments can comprise
approximately microspherical shapes. However,
collapsed, deformed or fractured particulates are also compatible. With this
caveat, it will further be appreciated that,
particularly preferred embodiments of the invention comprise spray dried
hollow, porous microspheres. In any case the
disclosed powders of perforated microstructures provide several advantages
including, but not limited to, increases in
suspension stability, improved dispersibility, superior sampling
characteristics, elimination of carrier particles and
enhanced aerodynamics.
To maximize dispersibility, dispersion stability and optimize distribution
upon administration, the mean geometric
particle size of the particulates or perforated microstructures is preferably
about 0.550 Nm, more preferably 1-30 Nm. It
will be appreciated that large particles (i.e. greater than 50,um) may not be
preferred in applications where a valve or small
orifice is employed, since large particles tend to aggregate or separate from
a suspension which could potentially clog
the device. In especially preferred embodiments the mean geometric particle
size (or diameter) of the perforated
microstructures is less than 20 ~m or less than lONm. More preferably the mean
geometric diameter is less than about 7
,um or 5 Nm, and even more preferably less than about 4 Nm or even 2.5 Nm.
Other preferred embodiments will comprise
preparations wherein the mean geometric diameter of the perforated
microstructures is between about 1,um and 5 Nm. In
especially preferred embodiments the perforated microstructures will comprise
a powder of dry, hollow, porous
microspherical shells of approximately 1 to l0,um or 1 to 5 Nm in diameter,
with shell thicknesses of approximately 0.1
,um to approximately 0.5,um. It is a particular advantage of the present
invention that the particulate concentration of the
dispersions and structural matrix components can be adjusted to optimize the
delivery characteristics of the selected
particle size.
As alluded to throughout the instant specification the porosity of the
microstructures may play a significant part
is establishing dispersibility (e.g. in DPIs) or dispersion stability (e.g.
for MDIs, jet guns or nebulizersl. In this respect, the
mean porosity of the perforated microstructures may be determined through
electron microscopy coupled with modem
imaging techniques. More specifically, electron micrographs of representative
samples of the perforated microstructures
may be obtained and digitally analyzed to quantify the porosity of the
preparation. Such methodology is well known in the
art and may be accomplished without undue experimentation.
For the purposes of the present invention, the mean porosity (i.e. the
percentage of the particle surface area that
is open to the interior andlor a central void) of the particulates or
perforated microstructures may range from
approximately 0.5% to approximately 80%. In more preferred embodiments, the
mean porosity will range from
approximately 2% to approximately 40%. Based on selected production
parameters, the mean porosity may be greater
than approximately, 2%, 59'0, 10°'0, 15%, 20%, 25% or 30% of the
microstructure surface area. In other embodiments,
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the mean porosity of the microstructures may be greater than about 40%, 50%,
60%, 70% or even BO%. As to the pores
themselves, they typically range in size from about 5 nm to about 400 nm with
mean pore sizes preferably in the range of
from about 20 nm to about 200 nm. In particularly preferred embodiments the
mean pore size will be in the range of from
about 50 nm to about 100 nm. As will be discussed in more detail below, it is
a significant advantage of the present
invention that the pore size and porosity may be closely controlled by careful
selection of the incorporated components and
production parameters.
In this regard, the particle morphology andlor hollow design of the
particulates or perforated microstructures also
plays an important role on the dispersibility or cohesiveness of the dry
powder formulations disclosed herein. That is, it
has been surprisingly discovered that the inherent cohesive character of fine
powders can be overcome by lowering the van
der Waals. electrostatic attractive and liquid bridging forces that typically
exist between dry particles. More specifically,
in concordance with the teachings herein, improved powder dispersibility may
be provided by engineering the particle
morphology and density, as well as control of humidity and charge. To that
end, preferred embodiments of the present
invention comprise perforated microstructures having pores, voids, hollows,
defects or other interstitial spaces which
reduce the surface contact area between particles thereby minimizing
interparticle forces. In addition, the use of
surfactants such as phospholipids and fluorinated blowing agents in accordance
with the teachings herein may contribute
to improvements in the flow properties of the powders by tempering the charge
and strength of the electrostatic forces as
well as moisture content.
Most fine powders (e.g. < 5 Nm) exhibit poor dispersibility which can be
problematic when attempting to
deliver, aerosolize andlor package the powders. In this respect the major
forces which control particle interactions can
typically be divided into long and short range forces. Long range forces
include gravitational attractive forces and
electrostatics, where the interaction varies as a square of the separation
distance or particle diameter. Important short
range forces for dry powders include van der Waals interactions, hydrogen
bonding and liquid bridges. The latter two short
range forces differ from the others in that they occur where there is already
contact between particles. It is a major
advantage of the present invention that these attractive forces may be
substantially attenuated or reduced through the use
of perforated microstructures as described herein.
Those skilled in the art will appreciate that the van der Waals IVDW)
attractive force occurs at short range and
depends, at least in part, on the surface contact between the interacting
particles. When two particles approach each
other the UDW forces increase with an increase in contact area. For two dry
particles, the magnitude of the IIDW
interaction force, F°,~"" can be calculated using the following
equation:
F, o -_ W y r 1 r2
8 ~cd o rl + r2
where A is Planck's constant, w is the angular frequency, do is the distance
at which the adhesional force is at a
maximum, and r, and rz are the radii of the two interacting particles.
Accordingly, it will be appreciated that one way to
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minimize the magnitude and strength of the VDW force for dry powders is to
decrease the interparticle area of contact. It
is important to note that the magnitude of do is a reflection of this area of
contact. The minimal area of contact between
two opposing bodies will occur if the particles are perfect spheres. In
addition, the area of contact will be further
minimized if the particles are highly porous. Accordingly, the perforated
microstructures of the present invention act to
reduce interparticle contact and corresponding VOW attractive forces. It is
important to note that this reduction in VDW
forces is largely a result of the unique particle morphology of the powders of
the present invention rather than an increase
in geometric particle diameter. In this regard, it will be appreciated that
particularly preferred embodiments of the present
invention provide powders having average or small particulates (e.g. mean
geometric diameter < 10 Nm) exhibiting
relatively low VDW attractive forces.
Further, as indicated above, the electrostatic force affecting powders occurs
when either or both of the particles
are electrically charged. This phenomenon will result with either an
attraction or repulsion between particles depending on
the similarity or dissimilarity of charge. In the simplest case, the electric
charges can be described using Coulomb's Law.
One way to modulate or decrease the electrostatic forces between particles is
if either or both particles have non-
conducting surfaces. Thus, if the perforated microstructure powders comprise
excipients, surfactants or active agents
that are relatively non-conducting, then any charge generated in the particle
will be unevenly distributed over the surface.
As a result, the charge half-life of powders comprising non-conducting
components will be relatively short since the
retention of elevated charges is dictated by the resistivity of the material.
Resistive or non-conducting components are
materials which will neither function as an efficient electron donor or
acceptor.
Derjaguin et al. (Muller, V.M., Yushchenko, V.S., and Derjaguin, B.V., J.
Colloid Interface Sci. 1980, 77, 115-
119), which is incorporated herein by reference, provide a list ranking
molecular groups for their ability to accept or donate
an electron. In this regard exemplary groups may be ranked as follows:
Donor: -NHZ > -OH > -OR > -COOR > -CH3 > -CsHs >
-halogen > -COOH > -CO > -CN Acceptor:
The present invention provides for the reduction of electrostatic effects in
the disclosed powders though the use
of relatively non-conductive materials. Using the above rankings, preferred
non-conductive materials would include
halogenated andlar hydrogenated components. Materials such as phospholipids
and fluorinated blowing agents (which
may be retained to some extent in spray dried powders) are preferred since
they can provide resistance to particle
charging. It will be appreciated that the retention of residual blowing agent
(e.g. fluorochemicals) in the particles, even at
relatively low levels, may help minimize charging of particulates or
perforated microstructures as is typically imparted
during spray drying and cyclone separation. Based on general electrostatic
principles and the teachings herein, one skilled
in the art would be able to identify additional materials that serve to reduce
the electrostatic forces of the disclosed
powders without undue experimentation. In this regard, highly charged agents
can be electrostatically modified and
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controlled through simple pH adjustments or chelation with oppositely charged
compounds, e.g. associating nucleic acids
with cationic lipids. Further, if needed, the electrostatic forces can also be
manipulated and minimized using efectrificatian
and charging techniques.
In addition to the surprising advantages described above, the present
invention further provides far the
attenuation or reduction of hydrogen and liquid bonding. As known to those
skilled in the art, both hydrogen bonding and
liquid bridging can result from moisture that is absorbed by the powder. In
general, higher humidifies produce higher
interparticle forces for hydrophilic surfaces. This is a substantial problem
in prior art pharmaceutical formulations for
inhalation therapies which tend to employ relatively hydrophilic compounds
such as lactose. However, in accordance with
the teachings herein, adhesion forces due to adsorbed water can be modulated
or reduced by increasing the hydrophobicity
of the contacting surfaces. One skilled in the art can appreciate that an
increase in particle hydrophobicity can be achieved
through excipient selection andfor use a post~production spray drying coating
technique such as employed using a fluidized
bed. Thus, preferred excipients include hydrophobic surfactants such as
phospholipids, fatty acid soaps and cholesterol.
In view of the teachings herein, it is submitted that a skilled artisan would
be able to identify materials exhibiting similar
desirable properties without undue experimentation.
Whether they are to be used as a dry powder or combined with a nonaqueous
suspension medium, the
particulates or perforated microstructures will preferably be provided in a
"dry" state. That is the microparticles will
possess a moisture content that allows the powder to remain chemically and
physically stable during storage at
ambient temperature and easily dispersible. As such, the moisture content of
the microparticles is typically less than
6% by weight, and preferably less 3% by weight. In some instances the moisture
content will be as low as 1 ~o by
weight. Of course it will be appreciated that the moisture content is, at
least in part, dictated by the formulation and
is controlled by the process conditions employed, e.g., inlet temperature,
feed concentration, pump rate, and blowing
agent type, concentration and post drying.
As known by those skilled in the art, methods such as angle of repose or shear
index can be used to assess the
flow properties of dry powders. The angle of repose is defined as the angle
formed when a cone of powder is poured onto
a flat surface. Powders having an angle of repose ranging from 45° to
20° are preferred and indicate suitable powder
flow. More particularly, powders which possess an angle of repose between
33° and 20° flow with relatively low shear
forces and are especially useful in pharmaceutical preparations for use in
inhalation therapies (e.g. OPlsl. The shear index,
though more time consuming to measure than angle of repose, is considered more
reliable and easy to determine. Those
skilled in the art will appreciate that the experimental procedure outlined by
Amidon and Houghton (G.E. Amidon, and M.E.
Houghton, Pharm. Manuf., 2, 20, 1985, incorporated herein by reference) can be
used estimate the shear index for the
purposes of the present invention. As described in S. Kocova and N. Pilpel, J.
Pharm. Pharmacol. 8, 33-55, 1973, also
incorporated herein by reference, the shear index is estimated from powder
parameters such as, yield stress, effective
angle of internal friction, tensile strength, and specific cohesion. In the
present invention powders having a shear index
less than about 0.98 are desirable. More preferably, powders used in the
disclosed compositions, methods and systems
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will have shear indices less than about 1.1. In particularly preferred
embodiments the shear index will be less than about
1.3 or even less than about 1.5. Of course powders having different shear
indices may be used provided the result in the
effective deposition of the active or bioactive agent at the site of interest.
It wilt also be appreciated that the flow properties of powders have been
shown to correlate well with bulk
density measurements. In this regard, conventional prior art thinking ~C.F.
Harwood, J. Pharm. Sci., 60, 161-163, 1971)
held that an increase in bulk density correlates with improved flow properties
as predicted by the shear index of the
material. Conversely, it has surprisingly been found that, for the perforated
microstructures of the present invention,
superior flow properties were exhibited by powders having relatively low bulk
densities. That is, the hollow porous
powders of the present invention exhibited superior flow properties over
powders substantially devoid of pores. To that
end, it has been found that it is possible to provide powders having bulk
densities of less than 0.5 glcm3 that exhibit
particularly favorable flow properties. More surprisingly, it has been found
that it is possible to provide perforated
microstructure powders having bulk densities of less than 0.3 glcm', less than
about 0.1 glcm' or even on the order of
0.05 glcm'that exhibit excellent flow properties. The ability to produce low
bulk density powders having superior
flowability further accentuates the novel and unexpected nature of the present
invention.
These low bulk densities are particularly advantageous when using the
disclosed powders in conjunction with
DPIs. Specifically, by affording powder formulations having extraordinarily
low bulk density, the present invention
allows for reduction of the minimal filling weight that is commercially
feasible for use in dry powder inhalation devices.
That is, most unit dose containers designed for DPIs are filled using fixed
volume or gravimetric techniques. Contrary
to many prior art formulations, the present invention provides powders wherein
bioactive agent and the incipients or
bulking agents make-up the entire inhaled particle. By providing particles
with very low bulk density, the minimum
powder mass that can be filled into a unit dose container is reduced, which
eliminates the need for carrier particles.
That is, the relatively low density of the powders of the present invention
provides for the reproducible administration
of relatively low dose pharmaceutical compounds without the use of carrier
particles. Moreover, the elimination of
carrier particles acts to minimize throat deposition and any "gag" effect,
since the large lactose particles of prior art
formulations tend to impact the throat and upper airways due to their size.
It will be appreciated that the reduced attractive forces (e.g. van der Waals,
electrostatic, hydrogen and
liquid bridging, etc.) and excellent flowability provided by the perforated
microstructure powders make them particularly
useful in preparations for inhalation therapies (e.g. in inhalation devices
such as DPIs, MDIs, nebulizersl. Along with the
superior flowability, the perforated ar porous andlor hollow design of the
microstructures also plays an important role
in the resulting aerosol properties of the powder when discharged. This
phenomenon holds true for particulates or
perforated microstructures aerosolized as a suspension, as in the case of an
MDI or a nebulizer, or delivery of
perforated microstructures in dry form as in the case of a DPI. In this
respect the perforated structure and relatively
high surface area of the dispersed microparticles enables them to be carried
along in the flow of gases during inhalation
with greater ease for longer distances than non-perforated particles of
comparable size.
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More particularly, because of their high porosity, the density of the
particles is significantly less than 1.0
glcm', typically less than 0.5 glcm', more often on the order of 0.1 glcm3,
and as low as 0.01 glcm'. Unlike the
geometric particle size, the aerodynamic particle size, doer, of the
perforated microstructures depends substantially
on the particle density, p: doer = dReoP, where dge~ is the geometric
diameter. For a particle density of 0.1
glcm3, doer will be roughly three times smaller than dge", leading to
increased particle deposition into the peripheral
regions of the lung and correspondingly less deposition in the throat. In this
regard, the mean aerodynamic diameter of
the perforated microstructures is preferably less than about 5 ,um, more
preferably less than about 3 ,um, and, in
particularly preferred embodiments, less than about 2 Nm. Such particle
distributions will act to increase the deep
lung deposition of the bioactive agent whether administered using a DPI, MDI
or nebulizer.
As will be shown subsequently in the Examples, the particle size distribution
of the aerosol formulations of
the present invention are measurable by conventional techniques such as, for
example, cascade impaction or by time of
flight analytical methods. In addition, determination of the emitted dose from
inhalation devices were done according
to the proposed U.S. Pharmacopeia method (Pharmacopeial Previews,
221199613065) which is incorporated herein
by reference. These and related techniques enable the °fine particle
fraction" of the aerosol, which corresponds to
those particulates that are likely to effectively deposited in the lung, to be
calculated. As used herein the phrase "fine
particle fraction" refers to the percentage of the total amount of active
medicament delivered per actuation from the
mouthpiece of a DPI, MDI or nebulizer onto plates 2-7 of an 8 stage Andersen
cascade impactor. Based on such
measurements the formulations of the present invention will preferably have a
fine particle fraction of approximately
20% or more by weight of the perforated microstructures (wlwl, more preferably
they will exhibit a fine particle
fraction of from about 25% to 90% wlw, and even more preferably from about 30
to 80% wlw. In selected
embodiments the present invention will preferably comprise a fine particle
fraction of greater than about 30%, 40°Yo,
50%, 60%, 709'0, 80% or even 909~o by weight.
Further, it has also been found that the formulations of the present invention
exhibit relatively low deposition
rates, when compared with prior art preparations, on the induction part and
onto plates 0 and 1 of the impactor.
Deposition on these components is linked with deposition in the throat in
humans. More specifically, most
commercially available MDIs and DPIs have simulated throat depositions of
approximately 40-70% (wlw) of the total
dose, while the formulations of the present invention typically deposit less
than about 209'o wlw. Accordingly,
preferred embodiments of the present invention have simulated throat
depositions of less than about 40%, 35%, 30%,
25%, 200, 15% or even 10% wlw. Those skilled in the art will appreciate that
significant decrease in throat
deposition provided by the present invention will result in a corresponding
decrease in associated local side-effects
such as throat irritation.
With respect to the advantageous deposition profile provided by the instant
invention it is well known that
MDI propellants typically force suspended particles out of the device at a
high velocity towards the back of the throat.
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Since prior art formulations typically contain a significant percentage of
large particles andfor aggregates, as much as
two-thirds ar more of the emitted dose may impact the throat. Moreover, the
undesirable delivery profile of
conventional powder preparations is also exhibited under conditions of low
particle velocity, as occurs with DPI
devices. In general, this problem is inherent when aerosolizing solid, dense,
particulates which are subject to
aggregation. Yet, as discussed above, the novel and unexpected properties of
the stabilized dispersions of the present
invention result in surprisingly low throat deposition upon administration
from inhalation device such as a DPI, MDI
atomizer or nebulizer.
While not wishing to be bound by any particular theory, it appears that the
reduced throat deposition
provided by the instant invention results from decreases in particle
aggregation and from the hollow andlor porous
morphology of the incorporated microstructures. That is, the hollow and porous
nature of the dispersed
microstructures slows the velocity of particles in the propellant stream (or
gas stream in the case of DPIs), just as a
hollowJporous whiffle ball decelerates faster than a baseball. Thus, rather
than impacting and sticking to the back of
the throat, the relatively slow traveling particles are subject to inhalation
by the patient. Moreover, the highly porous
nature of the particles allows the propellant within the perforated
microstructure to rapidly leave and the particle
density to drop before impacting the throat. Accordingly, a substantially
higher percentage of the administered
bioactive agent is deposited in the pulmonary air passages where it may be
efficiently absorbed.
E. Powder Formation
As seen from the passages above. various components may be associated with, or
incorporated in the
microparticulates of the present invention. Similarly, several techniques may
be used to provide particulates having the
desired morphology (e.g. a perforated or hallowlporous configuration),
dispersibility and density. Among other methods,
particulates compatible with the instant invention may be formed by techniques
including spray drying, vacuum drying,
solvent extraction, emulsification, lyophilization and combinations thereof.
It will further be appreciated that the basic
concepts of many of these techniques are well known in the prior art and would
not, in view of the teachings herein,
require undue experimentation to adapt them so as to provide the desired
particle configuration andlor density.
While several procedures are generally compatible with the present invention,
particularly preferred embodiments
typically comprise particulates or perforated microstructures formed by spray
drying. As is well known, spray drying is a
one-step process that converts a liquid feed to a dried particulate form. With
respect to pharmaceutical applications, it
will be appreciated that spray drying has been used to provide powdered
material for various administrative routes
including inhalation. See, for example, M. Sacchetti and M.M. Van Oort in:
Inhalation Aerosols: Physical and
Biological Basis for Therapy, A.J. Hickey, ed. Marcel Dekkar, New York, 1996,
which is incorporated herein by
reference.
In general, spray drying consists of bringing together a highly dispersed
liquid, and a sufficient volume of hot
air to produce evaporation and drying of the liquid droplets. The preparation
to be spray dried or feed (or feed stock)
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can be any solution, course suspension, slurry, colloidal dispersion, or paste
that may be atomized using the selected
spray drying apparatus. In preferred embodiments the feed stock will comprise
a colloidal system such as an emulsion,
reverse emulsion, microemulsion, multiple emulsion, particulate dispersion, or
slurry. Typically the feed is sprayed into
a current of warm filtered air that evaporates the solvent and conveys the
dried product to a collector. The spent air is
then exhausted with the solvent. Those skilled in the art will appreciate that
several different types of apparatus may
be used to provide the desired product. For example, commercial spray dryers
manufactured by Buchi Ltd. or Niro
Corp. will effectively produce particles of desired size, morphology and
density.
It will further be appreciated that these spray dryers, and specifically their
atomizers, may be modified or
customized for specialized applications, i.e. the simultaneous spraying of two
solutions using a double nozzle technique.
More specifically, a water-in-oil emulsion can be atomized from one nozzle and
a solution containing an anti-adherent
such as mannitol can be co-atomized from a second nozzle. In other cases it
may be desirable to push the feed solution
though a custom designed nozzle using a high pressure liquid chromatography
(HPLC) pump. Provided that
microstructures comprising the desired morphology andlor composition are
produced, the choice of apparatus is not critical
and would readily be apparent to the skilled artisan in view of the teachings
herein.
While the resulting spray-dried powders typically are approximately spherical
in shape, nearly uniform in size
and frequently are hollow, there may be some degree of irregularity in shape
depending upon the incorporated
medicament and the spray drying conditions. In many instances dispersion
stability and dispersibility of particulates or
perforated microstructures appears to be improved if an inflating agent (or
blowing agent) is used in their production.
Particularly preferred embodiments may comprise an emulsion with the inflating
agent as the disperse or continuous
phase. The inflating agent is preferably dispersed with a surfactant solution,
using, for instance, a commercially available
microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms
an emulsion, preferably stabilized by an
incorporated surfactant, typically comprising submicron droplets of water
immiscible blowing agent dispersed in an
aqueous continuous phase. The formation of such emulsions using this and other
techniques are common and well known
to those in the art. The blowing agent is preferably a fluorinated compound
(e.g. perfluorohexane, perffuorooctyl
bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the
spray-drying process, leaving behind, in
selected embodiments, relatively hollow, porous aerodynamically light
microspheres. As will be discussed in more
detail below, other suitable liquid blowing agents include nonfluorinated
oils, chloroform, Freons, ethyl acetate,
alcohols and hydrocarbons. Nitrogen and carbon dioxide gases are also
contemplated as suitable blowing agents.
Besides the aforementioned compounds, inorganic and organic substances which
can be removed under
reduced pressure by sublimation in a post-production step are also compatible
with the instant invention. These
sublimating compounds can be dissolved or dispersed as micronized crystals in
the spray drying feed solution and
include ammonium carbonate and camphor. Other compounds compatible with the
present invention comprise
rigidifying solid structures which can be dispersed in the feed solution or
prepared in-situ. These structures are then
extracted after the initial particle generation using a post-production
solvent extraction step. For example, latex
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particles can be dispersed and subsequently dried with other wall forming
compounds, followed by extraction with a
suitable solvent.
Although the particulates are preferably formed using a blowing agent as
described above, it will be
appreciated that, in some instances, no additional blowing agent is required
and an aqueous dispersion of the
medicament andlor excipients and surfactants) are spray dried directly. In
such cases, the formulation may be
amenable to process conditions (e.g., elevated temperatures) that may lead to
the formation of hollow, relatively
porous microparticles. Moreover, the medicament may possess special
physicochemical properties (e.g., high
crystallinity, elevated melting temperature, surface activity, etc.) that
makes it particularly suitable for use in such
techniques.
When a blowing agent is employed, the degree of porosity and dispersibility of
the resulting particulates
appears to depend, at least in part, on the nature of the blowing agent, its
concentration in the feed stock (e.g. as an
emulsion), and the spray drying conditions. With respect to controlling
porosity and, in suspensions, dispersibility it
has surprisingly been found that the use of compounds, heretofore
unappreciated as blowing agents, may provide
perforated microstructures having particularly desirable characteristics. More
particularly, in this novel and
unexpected aspect of the present invention it has been found that the use of
fluorinated compounds having relatively
high boiling points (i.e. greater than about 40°C) may be used to
produce particulates that are especially porous. Such
perforated microstructures are especially suitable for inhalation therapies.
In this regard it is possible to use
fluorinated or partially fluorinated blowing agents having boiling points of
greater than about 40°C, 50°C, 60°C,
70°C, 80°C, 90°C or even 95°C. Particularly
preferred blowing agents have boiling points greater than the boiling
point of water, i.e. greater than 100°C (e.g. perflubron,
perfluorodecalinl. In addition blowing agents with relatively
low water solubility ( < 10'6 MI are preferred since they facilitate the
production of stable emulsion dispersions with
mean weighted particle diameters less than 0.3 Vim.
As previously described, these blowing agents will preferably be incorporated
in an emulsified feed stock
prior to spray drying. For the purposes of the present invention this feed
stock will also preferably comprise one or
more bioactive agents, one or more surfactants or one or more excipients. Of
course, combinations of the
aforementioned components are also within the scope of the invention. While
high boiling ( > 100°C) fluorinated
blowing agents comprise one preferred aspect of the present invention, it will
be appreciated that nonfluorinated
blowing agents with similar boiling points ( > 100°C) may be also be
used to provide compatible particulates.
Exemplary nonfluorinated blowing agents suitable for use in the present
invention comprise the formula:
R'-X-RZ or R'-X
wherein: R' or RZ is hydrogen, alkyl, alkenyl, alkynl, aromatic, cyclic or
combinations thereof, X is any group containing
carbon, sulfur, nitrogen, halogens, phosphorus, oxygen and combinations
thereof.
While not limiting the invention in any way it is hypothesized that, as the
aqueous feed component
evaporates during spray drying it leaves a thin crust at the surface of the
particle. The resulting particle wall or crust
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formed during the initial moments of spray drying appears to trap any high
boiling blowing agents as hundreds of
emulsion droplets (ca. 200-300 nml. As the drying process continues, the
pressure inside the particulate increases
thereby vaporizing at least part of the incorporated blowing agent and forcing
it through the relatively thin crust. This
venting ar outgassing apparently leads to the formation of pores or other
defects in the microstructure. At the same
time remaining particulate components (possibly including some blowing agent)
migrate from the interior to the surface
as the particle solidifies. This migration apparently slows during the drying
process as a result of increased resistance
to mass transfer caused by an increased internal viscosity. Once the migration
ceases the particle solidifies, leaving
voids, pores, defects, hollows, spaces, interstitial spaces, apertures,
perforations or holes. The number of pores or
defects, their size, and the resulting wall thickness is largely dependent on
the formulation andlor the nature of the
selected blowing agent (e.g. boiling point), its concentration in the
emulsion, total solids concentration, and the spray-
drying conditions. As alluded to throughout the specification, this preferred
particle morphology appears to contribute,
at least in part, to the improved powder dispersibility, suspension stability
and aerodynamics.
It has been surprisingly found that substantial amounts of these relatively
high boiling blowing agents may be
retained in the resulting spray dried product. That is, spray dried
particulates as described herein may comprise as
I S much as 1 %, 3%, 5%, 10%, 20%, 30~ or even 40~o wlw of residual blowing
agent. In such cases, higher production
yields were obtained as a result an increased particle density caused by this
retained blowing agent. It will be
appreciated by those skilled in the art that retained fluorinated blowing
agent may alter the surface characteristics of
the particulates, thereby minimizing particle aggregation during processing
and further increasing dispersion stability.
Residual fluorinated blowing agent in the powders may also reduce the cohesive
forces between particles by providing
a barrier or by attenuating the attractive forces produced during
manufacturing (e.g., electrostaticsl. This reduction in
cohesive forces may be particularly advantageous when using the disclosed
microstructures in conjunction with dry
powder inhalers.
Furthermore, the amount of residual blowing agent can be controlled through
the process conditions (such as
outlet temperature), blowing agent concentration, or boiling point. If the
outlet temperature is at or above the boiling
point, the blowing agent escapes the particle and the production yield
decreases. Preferred outlet temperature will
generally be operated at 20, 30, 40, 50, 60, 70, 80, 90 or even 100°C
less than the blowing agent boiling point. More
preferably the temperature differential between the outlet temperature and the
boiling point will range from 50 to
150°C. It will be appreciated by those skilled in the art that particle
porosity, production yield, electrostatics and
dispersibility can be optimized by first identifying the range of process
conditions (e.g., outlet temperature) that are
suitable for the selected active agents andfor excipients. The preferred
blowing agent can be then chosen using the
maximum outlet temperature such that the temperature differential with be at
least 20 and up to 150°C. In same
cases, the temperature differential can be outside this range such as, for
example, when producing the particulates
under supercritical conditions or using lyophilization techniques. Those
skilled in the art will further appreciate that the
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preferred concentration of blowing agent can be determined without undue
experimentation using techniques similar to
those described in the Examples herein.
While residual blowing agent may be advantageous in selected embodiments it
may be desirable to
substantially remove any blowing agent from the spray dried product. In this
respect, the residual blowing agent can
$ easily be removed with a post-production evaporation step in a vacuum oven.
Moreover, such post production
techniques may be used to provide perforations in the particulates. For
example, pores may be formed by spray drying
a bioactive agent and an excipient that can be removed from the formed
particulates under a vacuum.
in any event, typical concentrations of blowing agent in the feed stock are
between 29'o and 50% vlv, and
more preferably between about 10% to 459'o vJv. In other embodiments blowing
agent concentrations will preferably
be greater than about 5~'°, 1096, 159°, 209'0, 25% or even 30%
vlv. Yet other feed stock emulsions may comprise 35~°,
40%, 45°l0 or even 50% vlv of the selected compound.
In preferred embodiments, another method of identifying the concentration of
blowing agent used in the feed
is to provide it as a ratio of the concentration of the blowing agent to that
of the stabilizing surfactant (e.g.
phosphatidylcholine or PC) in the precursor or feed emulsion. For fluorocarbon
blowing agents (e.g. perfluorooctyl
bromidel, and for the purposes of explanation, this ratio has been termed the
PFCIPC ratio. More generally, it will be
appreciated that compatible blowing agents andlor surfactants may be
substituted for the exemplary compounds
without falling outside of the scope of the present invention. In any event,
the typical PFCIPC ratio will range from
about 1 to about 60 and, mare preferably, from about 10 to about 50. For
preferred embodiments the ratio will
generally be greater than about 5, 10, 20, 25, 30, 40 or even 50. It should be
appreciated that the use of higher
PFCIPC ratios generally provides structures of a more hollow and porous
nature. More particularly, those methods
employing a PFCIPC ratio of greater than about 4.8 tended to provide
structures that are particularly compatible with
the dry power formulations and dispersions disclosed herein.
While relatively high boiling point blowing agents comprise one preferred
aspect of the instant invention, it will
be appreciated other blowing or inflating agents may also be used to provide
compatible microstructures. As such, the
blowing agent may comprise any volatile substance which can be incorporated
into the feed solution for the purpose of
producing the desired microstructures. The blowing agent may be removed during
the initial drying process or during a
post-production step such as vacuum drying or solvent extraction. Suitable
agents include:
1. Dissolved low-boiling (below 100°C) agents miscible with aqueous
solutions, such as methylene chloride,
acetone, ethyl acetate, and alcohols used to saturate the solution.
2. A gas, such as COZ or N2, or liquid such as Freons, CFCs, HFAs, PFCs, HFCs,
HFBs, fluoroalkanes and
hydrocarbons, used at elevated pressure.
3. Emulsions of immiscible low-boiling (below 100°C) liquids suitable
for use with the present invention are
generally of the formula:
R'-X-Rz or R'-X
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wherein: R' or RZ is hydrogen, alkyl, alkenyl, alkynl, aromatic, cyclic or
combinations thereof, X is any groups
containing carbon, sulfur, nitrogen, halogens, phosphorus, oxygen and
combinations thereof.
4. Dissolved or dispersed salts or organic substances which can be removed
under reduced pressure by
sublimation in a post-production step, such as ammonium salts, camphor, etc.
5 Dispersed solids which can be extracted after the initial particle
generation using a post-production solvent
extraction step.
With respect to lower boiling point inflating agents, they are typically added
to the feed stock in quantities of
about 1 % to 40% vlv of the surfactant solution. Approximately 15% vlv
inflating agent has been found to produce a spray
dried powder that may be used with the methods of the present invention.
Regardless of which blowing agent is ultimately selected, it has been found
that compatible particulates may
be produced using commercially available equipment such as a Biichi mini spray
drier (model B-191, Switzerland). As
will be appreciated by those skilled in the art, the inlet temperature and the
outlet temperature of the spray drier may
be adjusted to provide the desired particle size and to maintain the activity
of the incorporated bioactive agent. In this
regard, the inlet and outlet temperatures are adjusted depending on the
melting characteristics of the formulation
components and the composition of the feed stock. The inlet temperature may
thus be between 60°C and 170°C,
with the outlet temperatures of about 40°C to 120°C depending on
the composition of the feed and the desired
particulate characteristics. Preferably these temperatures will be from
90°C to 120°C for the inlet and from 60°C to
90°C for the outlet. The flow rate which is used in the spray drying
equipment will generally be about 3 ml per minute
to about 15 ml per minute. The atomizer air flow rate will vary between values
of 25 liters per minute to about 50
liters per minute. Commercially available spray dryers are well known to those
in the art, and suitable settings for any
particular dispersion can be readily determined through standard empirical
testing, with due reference to the examples that
follow.
Although the microparticulates are preferably formed using fluorinated blowing
agents in the form of an
emulsion, it will be appreciated that nonfluorinated oils may 6e used to
increase the loading capacity of the bioactive
agents without compromising the microstructure. In this case, selection of the
nonfluorinated oil is based upon the
solubility of the active or bioactive agent, water solubility, boiling point,
and flash point. The bioactive agent will be
dissolved in the oil and subsequently emulsified in the feed solution.
Preferably the oil will have substantial
solubilization capacity with respect to the selected agent, low water
solubility ( < 10-'M), boiling point greater than
water and a flash point greater than the drying outlet temperature. The
addition of surfactants, and co-solvents to the
nonfluorinated oil to increase the solubilization capacity is also within the
scope of the present invention.
In particularly preferred embodiments nonfluorinated oils may be used to
solubilize bioactive agents that have
limited solubility in aqueous compositions. The use of nonfluorinated oils is
of particular use for increasing the loading
capacity of hydrophobic peptides and proteins. Preferably the oil or oil
mixture for solubilizing these compounds will
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have a refractive index between 1.36 and 1.41 (e.g. ethyl butyrate, butyl
carbonate, dibutyl ether). In addition,
process conditions, such as temperature and pressure, may be adjusted in order
to boost solubility of the selected
agent. It will be appreciated that selection of an appropriate oil or oil
mixtures and processing conditions to maximize
the loading capacity of an agent are well within the purview of a skilled
artisan in view of the teachings herein and
may be accomplished without undue experimentation.
Particularly preferred embodiments of the present invention comprise spray
drying preparations comprising a
surfactant such as a phospholipid and at least one bioactive agent. Other
embodiments include spray drying preparations
that may further include an excipient comprising a hydrophilic moiety such as,
for example, a carbohydrate (i.e. glucose,
lactose, or starch) in addition to any selected surfactant. In this regard,
various starches and derivatized starches are
particularly suitable for use in the present invention. Other optional
components may include conventional viscosity
modifiers, buffers such as phosphate buffers or other conventional
biocompatible buffers or pH adjusting agents such as
acids or bases, and osmotic agents (to provide isotonicity, hyperosmolarity,
or hyposmolarity). Examples of suitable salts
include sodium phosphate (both monobasic and dibasic), sodium chloride,
calcium phosphate, calcium chloride and other
physiologically acceptable salts.
Whatever components are selected, the first step in particulate production
typically comprises feed stock
preparation. Preferably the selected drug is dissolved in water to produce a
concentrated solution. The drug may also
be dispersed directly in the emulsion, particularly in the case of water
insoluble agents. Alternatively, the drug may be
incorporated in the form of a solid particulate dispersion. The concentration
of the active or bioactive agent used is
dependent on the amount of agent required in the final powder and the
performance of the delivery device employed
(e.g., the fine particle dose for a MDI or DPI). As needed, cosurfactants such
as poloxamer 188 or span 80 may be
dispersed into this annex solution. Additionally, excipients such as sugars
and starches can also be added.
In selected embodiments an oil-in-water emulsion is then formed in a separate
vessel. The oil employed is
preferably a fluorocarbon (e.g., perfluorooctyl bromide, perfluorodecalin)
which is emulsified using a surfactant such as
a long chain saturated phospholipid. For example, one gram of phospholipid may
be homogenized in 150 g hot distilled
water (e.g., 60°C) using a suitable high shear mechanical mixer (e.g.,
Ultra-Turrax model T-25 mixer) at 8000 rpm for
2 to 5 minutes. Typically 5 to 25 g of fluorocarbon is added dropwise to the
dispersed surfactant solution while
mixing. The resulting perfluorocarbon in water emulsion is then processed
using a high pressure homogenizer to reduce
the particle size. For example, the emulsion may be processed at 12,000 to
18,000 psi, 5 discrete passes and kept at
50 to 80°C.
The bioactive agent solution and perfluorocarbon emulsion may then be combined
and fed into the spray
dryer. Typically the two preparations will be miscible as the emulsion will
preferably comprise an aqueous continuous
phase. While the bioactive agent is solubilized separately for the purposes of
the instant discussion it will be
appreciated that, in other embodiments, the active or bioactive agent may be
solubilized (or dispersed) directly in the
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emulsion. In such cases, the active or bioactive emulsion is simply spray
dried without combining a separate drug
preparation.
In any event, operating conditions such as inlet and outlet temperature, feed
rate, atomization pressure, flow
rate of the drying air, and nozzle configuration can be adjusted in accordance
with the manufacturer's guidelines in
order to produce the required particle size, and production yield of the
resulting dry microstructures- Exemplary
settings are as follows: an air inlet temperature between 60°C and
170°C; an air outlet between 40°C to 120°C; a
feed rate between 3 ml to about 15 ml per minute; and an aspiration air flow
of 300 Llmin. and an atomization air flow
rate between 25 to 50 Llmin. The selection of appropriate apparatus and
processing conditions are well within the
purview of a skilled artisan in view of the teachings herein and may be
accomplished without undue experimentation.
In any event, the use of these and substantially equivalent methods provide
for the formation of hollow porous
aerodynamically light microspheres with particle diameters appropriate for
aerosol deposition into the lung.
microstructures that are both hollow and porous, almost honeycombed or foam-
like in appearance. In especially
preferred embodiments the perforated microstructures comprise hollow, porous
spray dried microspheres.
Along with spray drying, perforated microstructures useful in the present
invention may be formed by
1 S lyophilization. Those skilled in the art will appreciate that
lyophilization is a freeze-drying process in which water is
sublimed from the composition after it is frozen. The particular advantage
associated with the lyophilization process is
that biologics and other pharmaceuticals that are relatively unstable in an
aqueous solution can be dried without
elevated temperatures (thereby eliminating the adverse thermal effects), and
then stored in a dry state where there are
few stability problems. With respect to the instant invention such techniques
are particularly compatible with the
incorporation of peptides, proteins, genetic material and other natural and
synthetic macromolecules in particulates or
perforated microstructures without compromising physiological activity.
Methods for providing lyophilized particulates
are known to those of skill in the art and it would clearly not require undue
experimentation to provide compatible
microstructures in accordance with the teachings herein. The lyophilized cake
containing a fine foam-like structure can
be micronized using techniques known in the art to provide particles having
mean diameters under 5 Nm or 10 Nm.
Accordingly, to the extent that lyophilization processes may be used to
provide microstructures having the desired
characteristics they are expressly contemplated as being within the scope of
the instant invention.
Besides the aforementioned techniques, the particulates and perforated
microstructures of the present
invention may also be formed using a method where a feed solution (either
emulsion or aqueous) containing wall
forming agents is rapidly added to a reservoir of heated oil (e.g. perflubron
or other high boiling FCs) under reduced
pressure. The water and volatile solvents of the feed solution rapidly boils
and are evaporated. This process may be
used to provide a perforated structure from wall forming agents similar to
puffed rice or popcorn. Preferably the wall
forming agents are insoluble in the heated oil. The resulting particles can
then separated from the heated oil using a
filtering technique and subsequently dried under vacuum.
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Additionally, the particles or perforated microstructures of the present
invention may also be formed using a
double emulsion method. In the double emulsion method the medicament is first
dispersed in a polymer dissolved in an
organic solvent (e.g. methylene chloride) by sonication or homogenization.
This primary emulsion is then stabilized by
forming a multiple emulsion in a continuous aqueous phase containing an
emulsifier such as polyvinylalcohol.
Evaporation or extraction using conventional techniques and apparatus then
removes the organic solvent. The
resulting microparticles are then washed, filtered and dried prior to use or
combining them with an appropriate
suspension medium in accordance with the present invention.
F. Administration
Whatever method is ultimately selected for production of the
microparticuiates, the resulting powders have a
number of advantageous properties that allow them to be effectively used in
either a powdered form or as a dispersion
comprising a nonaqueous suspension medium. In particularly preferred
embodiments the bioactive compositions,
whether in the form of a dry powder or dispersion, will be administered to the
mucosal surface of the respiratory tract
(i.e., the pulmonary and/or the nasal tract) via inhalation therapy. Such
administration may be effected using MDIs,
DPIs. nebulizers, nasal pumps, atomizers, spray bottles or by direct
instillation in the form of drops. However, while
inhalation therapies are extremely compatible with the present invention, it
will be appreciated that other forms andlor
routes of administration are also useful.
In this regard, the powders and stabilized dispersions of the present
invention may also be used for the
localized or systemic administration of compounds to any location of the body.
Accordingly, it should be emphasized
that, in preferred embodiments, the preparations may be administered using a
number of different routes including, but
not limited to, topical, intramuscular, transdermal, intradermal,
intraperitoneal, nasal, pulmonary, buccal, vaginal,
rectal, aural, oral or ocular administration. Preferred target sites may be
found in, for example, the gastrointestinal
tract, urogenital tract or respiratory tract. More generally, the stabilized
dispersions of the present invention may be
used to deliver agents topically or by administration to any body cavity. In
preferred embodiments the body cavity is
selected from the group consisting of the peritoneum, sinus cavity, rectum,
urethra, stomach, nasal cavity, vagina,
auditory meatus, oral cavity, buccal pouch and pleura. Those skilled in the
art will appreciate that the selected route
of administration will largely be determined by the choice of bioactive agent
and the desired response of the subject.
With regard to the delivery of the disclosed powders or stabilized
dispersions, another aspect of the present
invention is directed to systems for the administration of one or more
bioactive agents or biologics to a patient. As
alluded to above, exemplary inhalation devices compatible with the present
invention may comprise an atomizer, nasal
pump, a sprayer or spray bottle, a dry powder inhaler, a metered dose inhaler
or a nebulizer. In preferred embodiments,
these inhalation systems will deliver the bioactive agent to the desired
physiological site (e.g. a mucosal surface) as an
aerosol. For the purposes of the instant application the term "aerosolized"
shall be held to mean a gaseous suspension
of fine solid or liquid particles unless otherwise dictated by contextual
restraints. That is, an aerosol or aerosolized
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medicament may be generated, for example, by a dry powder inhaler, a metered
dose inhaler, an atomizer, a spray
bottle or a nebulizer. Of course, as explained in more detail below, the
compositions of the present invention may also
be delivered directly (e.g. by conventional injection or needleiess injection)
or using such techniques as liquid dose
instillation. As such, a further aspect of the present invention is directed
to needleless injectors (e.g. pressurized gas
guns) comprising the disclosed powders or dispersions.
F(il. Dry Powder Inhalers
With respect to inhalation therapies, those skilled in the art will appreciate
that the powders of the present
invention, particularly those comprising perforated microstructures, are
particularly useful in DPis. Conventional DPIs, or
dry powder inhalers, comprise powdered formulations and devices where a
predetermined dose of medicament, either
alone or in a blend with lactose carrier particles, is delivered as a fine
mist or aerosol of dry powder for inhalation. Useful
DPI medicaments are typically formulated so that they readily disperse into
discrete particles with a size rage between 0.5
to 20 Nm. The powder is actuated either by inspiration or by some external
delivery force, such as pressurized air. DPI
formulations are typically packaged in single dose units or they employ
reservoir systems capable of metering multiple
doses with manual transfer of the dose to the device.
DPIs are generally classified based on the dose delivery system employed. In
this respect, the two major types
of DPIs comprise unit dose delivery devices and bulk reservoir delivery
systems. As used herein, the term "reservoir" shall
be used in a general sense and held to encompass both configurations unless
otherwise dictated by contextual restraints.
In any event, unit dose delivery systems require the dose of powder
formulation presented to the device as a single unit.
With this system, the formulation is prefilled into dosing wells which may be
foihpackaged or presented in blister strips to
prevent moisture ingress. Other unit dose packages include hard gelatin
capsules. Most unit dose containers designed
for DPIs are filled using a fixed volume technique. As a result, there are
physical limitations (here density) to the
minimal dose that can be metered into a unit package, which is dictated by the
powder flowability and bulk density.
As previously alluded to, the powders of the present invention obviate many of
the difficulties associated with
prior art carrier preparations. That is, an improvement in DPI performance may
be provided by adjusting the particle size,
aerodynamics, morphology and density, humidity and charge as disclosed herein.
In this respect the present invention
provides for formulations wherein the medicament and the incipients or bulking
agents are preferably associated with
or comprise perforated microstructures. As set forth above, preferred
compositions according to the present invention
typically yield powders with bulk densities less than 0.1 glcm' and often less
than 0.05 glcm'. It will be appreciated
that providing powders having bulk densities an order of a magnitude less than
conventional DPI formulations allows
for much lower doses of the selected bioactive agent to be filled into a unit
dose container or metered via reservoir
based DPIs. The ability to effectively meter small quantities is significant
for relatively potent bioactive agents such
as hormones. Moreover, the ability to effectively deliver particulates without
associated carrier particles simplifies
product formulation, filling and reduces undesirable side effects.
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It will be appreciated that the powders of the present invention are
particularly effective at delivering
relatively high doses of bioactive agent in a single actuation. Unlike prior
art formulations, the powdered formulations
do not require the use of bulking agents for effective filling and delivery
and may therefore comprise higher levels of
bioactive agent on a weight by weight basis. Significantly, the disclosed
compositions may be used to deliver as much
as approximately 10 mg of bioactive agent in a single actuation. Such
advantages may be particularly important when
delivering, for example, immunomodulators or antibodies for passive
immunization, that may not be as potent as other
compatible agents. Of course, while the instant discussion is specifically
directed to the use of DPIs, this same
advantage is equally applicable to dispersion formulations and other forms of
administration such as MDIs, nasal
pumps and needleless injectors.
In addition to the aforementioned advantages, preferred embodiments of the
present invention exhibit
favorable aerodynamic properties that make them particularly effective for use
in DPIs. More specifically, the
perforated structure and relatively high surface area of the microparticles
enables them to be carried along in the flow of
gases during inhalation with greater ease and for longer distances than
relatively non~perforated particles of comparable
size. Because of their high porosity and low density, administration of
perforated microstructures with a DPI provides
for increased particle deposition at target sites such as mucosal surfaces in
the nasal passages and peripheral regions
of the lung with correspondingly less deposition in the throat. Such particle
distribution may be employed to increase
the deep lung deposition of the administered agent that is preferable for
systemic administration. Moreover, in a
substantial improvement over prior art DPI preparations the low-density,
highly porous powders of the present
invention preferably eliminate the need for carrier particles
flii). Stabilized Dispersions
Along with their use in a dry powder configuration, it will be appreciated
that the powders of the present
invention may be incorporated in a suspension medium to provide stabilized
dispersions. Preferably, the stabilized
dispersions will comprise a nonaqueous suspension medium. Among other uses,
the stabilized dispersions provide for
the effective delivery of bioactive agents to the pulmonary air passages of a
patient using MDIs, atomizers or spray
bottles, nasal pumps, needleless injectors, nebulizers or liquid dose
instillation (LDI techniques).
Those skilled in the art will appreciate the enhanced stability of the
disclosed dispersions or suspensions is
largely achieved by lowering the van der Waals attractive forces between the
suspended particles, and by reducing the
differences in density between the suspension medium and the particles. In
accordance with the teachings herein, the
increases in suspension stability may be imparted by engineering perforated
microstructures which are then dispersed
in a compatible suspension medium. As discussed above, the perforated
microstructures comprise pores, voids,
hollows, defects or other interstitial spaces that allow the fluid suspension
medium to freely permeate or perfuse the
particulate boundary. Particularly preferred embodiments comprise perforated
microstructures that are both hollow
and porous, almost honeycombed or foam-like in appearance. In especially
preferred embodiments the perforated
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microstructures comprise hollow, porous spray dried microspheres. Of course,
in other embodiments, including those
comprising relatively nonporous, solid particulates, enhanced stability may be
imparted through the selection of
particulate components (e.g. surfactantsl.
When perforated microstructures are placed in the suspension medium (i.e. a
hydrofluoroalkane propellant or
liquid fiuorocarbon), the suspension medium is able to permeate the particles,
thereby creating a "homodispersion",
wherein both the continuous and dispersed phases are indistinguishable. Since
the defined or "virtual" particles (i.e.
comprising the volume circumscribed by the microparticulate matrix) are made
up almost entirely of the medium in
which they are suspended, the forces driving particle aggregation
(flocculation) are minimized. Additionally, the
differences in density between the defined particles and the continuous phase
are minimized by having the
microstructures filled with the medium, thereby effectively slowing particle
creaming or sedimentation. As such, the
particulates and stabilized suspensions of the present invention are
particularly compatible with many aerosolization
techniques, such as MDI, atomization via a spray bottle, nasal pumps,
nebulization and the like. Moreover, the
stabilized dispersions are compatible with other routes of administration
including, but not limited to, liquid dose
instillation, needleless injection, conventional injection and topical
applications.
Unlike prior art compositions, preferred suspensions of the instant invention
are designed not to increase
repulsion between particles, but rather to decrease the attractive forces
between particles. In this respect it should be
appreciated that the principal forces driving flocculation in nonaqueous media
are van der Waals attractive forces. As
discussed above, VDW forces are quantum mechanical in origin, and can be
visualized as attractions between
fluctuating dipoles (i.e. induced dipole-induced dipole interactionsl.
Dispersion forces are extremely short-range and
scale as the sixth power of the distance between atoms. When two macroscopic
bodies approach one another the
dispersion attractions between the atoms sums up. The resulting force is of
considerably longer range, and depends on
the geometry of the interacting bodies.
More specifically, for two spherical particles, the magnitude of the VDW
potential, VA , can be approximated
by: ~ ~ - - A efr R, R z , where Ae~ is the effective Hamaker constant which
accounts for the nature of the
6H" (R, + R2)
particles and the medium, Ho is the distance between particles, and R~ and RZ
are the radii of spherical particles 1
and 2. The effective Hamaker constant is proportional to the difference in the
polarizabilities of the dispersed particles
and the suspension medium: A~ff = ( ASM - ApART )2 ~ where A~ and AP,~r are
the Hamaker constants
for the suspension medium and the particles, respectively. As the suspended
particles and the dispersion medium
become similar in nature, AS,,~ and AP,,Rr become closer in magnitude, and Ae~
and IAA become smaller. That is,
by reducing the differences between the Hamaker constant associated with
suspension medium and the Hamaker
constant associated with the dispersed particles, the effective Hamaker
constant (and corresponding van der Waals
attractive forces) may be reduced.
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One way to minimize the differences in the Hamaker constants is to create a
"homodispersion", that is make
bath the continuous and dispersed phases essentially indistinguishable as
discussed above. Besides exploiting the
morphology of the particles to reduce the effective Hamaker constant, the
components of the structural matrix
idefining the perforated microstructures) will preferably be chosen so as to
exhibit a Hamaker constant relatively close
to that of the selected suspension medium. In this respect, one may use the
actual values of the Hamaker constants
of the suspension medium and the particulate components to determine the
compatibility of the dispersion ingredients
and provide a good indication as to the stability of the preparation.
Alternatively, one could select relatively
compatible particulate or perforated microstructure components and suspension
mediums using characteristic physical
values that coincide with measurable Hamaker constants but are more readily
discernible.
In this respect, it has been found that the refractive index values of many
compounds tend to scale with the
corresponding Hamaker constant. Accordingly, easily measurable refractive
index values may be used to provide a
fairly good indication as to which combination of suspension medium and
particle excipients will provide a dispersion
having a relatively low effective Hamaker constant and associated stability.
It will be appreciated that, since
refractive indices of compounds are widely available or easily derived, the
use of such values allows for the formation
of stabilized dispersions in accordance with the present invention without
undue experimentation. For the purpose of
illustration only, the refractive indices of several compounds compatible with
the disclosed dispersions are provided in
Table I immediately below:
Table I
Compound Refractive Index
HFA-134a 1.172
HFA-227 1.223
CFC-12 1.287
CFC-114 1.288
PFOB 1.305
Mannitol 1.333
Ethanol 1.361
n-octane 1.397
DMPC 1.43
Pluronic F-68 1.43
Sucrose 1.538
Hydroxyethylstarch 1.54
Sodium chloride 1.544
Consistent with the compatible dispersion components set forth above, those
skilled in the art will appreciate
that, the formation of dispersions wherein the components have a refractive
index differential of less than about 0.5 is
preferred. That is, the refractive index of the suspension medium will
preferably be within about 0.5 of the refractive
index associated with the particles or perforated microstructures. It will
further be appreciated that, the refractive
index of the suspension medium and the particles may be measured directly or
approximated using the refractive
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indices of the major component in each respective phase. For the particulates
or perforated microstructures, the major
component may be determined on a weight percent basis. For the suspension
medium, the major component will
typically be derived on a volume percentage basis. In selected embodiments of
the present invention the refractive
index differential value will preferably be less than about 0.45, about 0.4,
about 0.35 or even less than about 0.3.
Given that lower refractive index differentials imply greater dispersion
stability, particularly preferred embodiments
comprise index differentials of less than about 0.28, about 0.25, about 0.2,
about 0.15 or even less than about 0.1. It
is submitted that a skilled artisan will be able to determine which excipients
are particularly compatible without undue
experimentation given the instant disclosure. The ultimate choice of preferred
excipients will also be influenced by
other factors, including biocompatibility, regulatory status, ease of
manufacture, cost.
As discussed above, minimization of density differences between the particles
and the continuous phase may
be achieved by using perforated andlor hollow microstructures, such that the
suspension medium constitutes most of
the particle volume. As used herein, the term "particle volume" corresponds to
the volume of suspension medium that
would be displaced by incorporated hollowlporous particles if they were solid,
i.e. the volume defined by the particle
boundary. For the purposes of explanation, and as discussed above, these fluid
filled particulate volumes may be
referred to as "virtual particles." Preferably, the average volume of the
bioactive agent(excipient shell or matrix (i.e.
the volume of medium actually displaced by the perforated microstructure)
comprises less than 80% of the average
particle volume (or less than 80% of the virtual particlel. More preferably,
the volume of the microparticulate matrix
comprises less than about 50°Yo, 400, 30% or even 200 of the average
particle volume. Even more preferably, the
average volume of the shell(matrix comprises less than about 10%, 5%, 3% or 1
% of the average particle volume.
Those skilled in the art will appreciate that such matrix or shell volumes
typically contribute little to the virtual particle
density which is overwhelmingly dictated by the suspension medium found
therein.
It will further be appreciated that, the use of such microstructures will
allow the apparent density of the
virtual particles to approach that of the suspension medium substantially
eliminating the attractive van der Waals
forces. Moreover, as previously discussed, the components of the
microparticulate matrix are preferably selected, as
much as possible given other considerations, to approximate the density of
suspension medium. Accordingly, in
preferred embodiments of the present invention, the virtual particles and the
suspension medium will have a density
differential of less than about 0.6 glcm'. That is, the mean density of the
virtual particles (as defined by the matrix
boundary) will be within approximately 0.6 glcm~ of the suspension medium.
More preferably, the mean density of the
virtual particles will be within 0.5, 0.4, 0.3 or 0.2 glcm' of the selected
suspension medium. In even more preferable
embodiments the density differential will be less than about 0.1, 0.05, 0.01,
or even less than 0.005 glcm3.
In addition to the aforementioned advantages, the use of the disclosed
particulates allows for the formation
of dispersions comprising much higher volume fractions of particles in
suspension. It should be appreciated that, the
formulation of prior art dispersions at volume fractions approaching
close~packing generally results in dramatic
increases in dispersion viscoelastic behavior. Rheological behavior of this
type is not appropriate for MDI or nebulizer
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applications. Those skilled in the art will appreciate that, the volume
fraction of the particles may be defined as the
ratio of the apparent volume of the particles (i.e. the particle volume) to
the total volume of the system. Each system
has a maximum volume fraction or packing fraction. For example, particles in a
simple cubic arrangement reach a
maximum packing fraction of 0.52 while those in a face centered
cubiclhexagonal close packed configuration reach a
maximum packing fraction of approximately 0.74. For non-spherical particles or
polydisperse systems, the derived
values are different. Accordingly, the maximum packing fraction is often
considered to be an empirical parameter for a
given system.
Here, it was surprisingly found that the preferred particulates of the present
invention do not exhibit
undesirable viscoelastic behavior even at high volume fractions, approaching
close packing. To the contrary, they
remain as free flowing, low viscosity suspensions having little or no yield
stress when compared with analogous
suspensions comprising solid particulates. The low viscosity of the disclosed
suspensions is thought to be due, at
least in large part, to the relatively low van der Waals attraction between
the fluid-filled hollow, porous particles. As
such. in selected embodiments the volume fraction of the disclosed dispersions
is greater than approximately 0.3.
Other embodiments may have packing values on the order of 0.3 to about 0.5 or
on the order of 0.5 to about 0.8, with
the higher values approaching a close packing condition. Moreover, as particle
sedimentation tends to naturally
decrease when the volume fraction approaches close packing, the formation of
relatively concentrated dispersions may
further increase formulation stability.
Although the methods and compositions of the present invention may be used to
form relatively concentrated
suspensions, the stabilizing factors work equally well at much lower packing
volumes and such dispersions are
contemplated as being within the scope of the instant disclosure. In this
regard, it will be appreciated that, dispersions
comprising low volume fractions are extremely difficult to stabilize using
prior art techniques. Conversely, dispersions
incorporating particulates comprising a bioactive agent as described herein
are particularly stable even at low volume
fractions. Accordingly, the present invention allows for stabilized
dispersions, and particularly respiratory dispersions,
to be formed and used at volume fractions less than 0.3. In some preferred
embodiments, the volume fraction is
approximately 0.0001 - 0.3, more preferably 0.001 - 0.01. Yet other preferred
embodiments comprise stabilized
suspensions having volume fractions from approximately 0.01 to approximately
0.1.
The perforated microstructures of the present invention may also be used to
stabilize dilute suspensions of
micronized bioactive agents. fn such embodiments the perforated
microstructures may be added to increase the
volume fraction of particles in the suspension, thereby increasing suspension
stability to creaming or sedimentation.
Further, in these embodiments the incorporated microstructures may also act in
preventing close approach
(aggregation) of the micronized drug particles. It should be appreciated that,
the perforated microstructures
incorporated in such embodiments do not necessarily comprise a bioactive
agent. Rather, they may be formed
exclusively of various excipients, including surfactants.
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Those skilled in the art will further appreciate that the stabilized
suspensions or dispersions of the present
invention may be prepared by dispersal of the microstructures in the selected
suspension medium which may then be
placed in a container or reservoir. In this regard, the stabilized
preparations of the present invention can be made by
simply combining the components in sufficient quantity to produce the final
desired dispersion concentration. Although
the microstructures readily disperse without mechanical energy, the
application of mechanical energy to aid in
dispersion (e.g. with the aid of sonication) is contemplated. Alternatively,
the components may be mixed by simple
shaking or other type of agitation. The process is preferably carried out
under anhydrous conditions to obviate any
adverse effects of moisture on suspension stability. Once formed, the
dispersion has a reduced susceptibility to
flocculation and sedimentation.
As indicated throughout the instant specification, the dispersions of the
present invention are preferably
stabilized. In a broad sense, the term "stabilized dispersion" will be held to
mean any dispersion that resists aggregation,
flocculation or creaming to the extent required to provide for the effective
delivery of a bioactive agent. Moreover, it is a
significant advantage of the instant invention that the disclosed dispersions
and powders are stable at room temperature
and do not require refrigeration or freezing to effectively maintain their
activity. Besides prolonging shelf life, this
remarkable temperature stability greatly simplifies shipping and
administration.
While those skilled in the art will appreciate that there are several methods
that may be used to assess the
stability of a given dispersion, a preferred method far the purposes of the
present invention comprises determination of
creaming or sedimentation time using a dynamic photosedimentation method. A
preferred method comprises subjecting
suspended particles to a centrifugal force and measuring absorbance of the
suspension as a function of time. A rapid
decrease in the absorbance identifies a suspension with poor stability. It is
submitted that those skilled in the art will
be able to adapt the procedure to specific suspensions without undue
experimentation.
For the purposes of the present invention the creaming time shall be defined
as the time for the suspended drug
particulates to cream to 112 the volume of the suspension medium. Similarly,
the sedimentation time may be defined as
the time it takes for the particulates to sediment in 112 the volume of the
liquid medium. Besides the photosedimentation
technique described above, a relatively simple way to determine the creaming
time of a preparation is to provide the
particulate suspension in a sealed glass vial. The vials are agitated or
shaken to provide relatively homogeneous
dispersions which are then set aside and observed using appropriate
instrumentation or by visual inspection. The time
necessary for the suspended particulates to cream to 112 the volume of the
suspension medium (i.e., to rise to the top half
of the suspension medium), or to sediment within 112 the volume (i.e., to
settle in the bottom 112 of the medium), is then
noted. Suspension formulations having a creaming time greater than 1 minute
are preferred and indicate suitable stability.
More preferably, the stabilized dispersions comprise creaming times of greater
than 1, 2, 5, 10, 15, 20 or 30 minutes. In
particularly preferred embodiments, the stabilized dispersions exhibit
creaming times of greater than about 1, 1.5, 2, 2.5,
or 3 hours. Substantially equivalent periods for sedimentation times are
indicative of compatible dispersions.
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It will also be understood that other components can be included in the
stabilized dispersions of the present
invention. For example, osmotic agents, stabilizers, chelators, buffers,
viscosity modulators, salts, and sugars can be
added to fine tune the stabilized dispersions for maximum life and ease of
administration. Such components may be
added directly to the suspension medium or associated with, or incorporated
in, the perforated microstructures.
Considerations such as sterility, isotonicity, and biocompatibility may govern
the use of conventional additives to the
disclosed compositions. The use of such agents will be understood to those of
ordinary skill in the art and, the specific
quantities, ratios, and types of agents can be determined empirically without
undue experimentation.
F(iiil. Metered Dose Inhalers
As discussed, the stabilized dispersions may preferably be administered to the
nasaf-or pulmonary air passages
of a patient via aerosolization, such as with a metered dose inhaler. The use
of such stabilized preparations provides for
superior dose reproducibility and improved deposition at the target site as
described above. MDIs are well known in the art
and could easily be employed for administration of the claimed dispersions
without undue experimentation. Breath
activated MDIs, as well as those comprising other types of improvements which
have been, or will be, developed are also
compatible with the stabilized dispersions and present invention and, as such,
are contemplated as being with in the scope
thereof.
MDI canisters generally comprise a container or reservoir capable of
withstanding the vapor pressure of the
propellant used such as, a plastic or plastic-coated glass bottle, or
preferably, a metal can or, for example, an aluminum
can which may optionally be anodized. lacquer-coated andlor plastic-coated,
wherein the container is closed with a
metering valve. The metering valves are designed to deliver a metered amount
of the formulation per actuation. The
valves incorporate a gasket to prevent leakage of propellant through the
valve. The gasket may comprise any suitable
elastomeric material such as, for example, low density polyethylene,
chlorobutyl, black and white butadiene-acrylonitrile
rubbers, butyl rubber and neoprene. Suitable valves are commercially available
from manufacturers well known in the
aerosol industry, for example, from Vafois, France (e.g. DFIO, DF30, DF 31150
ACT, DF601. Bespak plc, LTK (e.g. BK300,
BK3561 and 3M-Neotechnic Ltd., lIK (e.g. Spraymiserl.
Each filled canister is conveniently fitted into a suitable channeling device
or actuator prior to use to form a
metered dose inhaler for administration of the medicament into the lungs or
oral or nasal cavity of a patient. Suitable
channeling devices comprise for example a valve actuator and a cylindrical or
cone-like passage through which medicament
may be delivered from the filled canister via the metering valve, to the nose
or mouth of a patient e.g., a mouthpiece
actuator. Metered dose inhalers are designed to deliver a fixed unit dosage of
medicament per actuation such as, for
example, in the range of 10 to 5000 micrograms of bioactive agent per
actuation. Typically, a single charged canister will
provide for tens or even hundreds of shots or doses.
With respect to MDIs, it is an advantage of the present invention that any
biocompatible suspension medium
having adequate vapor pressure to act as a propellant may be used.
Particularly preferred suspension media are
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compatible with use in a metered dose inhaler. That is, they will be able to
form aerosols upon the activation of the
metering valve and associated release of pressure. In general, the selected
suspension medium should be biocompatible
(i.e. relatively non-toxic) and non-reactive with respect to the suspended
perforated microstructures comprising the
bioactive agent. Preferably, the suspension medium will not act as a
substantial solvent for any components incorporated
in the perforated microspheres. Selected embodiments of the invention comprise
suspension media selected from the
group consisting of fluorocarbons (including those substituted with other
halogens), hydrofluoroalkanes, perfiuorocarbons,
hydrocarbons, alcohols, ethers or combinations thereof. It will be appreciated
that, the suspension medium may comprise
a mixture of various compounds selected to impart specific characteristics.
Particularly suitable propellants for use in the MDI suspension mediums of the
present invention are those
propellant gases that can be liquefied under pressure at room temperature and,
upon inhalation or topical use, are safe,
toxicologically innocuous and free of side effects. !n this regard, compatible
propellants may comprise any hydrocarbon,
fluorocarbon, hydrogen-containing fluorocarbon ar mixtures thereof having a
sufficient vapor pressure to efficiently
form aerosols upon activation of a metered dose inhaler. Those propellants,
typically termed hydrofluoroalkanes or
HFAs, are especially compatible. Suitable propellants include, far example,
short chain hydrocarbons, C,.~ hydrogen
containing chlorofluorocarbons such as CHZCIF, CCIZFzCHCIF, CF3CHCIF,
CHFZCCIFZ, CHCIFCHF~, CF3CH2C1, and
CCIFZCH3; C,.~ hydrogen-containing fluorocarbons (e.g. HFAs) such as CHFZCHFZ,
CF3CHZF, CHFZCH3, and CF3CHFCF3;
and perfluorocarbons such as CF3CF3 and CF3CF2CF3. Preferably, a single
perfluorocarbon or hydrogen-containing
fluorocarbon is employed as the propellant. Particularly preferred as
propellants are 1,1,1,2-tetrafluoroethane
(CF3CHZF) (HFA-134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (CF3CHFCF3) (HFA-
2271, perfluoroethane,
monochlorodifluoromethane, 1,1-difluoroethane, and combinations thereof. It is
desirable that the formulations contain
no components that deplete stratospheric ozone. In particular it is desirable
that the formulations are substantially
free of chlorofluorocarbons such as CC13F, CCI2Fz, and CF3CCI3.
While preferred embodiments of the invention comprise ecologically benign
suspension media, traditional
chlorofluorocarbons and substituted fluorinated compounds may also be used as
suspension mediums in accordance
with the teachings herein. In this respect, FG11 (CCl.3F), FC-11B1 (CBrCI2F),
FC-11B2 (CBr2CIF), FC12B2 (CF2Br2?,
FC21 (CHCI2F1, FC21B1 (CHBrCIFI, FC-21B2 (CHBr2F), FC-31B1 (CH2BrF), FC113A
(CCI3CF31, FC-122
/CCIF2CHCI21, FC-123 (CF3CHCI21, FC-132 (CHCIFCHCIFI, FC-133 (CHCIFCHF2), FC-
141 (CH2CICHCIF1, FC-141B
(CGI2FCH31, FC-142 (CHF2CH2CI1, FC-151 (CH2FCH2CI), FC-152 (CH2FCH2F1, FC-1112
(CCIF-CCIFI, FC-1121
(CHCI-CFCI) and FC-1131 (CHCI-CHF) are all compatible with the teachings
herein despite possible attendant
environmental concerns. As such, each of these compounds may be used, alone or
in combination with other
compounds (i.e. less volatile fluorocarbons) to form stabilized respiratory
dispersions in accordance with the present
invention.
F(ivl. Nebulizers
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Along with the aforementioned embodiments, the stabilized dispersions of the
present invention may also be
used in conjunction with nebulizers to provide an aerosolized medicament that
may be administered to the pulmonary
air passages of a patient in need thereof. Nebulizers are well known in the
art and could easily be employed for
administration of the cla'uned dispersions without undue experimentation.
Breath activated nebulizers, as well as those
comprising other types of improvements which have been, or will be, developed
are also compatible with the stabilized
dispersions and present invention and are contemplated as being with in the
scope thereof.
Nebulizers work by forming aerosols, that is converting a bulk liquid into
small droplets suspended in a
breathable gas. Here, the aerosolized medicament to be administered
(preferably to the pulmonary air passages) will
comprise small droplets of suspension medium associated with perforated
microstructures comprising a bioactive agent.
In such embodiments, the stabilized dispersions of the present invention will
typically be placed in a fluid reservoir aperably
associated with a nebulizer. The specific volumes of preparation provided,
means of filling the reservoir, etc., will largely
be dependent on the selection of the individual nebufizer and is well within
the purview of the skilled artisan. Of course,
the present invention is entirely compatible with single~dose nebulizers and
multiple dose nebulizers.
The present invention overcomes these and other difficulties by providing
stabilized dispersions with a
suspension medium that preferably comprises a fluorinated compound (i.e. a
fluorochemical, fluorocarbon or
perfluorocarbon). Particularly preferred embodiments of the present invention
comprise fluorochemicals that are liquid at
room temperature. As indicated above, the use of such compounds, whether as a
continuous phase or, as a suspension
medium, provides several advantages over prior art liquid inhalation
preparations. In this regard, it is well established that
many fluorochemicals have a proven history of safety and biocompatibility in
the lung. Further, in contrast to aqueous
solutions, fluorochemicals do not negatively impact gas exchange following
pulmonary administration. To the contrary,
they may actually be able to improve gas exchange and, due to their unique
wettability characteristics. are able to carry an
aerosolized stream of particles deeper into the lung, thereby improving
systemic delivery of the desired pharmaceutical
compound. In addition, the relatively non-reactive nature of fluorochemicals
acts to retard any degradation (by proteolysis
or hydrolysis) of an incorporated bioactive agent.
In any event, nebulizer mediated aerosolization typically requires an input of
energy in order to produce the
increased surface area of the droplets and, in some cases, to provide
transportation of the atomized or aerosolized
medicament. One common mode of aerosolization is forcing a stream of fluid to
be ejected from a nozzle, whereby droplets
are formed. With respect to nebulized administration, additional energy is
usually imparted to provide droplets that will be
sufficiently small to be transported deep into the lungs. Thus, additional
energy is needed, such as that provided by a high
velocity gas stream or a piezoelectric crystal. Two popular types of
nebulizers, jet nebulizers and ultrasonic nebulizers, rely
on the aforementioned methods of applying additional energy to the fluid
during atomization.
In terms of pulmonary delivery of bioactive agents to the systemic circulation
via nebulization, recent research
has focused on the use of portable hand-held ultrasonic nebulizers, also
referred to as metered solutions. These devices,
generally known as single-bolus nebulizers, aerosolize a single bolus of
medication in an aqueous solution with a particle
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size efficient for deep lung delivery in one or two breaths. These devices
fall into three broad categories. The first
category comprises pure piezoelectric single-bolus nebulizers such as those
described by Mutterlein, et. al., (J. Aerosol
Med. 1988; 1:2311. In another category, the desired aerosol cloud may be
generated by microchannel extrusion single-
bolus nebulizers such as those described in U.S. Pat. No. 3,812,854. Finally,
a third category comprises devices
exemplified by Robertson, et. al., (WO 92111050) which describes cyclic
pressurization single-bolus nebulizers. Each of the
aforementioned references is incorporated herein in their entirety. Most
devices are manually actuated, but some devices
exist which are breath actuated- Breath actuated devices work by releasing
aerosol when the device senses the patient
inhaling through a circuit. Breath actuated nebulizers may also be placed in-
line on a ventilator circuit to release aerosol
into the air flow which comprises the inspiration gases for a patient.
Regardless of which type of nebulizer is employed, it is an advantage of the
present invention that biocompatible
nonaqueous compounds may be used as suspension mediums. Preferably, they will
be able to form aerosols upon the
application of energy thereto. In general, the selected suspension medium
should be biocompatible (i.e. relatively non-toxic)
and non-reactive with respect to the suspended perforated microstructures
comprising the bioactive agent. Preferred
embodiments comprise suspension media selected from the group consisting of
fluorochemicals, fluorocarbons (including
those substituted with other halogens), perfluorocarbons,
fluorocarbonlhydrocarbon diblocks, hydrocarbons, alcohols,
ethers, or combinations thereof. It will be appreciated that, the suspension
medium may comprise a mixture of various
compounds selected to impart specific characteristics.
In accordance with the teachings herein, the suspension media may comprise any
one of a number of different
compounds including hydrocarbons, fluorocarbons or hydracarbanlfluoracarbon
diblocks. In general, the contemplated
hydrocarbons or highly fluorinated or perfluorinated compounds may be linear,
branched or cyclic, saturated or unsaturated
compounds. Conventional structural derivatives of these fluorochemicals and
hydrocarbons are also contemplated as being
within the scope of the present invention as well. Selected embodiments
comprising these totally or partially fluorinated
compounds may contain one or more hetero-atoms andlor atoms of bromine or
chlorine. Preferably, these fluorochemicals
comprise from 2 to 16 carbon atoms and include, but are not limited to,
linear, cyclic or polycyclic perfluoroalkanes,
bislperfluoroalkyl)alkenes, perfluoroethers, perfluaroamines, perfluoroalkyl
bromides and perfluoroalkyl chlorides such as
dichlorooctane. Particularly preferred fluorinated compounds for use in the
suspension medium may comprise
perfluorooctyl bromide C8F"Br (PFOB or perflubronl, dichlorofluorooctane
CeF,sClz, and the hydrofluoroa(kane
perfluarooctyl ethane C8F"CZHS (PFOE). With respect to other embodiments, the
use of perfluarohexane or
perfluoropentane as the suspension medium is especially preferred.
More generally, exemplary fluorochemicals which are contemplated for use in
the present invention generally
include halogenated fluorochemicals ii.e. C,F2~,,X, XC~FZ~X, where n - 2-10, X
= Br, CI or 1) and, in particular, 1-bromo-F
butane n-C4FeBr, 1-bromo-F-hexane (n-CsF,3Br), 1-bromo-F-heptane (n-C,F,5Brl,
i,4-dibromo-F-butane and 1,6-dibromo-F-
hexane. Other useful brominated fluorochemicals are disclosed in US Patent No.
3,975,512 to Long and are incorporated
herein by reference. Specific fluorochemicals having chloride substituents,
such as perfluorooctyl chloride (n-CeF"CII, 1,8-
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dichloro-F-octane (n-CICeF,6Cl), 1,6-dichloro-F-hexane In-CICBF,2CI1, and 1, 4-
dichloro-F-butane (n-CIC4FeCl) are also
preferred.
Fluorocarbons, fluorocarbon-hydrocarbon compounds and halogenated
fluorochemicals containing other linkage
groups, such as esters, thioethers and amines are also suitable for use as
suspension media in the present invention. For
instance, compounds having the general formula, C~F2~,,OCmFZ~",, or C~F2~,,CH-
CHCmF2m,,, (as for example
C4F9CH-CHC4F9 /F-44E), i-C3F9CH=CHCBF,3 (F-i36E1, and CsF,3CH-CHC6F,3 (F-66EI)
where n and m ace the same or
different and n and m are integers from about 2 to about 12 are compatible
with teachings herein. Useful fluorochemical-
hydrocarbon diblock and triblock compounds include those with the general
formulas C~FZ~,,-CmH~", and C~Fz~,,C,~H~,.,,
where n - 2-12; m - 2-16 or CpH2p,,-C~F2~ CmH~",, where p = 1-12, m - 1-12 and
n = 2-12. Preferred compounds of
this type include CeF"C2H5, C6F,3C,oH2,, CBF"CeH", CsF"CH-CHCsH,3 and CBF"CH-
CHC,oHZ,. Substituted ethers or
polyethers (i.e. XC~FZ~OCmFZmX, XCFOC~FZ~OCFZX, where n and m - 1-4, X - Br,
CI or 1) and fluorochemical-hydrocarbon
ether diblocks or triblocks (i.e. C~F2~,,-0-CmHZm,,, where n - 2-10; m - 2-16
or CpHTp,,-0-C~Fz~ 0-CmHZm,,, where p - 2-
12, m - 1-12 and n - 2-12) may also used as well as C~Fz~,,O-CmF2mOCpHzp,,,
wherein n, m and p are from 1-12.
Furthermore, depending on the application, perfluoroalkylated ethers or
polyethers may be compatible with the claimed
dispersions.
Polycyclic and cyclic fluorochemicals, such as C,oF,e (F-decalin or
perfluorodecalin),
perfluoroperhydrophenanthrene, perfluorotetramethylcyclohexane (AP-144) and
perfluoro n-butyldecalin are also within the
scope of the invention. Additional useful fluorochemicals include
perfluorinated amines, such as F-tripropylamine ("FTPA")
and F-tributylamine ("FTBA"I. F-4-methyloctahydroquinalizine ("FMOO"), F-N-
methyl-decahydroisoquinoline ("FMIQ"), F-N-
methyldecahydroquinoline ("FHO"), F-N-cyclohexylpyrrolidine ("FCHP") and F-2-
butyltetrahydrofuran ("FC-75"or "FC-77").
Still other useful fluorinated compounds include perfluorophenanthrene,
perfluoromethyldecalin,
perfluorodimethylethylcyclohexane, perfluorodimethyldecalin,
perfluorodiethyldecalin, perfluoromethyladamantane,
perfluorodimethyladamantane. Other contemplated fluorochemicals having
nonfluorine substituents, such as,
perfluorooctyl hydride, and similar compounds having different numbers of
carbon atoms are also useful. Those skilled in
the art will further appreciate that other variously modified fluorochemicals
are encompassed within the broad definition of
fluorochemical as used in the instant application and suitable for use in the
present invention. As such, each of the
foregoing compounds may be used, alone or in combination with other compounds
to form the stabilized dispersions of the
present invention.
Additional exemplarly fluorocarbons, or classes of fluorinated compounds, that
may be useful as suspension
media include, but are not limited to, fluoroheptane, fluorocycloheptane
fluoromethylcycloheptane, fluorohexane,
fluorocyclohexane, fluoropentane, fluorocyclapentane,
fluoromethylcyclopentane, fluorodimethylcyclopentanes,
fluoromethylcyclobutane, fluorodimethylcyclobutane,
fluorotrimethylcyclobutane, fluorobutane, fluorocyclobutane,
fluoroprepane, fluoroethers, fluoropolyethers and fluorotriethylamines. Such
compounds are generally environmentally
sound and are biologically non-reactive.
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While any liquid capable of producing an aerosol upon the application of
energy may be used in conjunction with
a nebulizer, the selected suspension medium will preferably have a vapor
pressure less than about 5 atmospheres and more
preferably less than about 2 atmospheres. Unless otherwise specified, all
vapor pressures recited herein are measured at
25°C. In other embodiments, preferred suspension media compounds will
have vapor pressures on the order of about 5
tort to about 760 tort, with more preferable compounds having vapor pressures
on the order of from about 8 tort to about
600 tort, while still more preferable compounds will have vapor pressures on
the order of from about 10 tort to about 350
tort. Such suspension media may be used in conjunction with compressed air
nebulizers, ultrasonic nebufizers or with
mechanical atomizers to provide effective ventilation therapy. Moreover, more
volatile compounds may be mixed with
lower vapor pressure components to provide suspension media having specified
physical characteristics selected to further
improve stability or enhance the bioavailability of the dispersed bioactive
agent.
Other embodiments of the present invention directed to nebulizers will
comprise suspension media that boil at
selected temperatures under ambient conditions (i.e.1 atml. For example,
preferred embodiments will comprise suspension
media compounds that boil above 0°C, above 5°C, above
10°C, above 15°, or above 20°C. In other embodiments, the
suspension media compound may boil at or above 25°C or at or above
30°C. In yet other embodiments, the selected
suspension media compound may boil at or above human body temperature (i.e.
37°C), above 45°C, 55°C, 65°C, 75°C,
85°C or above 100°C.
F(v). Direct Administration
Along with MDIs and nebulizers, it will be appreciated that the stabilized
dispersions of the present invention
may be used to administer bioactive agents to a variety of target sites using
various routes of administration. For
example, the disclosed compositions may be delivered directly to the lungs in
conjunction with liquid dose instillation (LDI)
techniques. Alternatively, the stabilized dispersions could be effectively
delivered to mucosal surfaces in the nasal
passages using a nasal pump, spray bottle or atomizer. In yet other
embodiments, the disclosed dispersions could be
administered to a target site (e.g. intramuscularly or intradermally) using
conventional injections or through the use of
needleless injectors employing compressed gases. The latter are particularly
preferred in the case of needleless
inoculation. Still other embodiments are directed to the topical delivery of
the dispersions to target sites such as the eye
or the ear or, more preferably, mucosal surfaces such as those in the
urogenital tract or the gastrointestinal tract. Such
techniques may further employ ionophoresis to enhance penetration of the
incorporated bioactive agent. In any event, the
stabilized dispersions provide for excellent dose reproducibility while
preserving the activity of the incorporated agent.
Those skilled in the art will appreciate that suspension media compatible with
the aforementioned delivery
techniques are similar to those set forth above for use in conjunction with
nebulizers. That is, the stabilized
dispersions will preferably comprise suspension media selected from the group
consisting of fluorochemicals,
fluorocarbons (including those substituted with other halogens),
perfluorocarbons, fluorocarbonlhydrocarbon diblocks,
hydrocarbons, alcohols, ethers, or combinations thereof. More particularly,
for the purposes of the present application,
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compatible suspension media for such delivery techniques shall be equivalent
to those enumerated above in conjunction
with use in nebulizers. In particularly preferred embodiments the selected
suspension medium shall comprise a
fluorochemical that is liquid under ambient conditions.
It should be further be appreciated that liquid dose instillation involves the
direct administration of a stabilized
dispersion to the lung. In this regard, direct pulmonary administration of
bioactive compounds is particularly effective in
the treatment of disorders especially where poor vascular circulation of
diseased portions of a lung reduces the
effectiveness of intravenous drug delivery. With respect to LDI the stabilized
dispersions are preferably used in conjunction
with partial liquid ventilation or total liquid ventilation. Moreover, the
present invention may further comprise introducing a
therapeutically beneficial amount of a physiologically acceptable gas (such as
nitric oxide or oxygenl into the
pharmaceutical microdispersion prior to, during or following administration.
For LDI, the dispersions of the present invention may be administered to the
lung using a pulmonary delivery
conduit. Those skilled in the art will appreciate the term "pulmonary delivery
conduit", as used herein, shall be
construed in a broad sense to comprise any device or apparatus, or component
thereof, that provides for the instillation
or administration of a liquid in the lungs. In this respect a pulmonary
delivery conduit or delivery conduit shall be held
to mean any bore, lumen, catheter, tube, conduit, syringe, actuator,
mouthpiece, endotracheal tube or bronchoscope
that provides for the administration or instillation of the disclosed
dispersions to at least a portion of the pulmonary air
passages of a patient in need thereof. It will be appreciated that the
delivery conduit may or may not be associated
with a liquid ventilator or gas ventilator. In particularly preferred
embodiments the delivery conduit will comprise an
endotracheal tube or bronchoscope.
While the stabilized dispersions may be administered up to the functional
residual capacity of the lungs of a
patient, it will be appreciated that selected embodiments will comprise the
pulmonary administration of much smaller
volumes le.g. on the order of a milliliter or less. Far example, depending on
the disorder to be treated, the volume
administered may be on the order of 1, 3, 5, 10, 20, 50.100, 200 or 500
milliliters. In preferred embodiments the liquid
volume is less than 0.25 or 0.5 percent FRC. For particularly preferred
embodiments, the liquid volume is 0.1 percent
FRC or less. With respect to the administration of relatively low volumes of
stabilized dispersions it will be
appreciated that the wettability and spreading characteristics of the
suspension media (particularly fluorochemicals)
will facilitate the even distribution of the bioactive agent in the lung.
However, in other embodiments it may be
preferable to administer the suspensions a volumes of greater than 0.5, 0.75
or 0.9 percent FRC. Of course the
extraordinary wetting and spreading characteristics associated with at least
some fluorochemicals makes them
particularly compatible for administration to other mucosal surfaces such as
the nasal passages.
With regard to the powders and stabilized dispersions disclosed herein those
skilled in the art will appreciate
that they may be advantageously supplied to the physician or other health care
professional, in a sterile, prepackaged
or kit form. More particularly, the formulations may be supplied as stable
powders or preformed dispersions ready for
administration to the patient. Conversely, they may be provided as separate,
ready to mix components. When
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provided in a ready to use form, the powders or dispersions may be packaged in
single use containers or reservoirs, as
well as in multi-use containers or reservoirs. In either case, the container
or reservoir may be associated with the
selected inhalation or administration device and used as described herein.
When provided as individual components
(e.g., as powdered microspheres and as neat suspension medium) the stabilized
preparations may then be formed at
any time prior to use by simply combining the contents of the containers as
directed. Additionally, such kits may
contain a number of ready to mix, or prepackaged dosing units so that the user
can then administer them as needed.
G. Examples
The foregoing description will be more fully understood with reference to the
following Examples. Such
Examples, are, however, merely representative of preferred methods of
practicing the present invention and should not be
read or interpreted as limiting the scope of the invention in any manner.
I
Preparation of Hollow Porous Particles of HA Peptide by Spray-Drying.
Hollow porous HA 110-120 peptide (amino acid residues 110-120 of the
hemagglutinin of the influenza virus)
particles (PuImoSpheres~") were prepared by a spray drying technique with a B-
191 Mini Spray-Drier (Buchi, Flawil,
Switzerland) under the following conditions: aspiration: 1009'0, inlet
temperature: 85°C; outlet temperature: 51°C; feed
pump: 10%; NZ flow: 800 L[hr. The feed was prepared by mixing two
preparations, A and B, immediately prior to spray
drying. A 150 mesh stainless steel screen was placed in the cyclone exit port
to aid with the collection particles.
Preparation A: 5g of deionized water was used to dissolve l8mg of HA 110-120
peptide (Chiron Corp.,
Emeryville, CAI and 1 mg of hydroxyethyl starch (Ajinomoto, Japan).
Preparation B: A fluorocarbon-in-water emulsion stabilized by phospholipid was
prepared in the following
way. The phospholipid, 0.3g EPC-100-3 (Lipoid KG, Ludwigshafen, Germanyl, was
homogenized in 33g of hot
deionized water (T - 50 to 60°C) using an Ultra-Turrax mixer (model T-
25) at 8000 rpm for 2 to 5 minutes (T - 60-
70°CI. Eight grams of Perflubron (perfluorooctyl bromide: Atochem,
Paris, France) was added dropwise during mixing.
After the fluorocarbon was added, the emulsion was mixed for at least 4
minutes. The resulting coarse emulsion was then
passed through a high pressure homogenizes (Avestin, Ottawa, Canada) at 18,000
psi for 5 passes.
One eighth of preparation B was separated and added to preparation A. The
resulting HA peptide[perflubron
emulsion feed solution was fed into the spray dryer under the conditions
described above. The powder collected in the
cyclone, and sieving screen was washed into the collection jar using
perflubron. The HA suspension in perfiubron was
subsequently frozen at -60°C and lyophilized. A free flowing white
powder was obtained.
II
In Vitro Activity of Holiow Porous Particles Containing HA Peutide.
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The functionality of HA peptide in PuImoSpheres (HA-Pul) to activate antigen
presenting cells was compared
with neat HA peptide. HA peptide PuImoSpheres from Example I were incubated
with sterile PBS at a concentration of
5mglml (weight of formulationlvolume). Serial dilutions of the resultant HA-
Pul-PBS solution were added to microwells
containing M12 antigen presenting cells (1x1041we1p and HA specific TcH
(2x10'Iwell) in complete RPMI-10~ FCS.
The TcH cell line bears a reporter gene controlled by IL-2 promoter (IL-21-
gap.
After 12 hours incubation at 37°C, the microwell plate was centrifuged,
cells were fixed with
paraformaldehyde-glutaraldehyde for 5 minutes at 4°C, washed with PBS
and X-gal substrate was added overnight.
The number of activated TcH per 500 cells per well were counted using light
microscopy. The total number of
activated TcH per well was estimated by multiplying the total number of cells
with the percentage of blue cells. The
results, shown in Fig. 1, demonstrate the presence of active peptide in the
formulation. Comparison with a standard
activation curve (HA saline) showed that the concentration of active peptide
was approximately 5% (wtlwt), which
was in agreement with reverse phase-HPLC measurements.
HA Peptide PuImoSoheres Mechanism of Action
The requirement for internalization and processing of PuImoSphere
microparticles containing T cell epitope
HA 110-120 (HA-Pul) was examined. HA-Pul suspended in perflubron (500nMlwell
HA 110-120 peptide) were air-dried
and incubated with non-fixed or paraformaldehyde fixed M12 antigen presenting
cells (APC) cells in the presence of
specific TcH cells in complete RPMI-1096 FCS, and compared with HA-Pul
suspended in PBS and neat HA peptide at
similar concentrations. Sucrose-purified AIPRI8134 (H1N1) virus (15 gimp was
used as the positive control, since it
does that require intracellular processing. Negative controls comprised a
formulation of NP 147-155 peptide, non-
formulated NP peptide and an irrelevant virus. The number of cells and culture
conditions described in Example II were
followed. The cells were fixed and exposed to an X-gal substrate. The results
were expressed as ~ of activated TcH.
Fig. 2 shows that both fixed and non-fixed APC were able to present neat HA
peptide and HA-Pul. In
contrast, only live APC were able to present HA peptide from the viral
context. Furthermore, formulated or neat NP
peptide as well as BILee virus did not activate the specific TcH. The results
indicate that internalization and processing
of HA-Pul is not a prerequisite for the activation of TeH. Rather, the HA-
peptide is readily released from the
PuImoSpheres and binds to MHC class II molecules (I-E") on M12 APC, resulting
in the engagement of TCR and
activation of TcH. This processing step was observed for neat HA peptide as
well as HA-Pul delivered in PBS or
perflubron. Moreover, these results demonstrate that HA 110-120 peptide
formulated in PulmoSpheres and stabilized
in perflubron retains its immunogenicity.
IV
Preuaration of Fluorescent-Labeled Holiow Porous
HA Peptide Particles by Spray-Drvinn.
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Hollow porous HA-fluoroscein 110-120 peptidelTexas Red DHPE particles were
prepared by a spray drying
technique with a B-191 Mini Spray-Drier (Buchi, Flawil, Switzerland) under the
following conditions: aspiration: 100%,
inlet temperature: 85°C; outlet temperature: 51°C; feed pump:
10%; NZ flow: 800 Llhr. The feed was prepared by
S mixing two solutions A and B immediately prior to spray drying. A 150 mesh
stainless steel screen was placed in the
cyclone exit port to aid with the collection particles.
Preparation A: 5g of deionized water was used to dissolve 20mg of HA-
fluoroscein 110-120 peptide (Chiron
Corp., Emeryville, CAI and 1mg of hydroxyethyl starch (Ajinomota, Japan).
Preparation B: A fluorocarbon-in-water emulsion stabilized by phospholipid was
prepared in the following
way. The phospholipid, 0.3g EPC-100-3 (Lipoid KG, ludwigshafen, Germany), and
0.3 mg fluorescent dye, Texas Red
DHPE, (Molecular Probes, Eugene, OR, 3mg) were first dissolved in chloroform.
The chloroform was then removed using
a Buchi Rotollap. The E100-3lTexas Red DHPE thin film was then dispersed into
33 ml hot deionized water (60 to 70°C).
The surfactants were then further processed in the aqueous phase using an
Ultra-Turrax mixer (model T-25) at 10,000 rpm
for approximately 2 minutes (T - 50 to 60 CI. 8g of perflubron (Atochem,
Paris, France) was added dropwise during
mixing. After the fluorocarbon was added, the emulsion was mixed for at least
4 minutes. The resulting coarse emulsion
was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada)
at 18.000 psi for 5 passes.
One eighth of preparation B was separated and added to preparation A. The
resulting HA-ffuoroscein
peptidelTexas Red DHPEIPerflubron emulsion feed solution was fed into the
spray dryer under the conditions described
above. The powder collected in the cyclone, and sieving screen was washed into
the collection jar using Perflubron.
The HA suspension in Perflubron was subsequently frozen at -60°C and
lyophilized. A free flowing fluorescent
fuschsia-colored powder was obtained.
U
Bioavailabilitv of Fluorescent-Labeled HA PuImoSnheres
A formulation comprising fluoroscein-HA peptide (20%wtlwt) PuImoSpheres (f-HA-
Pul) prepared as in
Example IU was suspended in perflubron. Metofane anesthetized mice were
inoculated intranasally (in.) with a 70 I
volume of f-HA-Pul in perflubron, corresponding to 70 g of peptide dose. Blood
samples were collected by ocular
bleeding in heparin-treated tubes, the plasma was separated and the
concentration of the peptide was measured by
fluorometry. As a control, an intravenous (i.v.) inoculation of 70 g of f-HA
peptide in 70 I of sterile saline (n~4 for all
groups) was used.
Fig. 3 depicts the serum concentration of f-HA peptide over time. The absolute
bioavailability for the i.n.
delivered f-HA peptide was approximately 5%, with Tm" occurring at 20 minutes.
The pharmacokinetic profile differed
between the two routes of administration, with a continuous logarithmic decay
for the i.v. administration and a
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transient increase followed by an exponential decay in the case of i.n.
administration. Elimination of f-HA occurs via
urine (not shown), with total clearance by 6 hours.
This Example shows that in, administration of T cell epitopes (having a
molecular weight of approximately
l.4kDa) formulated in Pul is compatible with systemic delivery.
vl
Preparation of Hollow Porous Particles of Human IeG by Snray-Dryina.
Hollow porous Human igG particles were prepared by a spray drying technique
with a B-191 Mini Spray-Drier
(Buchi, Flawil, Switzerland) under the following conditions: aspiration: 100%,
inlet temperature: 85°C; outlet
temperature: 61°C; feed pump: 10%; NZ flow: 800 Llhr. The feed was
prepared by mixing two solutions A and B
immediately prior to spray drying.
Preparation A: 2g of normal saline (Baxter, Chicago, Il.) was used to dissolve
55mg of human IgG (Sigma
Chemicals. St. Louis, MO) and 3.2mg of hydroxyethyl starch (Ajinomoto, Japanl.
1 S Preparation B: A fluorocarbon-in-water emulsion stabilized by phospholipid
was prepared in the following
way. The phospholipid, 0.4158 EPC-100-3 (Lipoid KG, Ludwigshafen, Germany),
was homogenized in 40.38 of hot
deionized water (T - 50 to 60°C) using an Ultra-Turrax mixer (model T-
25) at 8000 rpm for 2 to 5 minutes (T - 60-
70°C). 5.28 of perflubron (Atochem, Paris, France) was added dropwise
during mixing. After the fluorocarbon was
added, the emulsion was mixed for at least 4 minutes. The resulting coarse
emulsion was then passed through a high
pressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes.
One eighth of preparation B by volume was separated and added to preparation
A. The resulting
IgGlperflubron emulsion feed solution was fed into the spray dryer under the
conditions described above. The powder
collected in the cyclone, and sieving screen was washed into the collection
jar using perflubron. The IgG suspension in
perflubron was subsequently frozen at -60°C and lyophilized. A free
flowing white powder was obtained. The hollow
porous IgG particles had a volume-weighted mean aerodynamic diameter of 2.373
~ 1.88 Nm as determined by a time-
of-flight analytical method (Aerosizer, Amherst Process Instruments, Amherst,
MA).
vn
In Uitra Activity of nolvclonal human IaG PuImoSuheres
A formulation of polyclonal human IgG PuImoSpheres (hlgG-Pup from Example VI
was characterized for
activity using a capture hlgG ELISA. A 5 mglml hlgG-Pul suspension in
perflubron was prepared, pipetted into the
wells and air dried. PBS was added to the dried hlgG-Pul and allowed to
incubate overnight. The hydrated hlgG-Pul
solution was diluted and transferred to an ELISA plate coated with mouse anti-
human k chain monoclonal antibody in
coating buffer (dil. 1:1000, Sigma Immunochemical), and subsequently blocked
with PBS containing 15% goat serum
for 2 hours at room temperature. The wells were washed and the assay was
developed using goat anti-human IgG
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alkaline phosphatase conjugate 11:1000 in PBS-15% goat serum 0.05% Tween),
followed by addition of pNPP
substrate. The optical density (OD) was read using at an automatic plate
reader set at =405nm. hlgG in saline
(standard) and hlgG mixed with blank PuImoSpheres were employed as controls to
rule out an effect of the lipid on the
assay. The blank PuImoSpheres were comprised of only phospholipid and starch.
Fig. 4 depicts the calibration curves for the hlgG-Pul, hlgG and hlgG + blank
PuImoSpheres. The hlgG-Pul
formulation was determined to comprise approximately 20% hlgG by weight. In
addition, the hIgGPuI retained the
expression of k light chain and heavy chain epitopes.
vm
Dissolution Kinetics of HA 110-120 oentide
and human IaG from PuImoSphere Formulations
The kinetics of antigen and hlgG release from PuImoSpheres was measured using
dissolution chambers
equipped with 0.2Nm diameter filters, and adapted in 24-well flat bottom cell
culture plates. Approximately 3mg of
PuImoSphere powder from Examples I and vl were placed in the lower compartment
of the dissolution chamber and
exposed simultaneously to sterile PBS (l.3mllwell). The plates were placed an
a horizontal shaker (30 RPM) at 37°C,
to simulate the breathing pattern. 25,u1 samples were collected from the upper
compartment and analyzed by capture
ELISA in the case of hlgG (Fig. 5A) or bioassay in the case of fluorascein-
labeled HA peptide formulation (f-HA) (Fig.
5B). The results for f-HA were independently confirmed by fluorometry (not
shown). The dissolution kinetics of the
hlgG and HA peptide PuImoSphere formulations were compared with their
respective aqueous controls.
The results depicted in Figs. 5A and 5B and were expressed as percent release.
A rapid diffusion-controlled
release was observed for HA peptide formulation, with no difference between
the aqueous control. Complete
dissolution occurred within 2 hours. In contrast, a slower erosion-controlled
kinetics was observed for the hlgG
formulation. Complete dissolution required more than 6 hours as compared with
1 hour for the aqueous IgG control.
The results described herein demonstrate that the dissolution kinetics from
PuImoSpheres depends, at least in part, on
the molecular weight of the formulated compound (1.4 kDa and 150 k0a,
respectivelyl. It will also be appreciated that
differences in hydrophilicity or hydrophobicity may have similar effects.
IX
Bioavailability of hlaG PuImoSnheres
Human IgG PuImoSpheres (hlgG-Pul) from Example vl were administered either
intratracheally (20 g hlgG in
20 I of perflubron) to mice anesthetized with ketaminelxylazine, or via the
nasal route (70 g hlgG in 70,u1 of perflubron)
to mice anesthetized with metofane. An identical volume of hlgG in sterile PBS
was administered intravenously in the
control group. The mice were bled at various time intervals and the serum
concentration of hlgG was assessed by
capture ELISA in all groups (n-3). Absolute bioavailability was determined
from the areas under the serum
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concentration-time curve (AUCI as compared with the i.v, control. AUC values
were calculated using the trapezoid
rule.
The plamsa hlgG concentration curves are depicted in Figs. 6A (i.t.), and 6B
(in.). The absolute
bioavailability for the intratracheal delivery of hlgG-Pul was 270, and 1.5%
for intranasal inoculation. In both cases,
the Tm,I occurred at approximately 2 days. Western blotting showed that the
molecular weight for the circulating hlgG
after delivery via the respiratory tract was indistinguishable from the neat
material. The hlgG was observed to persist
in the circulation more than 14 days.
X
Antibody resoonse to hlgG PuImoSnheres
delivered via the tracheal route
The humoral response in the blood and bronchoalveolar lavage (BAL) in mice
treated with hlgG-PuImoSpheres
(hlgG-Pull from Example UI suspended in perflubron via intratracheal
administration (20 g dose of hlgG). Mice were
also treated with the following controls: 20ug hlgG in saline i.t., 100,ug
hlgG in saline i.v. and i.t., 100Ng in complete
Freund's Adjuvant (CFA) subcutaneous and saline i.t. Each group was done in
triplicate. Blood and BAL were collected
2 weeks after immunization.
The titer of anti-hlgG mouse IgG was measured using ELISA plates coated with
hlgG or with 0.19'o BSA. The
wells were blocked with PBS-15% goat serum and incubated for two hours using
various dilutions of sera or BAL.
After washing, the assay was developed with goat anti-mouse IgG alkaline
phosphatase conjugate, followed by the
addition of pNPP substrate. The optical density (OD) of the plates were
analyzed at 550nm using an automatic plate
reader and the results were expressed as endpoint dilution titers in the case
of serum IgG (Fig. 7A) or mean OD for BAL
IgG (Fig. 7B).
The results show an increased systemic and local humoral responses in mice
treated with hlgG-Pul via the
intratracheal route, as compared with the doselroute matched group that
received hlgG in saline. Moreover, the
response was enhanced as compared with mice that received higher doses of hlgG
in saline, via intratracheal or
intravenous routes. The titer of serum antibodies was similar to that measured
in mice immunized s.c. with hlgG in
CFA. Interestingly, the humoral response did not correlate with the systemic
bioavailability (data not shown), implying
the participation of local immunity.
XI
T cell response to hlcG PuImoSuheres delivered via tracheal route.
The level of T cell immunity induced in the spleens of mice immunized with
hlgG-PuImoSpheres (hlgG-Pul)
ftom Example VI suspended in perflubron by the tracheal route. The spleens
were dissociated into single cell
suspensions that were treated with hypotonic buffer to remove the red blood
cells. The splenocytes were resuspended
in complete RPM1~109~o FCS at 4x106cellslml and incubated in 24-well flat
bottom plates (lmllwellh in the presence of
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6 glml of hlgG. After 72 hours incubation, the supernatants were collected and
the concentration of IL-2, IFN- and IL-
4 determined by ELISA (Biosource International. Camarillo - CA).
The results (Fig. 8) were expressed as mean values of cytokine concentration
among individual mice in each
group, and showed enhanced production of all three cytokines in mice immunized
with hlgG-Pul as compared with the
hlgG saline controls.. The production of cytokines by splenic T cells for the
hlgG-Pul treated group was comparable
with that observed for the i.v. hlgG in saline group. These results strongly
suggest systemic migration of memory T
cells primed in the lung.
XII
Antibody Response to hlaG PuImoSpheres delivered via the nasal route.
The humoral response of mice that received hlgG via intranasal instillation
(20 gl either formulated as
PuImoSpheres (hlgG-Pul) from Example UI suspended in perflubron or dissolved
in saline was characterized. Sera was
obtained at various time intervals after immunization and the titer of
specific mouse IgG raised against the hlgG was
measured using the ELISA procedure described at Example X. The results (Fig.
9) were expressed as mean endpoint
titers (n=31, and showed that the kinetics of onset was faster, the magnitude
was higher and the intersubject
reproducibility of immune responses was lower in mice treated with hIgGPuI as
compared to saline.
XIII
Antibody Response to hlaG PuImoSuheres delivered via Peritoneal Route.
The humoral response of mice treated with higG-Pulmospheres IhIgG-Pul) from
Example VI suspended in
perflubron via the peritoneal (ip.) route (100 g dose of hlgG). Mice were also
treated ip, with 100,ug hlgG in the
following controls: in saline, in a multilamellar
dipalmitoylphospahtidylcholine (DPPC) liposome saline solution (+ml lip),
in a unilamellar DPPC liposome saline solution (+ ul lip) and in a blank
PuImoSphere saline solution (+ empty Pul) An
additional control group of blank PuImoSphere solution devoid of hlgG was also
tested. The particle median diameter
of ml lip ( > l0,um) and ul lip (90nm) were determined using a laser light
scattering technique. Each group was done in
triplicate. The IgG humoral immune response in sera, at 7 and 14 days was
measured using the same ELISA technique
described in Example X.
The results were expressed as means of endpoint titers and showed a consistent
increase in antibody titers
for animals that were inoculated with hlgG-Pul. More particularly Figs. 10A
and 108 show endpoint titers at 7 and 14
days respectively. hlgG added to empty Pul induced titers similar to hlgG in
saline. Furthermore, addition of either
DPPC liposome preparation to hlgG did not restore the increased immunity
observed with hlgG-Pul. Thus, these results
demonstrate that an: (1) enhanced immunity hlgG-Pul is not a route dependent
phenomenon (see Examples X and XII);
(2) formulation of hlgG-Pul is a prerequisite for the enhanced immunogenicity
of hlgG; and (3) DPPC or other
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components of Pul do not have an independent adjuvant effect. Moreover, these
results elucidate the importance of
the route of delivery as well as other factors responsible for the enhanced
immunity elicited by hlgG-Pul.
xlv
Preparation of Hollow Porous Particles of
Influenza Virus AIWSNI32 (H1N1) by Snrav-Drvinu.
Hollow porous Influenza Virus (AIWSNI32 H1N11, which comprises a relatively
complex enveloped virus
comprising 8 structural protein complexes and 8 negatively charged RNA
segments, were successfully incorporated in
microparticles prepared by a spray drying technique with a B-191 Mini Spray-
Drier (Buchi, Flawil, Switzerland) under
the following conditions: aspiration: 100%, inlet temperature: 85°C;
outlet temperature: 61 °C; feed pump: 100; Nz
flow: 800 Llhr. The feed was prepared by mixing two preparations A and B
immediately prior to spray drying. Prior to
formulation, the virus was live and had been purified by sucrose-gradient
centrifugation.
Preparation A: Weighed 1 mg hydroxyethyl starch (Ajinomoto, Japan) and
transferred to tube containing 0.6
mg Influenza Virus in saline.
Preparation B: A fluorocarbon-in-water emulsion stabilized by phospholipid was
prepared in the following
way. The phospholipid, 0.111 g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany),
was homogenized in 20g of hot
deionized water (T = 50 to 60°C) using an Ultra-Turrax mixer (model T-
25) at 6000 rpm for 2 to 5 minutes (T = 60-
70°C). 4.4g of perfiubron (Atochem, Paris, France) was added dropwise
during mixing. After the fluorocarbon was
added, the emulsion was mixed for at least 4 minutes. The resulting coarse
emulsion was then passed through a high
pressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes.
One eighth of preparation B by volume was separated and added to preparation
A. The resulting Influenza
Viruslperflubron emulsion feed solution was fed into the spray dryer under the
conditions described above. The powder
collected in the cyclone, and the sieving screen was washed into the
collection jar using perflubron. The Influenza
Virus suspension in perflubron was subsequently frozen at -60°C and
lyophilized. A free flowing white powder was
obtained.
XV
In Vitro Activity of Influenza Virus AIWSN1321H1N1) PuImoSpheres
The incorporation of live viral antigen into spray-dried particles was
characterized using the following
technique: Influenza Virus AIWSNI32 (H1N1) PuImoSpheres (WSN-Pull from Example
XIV were dissolved in sterile PBS
at a concentration of 5mg(ml for 6 hours at 40°C. The hydrated WSN-Pul
was then incubated at various dilutions
with non-fixed or paraformaldehyde-fixed M12 antigen presenting cells (APC)
for 1 hour at 37°C, in 96-well plates.
After antigen pulsing, the APCs were washed and incubated for four hours with
TcH. The formaldehyde-
glutaraldehyde fixed cells were incubated with X-gal substrate, and positive
cells were counted.
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Results were expressed as percent activated TcH (Fig. 11 A). Various
concentrations of sucrose-purified live
WSN virus were used as controls (Fig. 11 B). The WSN-Pul formulation was
determined to contain approximately 5%
influenza virus by weight. Only the unfixed APC's could activate the virus,
indicating that the antigens had not
degraded. Titration of infectious virus was determined by MDCK (Madine Darby
kidney carcinoma cells) assay (Fig.
11 CI. and showed that approximately 1 % of the total virus was still able to
infect and replicate in the permissive cells.
Together, these results demonstrate successful incorporation of relatively
large influenza virus antigens in
PuImoSphere powders.
xvl
Antibody Resuonse to Influenza Virus AIWSNI32 IH1N1)
PuImoSoheres Delivered via Nasal Route.
The induction of virus-specific IgG antibody response against WSN virus after
intranasal inoculation of
BALBIc mice with an Influenza Virus AIWSNI32 (H1N1) PuImoSphere (WSN-Pul)
formulation containing 5 g of virus and
2x10' TCIDSO of live virus f1% of the total antigen load corresponding to the
amount of live virus) was measured.
Control mice were immunized mice with 2x10' TCIDSO live virus (corresponding
to 0.05 g of total virus) or UV-killed
WSN virus (5 g). Sera from mice treated with hlgG was used as negative
control. The antibody response was
measured in sera using the following ELISA technique: wells were coated with
sucrose purified WSN virus in coating
buffer, blocked with non-mammalian proteins (SeraBlock) and incubated with
serial difutions of serum samples. The
samples were washed, and the assay was developed with biotin conjugated rat
anti-mouse mAb followed by
strptavidin-alkaline phosphatase and pNPP substrate. The results were
expressed as geometrical means of reciprocal
endpoint titers. The number of mice per inoculation group was three.
The results depicted in Fig, 12 show the induction of high titers of IgG
antibodies in mice immunized with
WSN~PuI or live WSN virus in saline (WSNhoI at 7 and 14 days. In contrast,
only small titers of specific IgG were
detected in mice immunized with killed virus in saline.
XVII
T cell Response to Influenza Virus AIWSNI32 IH1N11
PuImoSoheres Delivered via Nasal Route.
The T cell response was defined in terms of virus and epitope-specific
cytokine production of lymphocytes
from mice immunized as described above (Example XVII. The induction of T-cell
response after intranasal inoculation of
BALBIc mice with a Influenza Virus AIWSNI32 (H1N1) PuImoSpheres (WSN-Pul)
formulation containing 5 g of virus and
2x10' TCIDSO of live virus (1 % of the total antigen load corresponding to the
amount of live virus) was measured.
Control mice were immunized mice with 2x10' TCIDso live virus (corresponding
to 0.05 g of total virus) or UV-killed
WSN virus (5 g). The antigens examined were sucrose-purified WSN virus, HA 110-
120 peptide and NP 147155
peptide. An untreated saline group was included as control.
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Peripheral blood mononuclear cells (PBMCI were isolated from blood at day 10
after immunization, by Ficoll
gradient centrifugation. Various numbers of responder cells were incubated in
nitrocelluloselanti-IFN or anti-IL-4
(PharMingen) ELISPOT plates (Millipore) at 3x105 cellstwell in complete RPMI-
10% FCS. Stimulator cells (mytomicin
treated splenocytes, 5x105fwelll, antigens and human rIL-2 (20UIm11 were added
and the plates were co-incubated for
48 hours. The cells were then washed with PBS-0.05% Tween, anti-cytokine
antibodies (PharMingen) were incubated
overnight and the assay was developed using HRP-streptavidin conjugate
followed by insoluble substrate (Vector
Laboratories). The assay was stopped with water, the wells were air-dried and
the spats were counted using a
stereomicroscope.
The results were expressed as the frequency of specific cells that produce IFN-
or iL-4 l 106 PBMC, after
subtracting the background signal. The background was reproducibly below
61106. PBMC were pooled from the mice
in each group. The results in Figs. 13A, 13B and 13C show that vaccination
with WSN-Pul and WSN virus generally
induced HA-, NP- and WSN-specific T cells producing IFN- and IL-4. In
contrast, immunization with killed virus induced
predominantly IL-4 producing T cells. Moreover, the immunization with killed
virus induced an enhanced subpopulation
of IL-4 producing Tc2 cells, specific for the NP 147-155 peptide. These data
indicate that the T cell response
provoked by the live control and formulated virus (i.e. comprising live and
killed virus) was more effective the response
provoked by the killed virus control corresponding to typical conventional
vaccines.
xvul
Protection Anainst Infectious Chalfenee of Mice Immunized with
Influenza Virus AIWSNI32 (H1N1) PuImoSpheres Delivered via Nasal Route.
Mice immunized as described in Example XVII were challenged at three weeks
after immunization with
1.2x106 influenza virus delivered via the nasal route. The protection in terms
of virus shedding and variation of body
weight were defined at day 4 after the challenge. The results are shown in
Figs.14A and 14B.
Measurement of virus titers in the nasal wash was determined by titrating the
live virus in the MDCK assays.
Results showed the absence of infectious virus in mice previously immunized
with Influenza Virus AIWSNt32 (H1N1)
PuImoSpheres (WSN-Pull or control live WSN virus (Fig. 14A1. Mice immunized
with UV killed WSN virus or naive mice
displayed significant titers of influenza virus in the nasal wash. In
addition, the mice immunized with WSN-Pul ar WSN
virus how dose of live virus) retained their body weight following the
challenge (Fig. 1481. Whereas the non-immunized
mice and those immunized with UV killed WSN virus displayed significant
reduction of body weight followed by death
(213 in each group by day 71. These results demonstrated that the WSN-Pul can
provide effective vaccination
efficiency upon mucosal delivery.
xlx
Preparation of Hollow Porous Particles of TA7 Retrovirus by Spray-Dryin4.
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Hollow porous TA7 Retrovirus particles were prepared by a spray drying
technique with a B-191 Mini Spray-
Drier (Buchi, Flawil, Switzerland) under the following conditions: aspiration:
10090, inlet temperature: 85°C; outlet
temperature: 61°C; feed pump: 10%; NZ flaw: 800 Lfhr. The feed was
prepared by mixing two solutions A and B
immediately prior to spray drying.
Preparation A: 2g of deionized water was used to dissolve 1mg of TA7
Retrovirus.
Preparation B: A fluorocarbon-in-water emulsion stabilized by phospholipid was
prepared in the following
way. The phospholipid, 0.3g EPC-100-3 (Lipoid KG, Ludwigshafen, Germanyl, was
homogenized in 16.58 of hot
deionized water (T ~ 50 to 60°C) using an Ultra-Turrax mixer (model T-
25) at 8000 rpm for 2 to 5 minutes (T = 60-
70°C?. B.Og of perflubron (Atochem, Paris, France) was added dropwise
during mixing. After the fluorocarbon was
added, the emulsion was mixed for at least 4 minutes. The resulting coarse
emulsion was then passed through a high
pressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes.
One eighth of preparation B by volume was separated and added to preparation
A. The resulting TA7
Retroviruslperflubron emulsion feed solution was fed into the spray dryer
under the conditions described above. The
powder collected in the cyclone, and sieving screen was washed into the
collection jar using Perflubron. The TA7
Retrovirus suspension in perflubron was subsequently frozen at -60°C
and lyophilized. A free flowing white powder
was obtained.
XX
In Vitro Activity of TA7 Retrovirus Spray-Dried Particles
The activity of TA7 Retravirus following incorporation into the spray-dried
particles prepared in Example XIX
was examined. Spray-dried TA7 Retrovirus particles were dissolved in saline
and applied to Hela cells for 1 hour. 24h
hours post inoculation, the cells were then assayed for transgenic expression
using ~i-gal. No difference was observed
between the neat and spray-dried TA7 Retrovirus I particles. These results
demonstrate that the TA7 Retrpvirus, a
relatively large and complex entity, can be effectively incorporated in spray-
dried particles with no apparent loss of
activity.
XXI
Preparation of Hollow Porous Particles
of Bovine Gamma Globulin by Spray-Dryinq
Hollow porous bovine gamma globulin (BGG) particles were prepared by a spray
drying technique with a B-
191 Mini Spray-Drier (Buchi, Flawil, Switzerland) under the following
conditions: aspiration: 100%, inlet temperature:
85°C; outlet temperature: 61°C; feed pump: 109'0; N2 flow: 800
Llhr. The feed was prepared by mixing two solutions
A and B immediately prior to spray drying.
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CA 02335940 2000-12-22
WO 00/00215 PCT/US99/06855
Preparation A: 21g of 0.2% saline solution was used to dissolve O.6g of BGG
(CaIBiochem San Diego, CA),
0.42 g Lactose (Sigma Chemicals, St. Louis, M0) and 25mg of Pluronic F-68, NF
grade (BASF, Parsippany, NY I.
Preparation B: A fluorocarbon-in-water emulsion stabilized by phospholipid was
prepared in the following
way. The phospholipid, 1.02g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), was
homogenized in 30g of hot
deionized water (T a 50 to 60°C) using an Ultra-Turrax mixer (model T-
251 at 8000 rpm for 2 to 5 minutes (T - 60-
70°C1. 35g of F-decalin (Air Products, Allentown, PA) was added
dropwise during mixing. After the fluorocarbon was
added, the emulsion was mixed for at least 4 minutes. The resulting coarse
emulsion was then passed through a high
pressure homogenizer (Avestin, Ottawa, Canada) at 18,000 psi for 5 passes.
Preparations A and B were combined and fed into the spray-dryer under the
conditions described above. A
free flowing white powder was collected at the cyclone separator. The hollow
porous particles had a volume-weighted
mean aerodynamic diameter of 1.27 ~ 1.42 Nm as determined by a time-of-flight
analytical method (Aerosizer,
Amherst Process Instruments, Amherst, MAI.
XXII
Andersen Cascade Imuactor Results for
Bovine Gamma Globulin MDI Formulations
The inhalation properties of a metered dose inhaler (MDp formulated with
hollow porous particles of BGG
was prepared according to Example XXI was assessed using an Andersen Cascade
impactor. 83 mg of the hollow
porous BGG particles was weighed a into 10 ml aluminum can, and dried in a
vacuum oven under the flow of nitrogen
for 3 - 4 hours at 40°C. The can was crimp sealed using a DF31~50act 50
I valve (Valois of America, Greenwich, CT)
and filled with 9.648 HFA-134a (DuPont, Wilmington, DE) propellant by
overpressure through the stem.
Upon actuation of the apparatus, a fine particle fraction of 619'o and fine
particle dose of 68,ug were
observed (Fig. 151. The instant example illustrates that a relatively large
bioactive agent such as BGG can be
formulated and effectively delivered from a MDI.
Those skilled in the art will further appreciate that the present invention
may be embodied in other specific forms
without departing from the spirit or central attributes thereof. In that the
foregoing description of the present invention
discloses only exemplary embodiments thereof, it is to be understood that,
other variations are contemplated as being
within the scope of the present invention. Accordingly, the present invention
is not limited to the particular embodiments
that have been described in detail herein. Rather, reference should be made to
the appended claims as indicative of the
scope and content of the invention.
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