U.S. patent application number 09/752106 was filed with the patent office on 2002-05-02 for particles for inhalation having sustained release properties.
This patent application is currently assigned to Massachusetts Institute of Technology The Penn State Research Foundation. Invention is credited to Chen, Donghao, Edwards, David A., Langer, Robert S., Mintzes, Jeffrey, Vanbever, Rita, Wang, Jue.
Application Number | 20020052310 09/752106 |
Document ID | / |
Family ID | 27369571 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052310 |
Kind Code |
A1 |
Edwards, David A. ; et
al. |
May 2, 2002 |
Particles for inhalation having sustained release properties
Abstract
The invention generally relates to a method for pulmonary
delivery of therapeutic, prophylactic and diagnostic agents to a
patient wherein the agent is released in a sustained fashion, and
to particles suitable for use in the method. In particular, the
invention relates to a method for the pulmonary delivery of a
therapeutic, prophylactic or diagnostic agent comprising
administering to the respiratory tract of a patient in need of
treatment, prophylaxis or diagnosis an effective amount of
particles comprising a therapeutic, prophylactic or diagnostic
agent or any combination thereof in association with a charged
lipid, wherein the charged lipid has an overall net charge which is
opposite to that of the agent upon association with the agent.
Release of the agent from the administered particles occurs in a
sustained fashion.
Inventors: |
Edwards, David A.; (Boston,
MA) ; Langer, Robert S.; (Newton, MA) ;
Vanbever, Rita; (Brussels, BE) ; Mintzes,
Jeffrey; (Brighton, MA) ; Wang, Jue; (Clifton,
NJ) ; Chen, Donghao; (Quincy, MA) |
Correspondence
Address: |
Carolyn S. Elmore
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
Massachusetts Institute of
Technology The Penn State Research Foundation
|
Family ID: |
27369571 |
Appl. No.: |
09/752106 |
Filed: |
December 29, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09752106 |
Dec 29, 2000 |
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09394233 |
Sep 13, 1999 |
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09394233 |
Sep 13, 1999 |
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08971791 |
Nov 17, 1997 |
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5985309 |
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60059004 |
Sep 15, 1997 |
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Current U.S.
Class: |
424/45 ; 424/43;
514/1.1; 514/5.9 |
Current CPC
Class: |
A61K 38/28 20130101;
A61K 9/1617 20130101; A61K 31/137 20130101; A61K 47/6927 20170801;
A61K 47/12 20130101; A61K 9/1694 20130101; A61K 9/0073 20130101;
A61K 47/24 20130101; A61K 9/0075 20130101; A61K 9/1623 20130101;
A61K 31/566 20130101; A61K 9/1658 20130101; A61K 31/135 20130101;
A61K 38/38 20130101; A61K 9/16 20130101; A61K 9/1647 20130101; A61K
9/1641 20130101 |
Class at
Publication: |
514/2 ;
424/43 |
International
Class: |
A61K 038/17; A61K
009/00 |
Claims
What is claimed is:
1. A method for delivery via the pulmonary system comprising:
administering to the respiratory tract of a patient in need of
treatment, prophylaxis or diagnosis an effective amount of
particles comprising: a bioactive agent in association with a
charged lipid wherein the charged lipid has an overall net charge
which is opposite to the overall net charge of the agent upon
association and wherein release of the agent is sustained.
2. The method of claim 1, wherein association of the agent and
charged lipid comprises an ionic complexation.
3. The method of claim 2, wherein association of the lipid and
agent further comprises hydrogen bonding.
4. The method of claim 1, wherein the charge ratio of lipid to
bioactive agent is from about 0.25:1 to about 1:0.25.
5. The method of claim 4, wherein the charge ratio of lipid to
bioactive agent is from about 0.5:1 to about 1:0.5.
6. The method of claim 5 wherein the charge ratio of lipid to
bioactive agent is about 1:1.
7. The method of claim 1 wherein the bioactive agent is a
protein.
8. The method of claim 7 wherein the protein is insulin.
9. The method of claim 8, wherein the sustained release is at least
about 6 hours post administration.
10. The method of claim 1 wherein the bioactive agent is estrone
sulfate.
11. The method of claim 1, wherein the bioactive agent is albuterol
sulfate.
12. The method of claim 1, wherein the lipid possesses and overall
net negative charge.
13. The method of claim 12, wherein the lipid is a
1,2-diacyl-sn-glycero-3- -[phospho-rac-( 1-glycerol)] and a
1,2-diacyl-sn-glycerol-3-phosphate.
14. The method of claim 13, wherein the
1,2-diacyl-sn-glycero-3-[phospho-r- ac-(1-glycerol)] lipid is
represented by Formula I: 7wherein, R.sub.1 and R.sub.2 are
independently an aliphatic group having from about 3 to about 24
carbons;
15. The method of claim 13 wherein the
1,2-diacyl-sn-glycero-3-[phospho-ra- c-(1-glycerol)] lipid is
1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glyce- rol)] (DSPG),
1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG),
1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)] (DPPG),
1,2-dilauroyl-sn -glycero-3-[phospho-rac-(1-glycerol)] (DLPG),
1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or any
combination thereof.
16. The method of claim 13, wherein the
1,2-diacyl-sn-glycerol-3-phosphate is represented by the Formula II
8wherein, R.sub.1 and R.sub.2 are independently an aliphatic group
having from about 3 to about 24 carbons;
17. The method of claim 13 wherein the
1,2-diacyl-sn-glycerol-3-phosphate lipid is
1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),
1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),
1,2-dilauroyl-sn-glycero-3- -phosphate (DLPA), 1,2-dioleoyl
-sn-glycero-3-phosphate (DOPA),
1,2-distearoyl-sn-glycero-3-phosphate (DSPA) or any combination
thereof.
18. The method of claim 1 wherein the particles have a tap density
less than about 0.4 g/cm.sup.3.
19. The method of claim 18, wherein the particles have a tap
density less than about 0.1 g/cm.sup.3.
20. The method of claim 1, wherein the particles have a median
geometric diameter of from about 5 micrometers and about 30
micrometers.
21. The method of claim 1, wherein the particles have an
aerodynamic diameter of from about 1 to about 5 microns.
22. The method of claim 2 1, wherein the particles have an
aerodynamic diameter of from about 1 to about 3 microns.
23. The method of claim 22, wherein the particles have an
aerodynamic diameter of from about 3 to about 5 microns.
24. The method of claim 1, wherein d elivery to the pulmonary
system includes delivery to the deep lung.
25. The method of claim 1, wherein delivery to the pulmonary system
includes delivery to the central airways.
26. The method of claim 1, wherein delivery to the pulmonary system
includes delivery to the upper airways.
27. The method of claim 1, wherein the particles further comprise a
lipid having no overall net charge.
28. The method of claim 1 wherein the particles further comprise a
carboxylic acid or salt thereof.
29. The method of claim 28, wherein the carboxylic acid includes at
least two carboxyl groups.
30. The method of claim 1, wherein the particles further comprise a
multivalent metal salt or ionic components thereof.
31. The method of claim 30, wherein the multivalent salt is a salt
of an alkaline earth metal.
32. The method of claim 1, wherein the particles further comprise
an amino acid.
33. The method of claim 32, wherein the amino acid is
hydrophobic.
34. The method of claim 33, wherein the hydrophobic amino acid is
leucine, isoleucine, alanine, valine, phenylalanine or any
combination thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/394,233 filed on Sep. 13, 1999, which is a
continuation-in-part of application Ser. No. 08/971,791 filed on
Nov. 17, 1997, now U.S. Pat. No. 5,985,309.
[0002] This application also relates to application Ser. Nos.
09/337,245, filed on Jun. 22, 1999, 09/383,054, filed on Aug. 25,
1999, 09/382,959, filed on Aug. 25, 1999, 09/644,320, filed on Aug.
23, 2000, 09/665,252 filed on Sep. 19, 2000, 09/644,105, filed on
Aug. 23, 2000, 09/644,736 filed on Aug. 23, 2000 and 09/591,307
filed on Jun. 9, 2000.
[0003] The entire contents of the above-reference applications are
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0004] Pulmonary delivery of bioactive agents, for example,
therapeutic, diagnostic and and prophylactic agents provides an
attractive alternative to, for example, oral, transdermal and
parenteral administration. That is, pulmonary administration can
typically be completed without the need for medical intervention
(self -administration), the pain often associated with injection
therapy is avoided, and the amount of enzymatic and pH mediated
degradation of the bioactive agent, frequently encountered with
oral therapies, can be significantly reduced. In addition, the
lungs provide a large mucosal surface for drug absorption and there
is no first-pass liver effect of absorbed drugs. Further, it has
been shown that high bioavailability of many molecules, for
example, macromolecules, can be achieved via pulmonary delivery or
inhalation. Typically, the deep lung, or alveoli, is the primary
target of inhaled bioactive agents, particularly for agents
requiring systemic delivery.
[0005] The release kinetics or release profile of a bioactive agent
into the local and/or systemic circulation is a key consideration
in most therapies, including those employing pulmonary delivery.
That is, many illnesses or conditions require administration of a
constant or sustained levels of a bioactive agent to provide an
effective therapy. Typically, this can be accomplished through a
multiple dosing regimen or by employing a system that releases the
medicament in a sustained fashion.
[0006] However, delivery of bioactive agents to the pulmonary
system typically results in rapid release of the agent following
administration. For example, U.S. Pat. No. 5,997,848 to Patton et
al. describes the rapid absorption of insulin following
administration of a dry powder formulation via pulmonary delivery.
The peak insulin level was reached in about 30 minutes for primates
and in about 20 minutes for human subjects. Further, Heinemann,
Traut and Heise teach in Diabetic Medicine 14:63-72 (1997) that the
onset of action, assessed by glucose infusion rate, in healthy
volunteers after inhalation was rapid with the half-maximal action
reached in about 30 minutes.
[0007] As such, a need exists for formulations suitable for
inhalation comprising bioactive agents and wherein the bioactive
agent of the formulation is released in a sustained fashion into
the systemic and/or local circulation.
SUMMARY OF THE INVENTION
[0008] This invention is based upon the unexpected discovery that
combining a charged agent with a lipid carrying an opposite charge
results in a sustained release profile of the agent.
[0009] The invention generally relates to a method for pulmonary
delivery of therapeutic, prophylactic and diagnostic agents to a
patient wherein the agent is released in a sustained fashion, and
to particles suitable for use in the method. In particular, the
invention relates to a method for the pulmonary delivery of a
therapeutic, prophylactic or diagnostic agent comprising
administering to the respiratory tract of a patient in need of
treatment, prophylaxis or diagnosis an effective amount of
particles comprising a therapeutic, prophylactic or diagnostic
agent or any combination thereof in association with a charged
lipid, wherein the charged lipid has an overall net charge which is
opposite to that of the agent upon association with the agent.
Release of the agent from the administered particles occurs in a
sustained fashion.
[0010] In one embodiment, the association of the therapeutic,
prophylactic or diagnostic agent and the oppositely charged lipid
can result from ionic complexation. In another embodiment,
association of the therapeutic, prophylactic or diagnostic agent
and the oppositely charged lipid can result from hydrogen
bonding.
[0011] In yet a further embodiment, the association of the
therapeutic, prophylactic or diagnostic agent and the oppositely
charged lipid can result from a combination of ionic complexation
and hydrogen bonding.
[0012] The particles suitable for use in the method can comprise a
therapeutic, prophylactic or diagnostic agent in association with a
charged lipid having a charge opposite to that of the agent. The
charges are opposite upon association, prior to administration. In
a preferred embodiment, the charges of the agent and lipid upon
association, prior to administration, are those which the agent and
lipid possess at pulmonary pH.
[0013] For example, the particles suitable for pulmonary delivery
can comprise a therapeutic, prophylactic or diagnostic agent which
possesses an overall net negative charge, in association with a
lipid which possesses an overall net positive charge. For example,
the agent can be insulin which has an overall net charge which is
negative and the lipid can be
1,2-dipalmitoyl-sn-glycero-3-ethylphosphatidylcholine (DPePC).
[0014] Alternatively, the particles suitable for pulmonary delivery
can comprise a therapeutic, prophylactic or diagnostic agent which
possesses an overall net positive charge in association with a
lipid which possesses an overall net negative charge. For example,
the agent can be albuterol which possesses an overall positive
charge and the lipid can be
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](DPPG) which
possesses an overall net negative charge.
[0015] Further, the particles suitable for pulmonary delivery can
comprise a therapeutic, prophylactic or diagnostic agent which has
an overall net charge which can be modified by adjusting the pH of
a solution of the agent, prior to association with the lipid. For
example, at a pH of about 7.4 insulin has an overall net charge
which is negative. Therefore, insulin and a positively charged
lipid can be associated at this pH prior to administration to
prepare a particle having an agent in association with a charged
lipid wherein the charged lipid has a charge opposite to that of
the agent. However, the charges on insulin can also be modified,
when in solution, to possess an overall net charge which is
positive by modifying the pH of the solution to be less than the pI
of insulin (pI=5.5). As such, when insulin is in solution at a pH
of about 4, for example, it will possess an overall net charge
which is positive. As this is the case, the positively charged
insulin can be associated with a negatively charged lipid, for
example, 1,2-distearoyl-sn-glycero-3-[phosp-
ho-rac-(1-glycerol)](DSPG).
[0016] Modification of the charge of the therapeutic, prophylactic
or diagnostic agent prior to association with the charged lipid,
can be accomplished with many agents, particularly, proteins. For
example, charges on proteins can be modulated by spray drying feed
solutions below or above the isoelectric points (pI) of the
protein. Charge modulation can also be accomplished for small
molecules by spray drying feed solutions below or above the pKa of
the molecule.
[0017] In a particular embodiment, the particles of the invention
comprise more than one lipid, more than one bioactive agent or
both. Also charged lipids can be combined with lipids without a net
charge.
[0018] The particles, can further comprise a carboxylic acid which
is distinct from the bioactive agent and lipid. In one embodiment,
the carboxylic acid includes at least two carboxyl groups.
Carboxylic acids, include the salts thereof as well as combinations
of two or more carboxylic acids and/or salts thereof. In a
preferred embodiment, the carboxylic acid is a hydrophilic
carboxylic acid or salt thereof. Citric acid and citrates, such as,
for example sodium citrate, are preferred. Combinations or mixtures
of carboxylic acids and/or their salts also can be employed.
[0019] The particles suitable for use in the invention can further
comprise a multivalent salt or its ionic components. In a preferred
embodiment, the salt is a divalent salt. In another preferred
embodiment, the salt is a salt of an alkaline-earth metal, such as,
for example, calcium chloride. The particles of the invention can
also include mixtures or combinations of salts and/or their ionic
components.
[0020] The particles suitable for use in the invention can further
comprise an amino acid. In a preferred embodiment the amino acid is
hydrophobic.
[0021] The particles, also referred to herein as powder, can be in
the form of a dry powder suitable for inhalation. The particles can
have a tap density of less than about 0.4 g/cm.sup.3, preferably
less than about 0.1 g/cm.sup.3. Further, the particles suitable for
use in the invention can have a median geometric diameter of from
about 5 micrometers to about 30 micrometers. In yet another
embodiment, the particles suitable for use in the invention have an
aerodynamic diameter of from about 1 to about 5 micrometers.
[0022] The invention has numerous advantages. For example,
particles suitable for inhalation can be designed to possess a
sustained release profile. This sustained released profile provides
for prolonged residence of the administered bioactive agent in the
lung and increases the amount of time in which therapeutic levels
of the agent are present in the local environment or systemic
circulation. The sustained release of agent provides a desirable
alternative to injection therapy currently used for many
therapeutic, diagnostic and prophylactic agent requiring sustained
release of agent, such as insulin for the treatment of diabetes. In
addition, the invention provides a method of delivery to the
pulmonary system wherein the high initial release of agent
typically seen in inhalation therapy is reduced. Consequently,
patient compliance and comfort can be increased by not only
reducing frequency of dosing, but by providing a therapy which is
more amenable to patients.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a graph showing the in vivo release profile of dry
powder formulations comprising insulin and either a lipid (DPPC)
with no overall net a charge or lipid having an overall net charge
opposite to that of insulin (DSePC and DPePC).
[0024] FIG. 2 is a graph of the in vivo release profile of dry
powder formulations comprising insulin in combination with a lipid
with no overall net charge (DPPC) or insulin in combination with a
charged lipid having an overall negative charge (DPPG) and spray
dried with the active agent at either pH 4 or 7.4.
[0025] FIG. 3 is a graph showing the in vivo release profile of dry
powder formulation comprising estrone sulfate (-) and either a
lipid with no overall net charge (DPPC) or a lipid having an
overall charge opposite (DPePC, +) to that of the estrone
sulfate.
[0026] FIG. 4 is a graph of percent of PenH above baseline versus
time following administration of dry powder formulations of
albuterol sulfate and lipid in animals which have been challenged
repeatedly over time with methacholine given by nebulization.
[0027] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings. The drawings are not
necessarily to scale, emphasis instead being placed upon
illustrating the principles of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0028] A description of preferred embodiments of the invention
follows.
[0029] Therapeutic, prophylactic or diagnostic agents, can also be
referred to herein as "bioactive agents", "medicaments" or
"drugs".
[0030] The invention relates to a method for the pulmonary delivery
of therapeutic, prophylactic and diagnostic agents comprising
administering to the respiratory tract of a patient in need of
treatment, prophylaxis or diagnosis an effective amount of
particles comprising a therapeutic, prophylactic or diagnostic
agent or any combination thereof in association with a charged
lipid, wherein the charged lipid has an overall net charge which is
opposite to that of the agent. The agent is released from the
administered particles in a sustained fashion.
[0031] The particles of the invention release bioactive agent in a
sustained fashion. As such, the particles possess sustained release
properties. "Sustained release", as that term is used herein,
refers to a release of active agent in which the period of release
of an effective level of agent is longer than that seen with the
same bioactive agent which is not associated with an oppositely
charged lipid, prior to administration. In addition, a sustained
release also refers to a reduction in the burst of agent typically
seen in first two hours following administration, and more
preferably in the first hour, often referred to as the initial
burst. In a preferred embodiment, the sustained release is
characterized by both the period of release being longer in
addition to a decreased burst. For example, a sustained release of
insulin can be a release showing elevated levels out to at least 4
hours post administration, such as about 6 hours or more.
[0032] "Pulmonary delivery", as that term is used herein refers to
delivery to the respiratory tract. The "respiratory tract", as
defined herein, encompasses the upper airways, including the
oropharynx and larynx, followed by the lower airways, which include
the trachea followed by bifurcations into the bronchi and
bronchioli (e.g., terminal and respiratory). The upper and lower
airways are called the conducting airways. The terminal bronchioli
then divide into respiratory bronchioli which then lead to the
ultimate respiratory zone, namely, the alveoli, or deep lung. The
deep lung, or alveoli, are typically the desired the target of
inhaled therapeutic formulations for systemic drug delivery.
[0033] In one embodiment, the therapeutic, prophylactic or
diagnostic agent and the oppositely charged lipid can be in
association primarily as a result of ionic bonding, for example,
ionic complexation. In another embodiment, the therapeutic,
prophylactic or diagnostic agent and the oppositely charged lipid
can be in association primarily as a result of hydrogen bonding. It
is understood that a combination of ionic and hydrogen bonding can
contribute to the association of the bioactive and charged
lipid.
[0034] Ionic bonding is bonding which occurs via charge/charge
interactions between atoms or groups of atoms. Since opposite
charges attract, the atoms in an ionic compound are held together
by this attraction.
[0035] Hydrogen bonding refers to bonding wherein a hydrogen atom
is shared between two molecules. For example, a hydrogen atom
covalently attached to an electronegative atom such as nitrogen,
oxygen, sulfur or phosphorous shares its partial positive charge
with a second electronegative atom, for example, nitrogen, oxygen,
sulfur or phosphorous.
[0036] The particles suitable for use in the method can comprise a
therapeutic, prophylactic or diagnostic agent in association with a
charged lipid having a charge opposite to that of the agent upon
association, prior to administration. In a preferred embodiment,
the charges possessed by the agent and lipid, upon association, are
the same as the charges which the agent and lipid possess at
pulmonary pH following administration.
[0037] For example, the particles suitable for pulmonary delivery
can comprise a therapeutic, prophylactic or diagnostic agent which
possesses an overall net negative charge in association with a
lipid which possesses an overall net positive charge. For example,
the agent can be insulin and the lipid can be an
alkylphosphatidylcholine, such as
1,2-dipalmitoyl-sn-glycero-3-ethylphosphatidylcholine (DPePC).
[0038] Alternatively, the particles suitable for pulmonary delivery
can comprise a therapeutic, prophylactic or diagnostic agent which
possesses an overall net positive charge in association with a
lipid which possesses an overall net negative charge, preferably in
the pulmonary pH range. For example, the agent can be albuterol
sulfate which possesses an overall positive charge and the lipid
can be 1,2-dipalmitoyl-sn -glycero-3-[phospho-rac-(1-glycerol)]
(DPPG) which possesses an overall net negative charge.
[0039] Further, the particles suitable for pulmonary delivery can
comprise a therapeutic, prophylactic or diagnostic agent which has
an overall net charge which can be modified by adjusting the pH of
a solution of the agent prior to association with the charged
lipid. For example, at a pH of about 7.4 insulin has an overall net
charge which is negative. Therefore, insulin and a positively
charged lipid can be associated at this pH, prior to
administration, to prepare a particle having an bioactive agent in
association with a charged lipid wherein the charged lipid has a
charge opposite to that of the agent upon association. However,
insulin can also be modified when in solution to possess an overall
net charge which is positive by modifying the pH of the solution to
be less than the pI of insulin (pI=5.5). As such, when insulin is
in solution at a pH of 4, for example, it will possess an overall
net charge which is positive. As this is the case, the positively
charged insulin can be associated with a negatively charged lipid,
for example, 1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
(DSPG). Modification of the charge of the therapeutic, prophylactic
or diagnostic agent is applicable to many agents, particularly,
proteins.
[0040] "Pulmonary pH range", as that term is used herein, refers to
the pH range which can be encountered in the lung of a patient.
Typically, in humans, this range of pH is from about 6.4 to about
7.0, such as from 6.4 to about 6.7. pH values of the airway lining
fluid (ALF) have been reported in "Comparative Biology of the
Normal Lung", CRC Press, (1991) by R. A. Parent and range from 6.44
to 6.74)
[0041] "Charged lipid" as that term is used herein, refers to
lipids which are capable of possessing an overall net charge. The
charge on the lipid can be negative or positive. The lipid can be
chosen to have a charge opposite to that of the active agent when
the lipid and active agent are associated. In a preferred
embodiment the charged lipid is a charged phospholipid. Preferably,
the phospholipid is endogenous to the lung or can be metabolized
upon administration to a lung endogenous phospholipid. Combinations
of charged lipids can be used. The combination of charged lipid
also has an overall net charge opposite to that of the bioactive
agent upon association.
[0042] The charged phospholipid can be a negatively charged lipid
such as, a 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and a
1,2-diacyl-sn-glycerol-3-phosphate.
[0043] The 1,2-diacyl-sn-glycero-3-[phospho-rac-(l -glycerol)]
phospholipids can be represented by the Formula I: 1
[0044] wherein R.sub.1 and R.sub.2 are independently aliphatic
groups having from about 3 to about 24 carbon atoms, preferably
from about 10 to about 20 carbon atoms.
[0045] Aliphatic group as that term is used herein in Formulas I-VI
refers to substituted or unsubstituted straight chained, branched
or cyclic C.sub.1-C.sub.24 hydrocarbons which can be completely
saturated, which can contain one or more heteroatoms such as
nitrogen, oxygen or sulfur and/or which can contain one or more
units of unsaturation.
[0046] Suitable substituents on an aliphatic group include --OH,
halogen (--Br, --Cl, --I and --F) --O(aliphatic, substituted),
--CN, --NO.sub.2, --COOH, --NH.sub.2, --NH(aliphatic group,
substituted aliphatic), --N(aliphatic group, substituted aliphatic
group).sub.2, --COO(aliphatic group, substituted aliphatic group),
--CONH.sub.2, --CONH(aliphatic, substituted aliphatic group), --SH,
--S(aliphatic, substituted aliphatic group) and
--NH--C(.dbd.NH)--NH.sub.2. A substituted aliphatic group can also
have a benzyl, substituted benzyl, aryl (e.g., phenyl, naphthyl or
pyridyl) or substituted aryl group as a substituent. A substituted
aliphatic can have one or more substituents.
[0047] Specific examples of this type of negatively charged
phospholipid include, but are not limited to,
1,2-distearoyl-sn-glycero-3-[phospho-rac- -(1-glycerol)] (DSPG),
1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycer- ol)] (DMPG),
1,2-dipalmitoyl-sn -glycero-3-phospho-rac-(1-glycerol)] (DPPG),
1,2-dilauroyl-sn-glycero-3-[phospho-rac -(1-glycerol)] (DLPG), and
1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG).
[0048] The 1,2-diacyl-sn-glycerol-3-phosphate phospholipids can be
represented by the 2
[0049] R.sub.1 and R.sub.2 are independently an aliphatic group
having from about 3 to about 24 carbon atoms, preferably from about
10 to about 20 carbon atoms.
[0050] Specific examples of this type of phospholipid include, but
are not limited to, 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),
1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),
1,2-dioleoyl-sn-glycero-3-- phosphate (DOPA), 1,2-distearoyl-sn
-glycero-3-phosphate (DSPA), and
1,2-dilauroyl-sn-glycero-3-phosphate (DLPA).
[0051] The charged lipid can be a positively charged lipid such as
a 1,2-diacyl-sn -glycero-3-alkylphosphocholine and a
1,2-diacyl-sn-glycero-3-alkylphosphoalkanolamine.
[0052] The 1,2-diacyl-sn-glycero-3-alkyllphosphocholine
phospholipids can be represented by the Formula III: 3
[0053] wherein R.sub.1 and R.sub.2 are independently an aliphatic
group having from about 3 to about 24 carbon atoms, preferably from
about 10 to about 20 carbon atoms. R.sub.3 is an aliphatic group
having from about I to about 24 carbons, for example, methyl,
ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and the like.
R.sub.4 is independently hydrogen, or an aliphatic group having
from about 1 to about 6 carbon atoms.
[0054] Specific examples of this type of positively charged
phospholipid include, but are not limited to, 1
,2-dipalmitoyl-sn-glycero-3-ethylphosp- hocholine(DPePC),
1,2-dimyristoyl-sn-glycero-3-ethylpho sphocholine(DMeP C), 1
,2-distearoyl-sn-glycero-3-ethylphosphocholine(DSePC), 1
,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLeP C), and 1
,2-dioleoyl-sn-glycero-3-ethylphosphocholine(DOePC).
[0055] The 1,2-diacyl-sn-glycero-3-alkylphosphoalkanolamine
phospholipids can be represented by the Formula IV: 4
[0056] wherein R.sub.1 and R.sub.2 are independently an aliphatic
group having from about 3 to about 24 carbon atoms, preferably from
about 10 to about 20 carbon atoms. R.sub.3 is an aliphatic group
having from about 1 to about 24 carbons, for example, methyl,
ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and the like.
R.sub.4 is independently hydrogen, or an aliphatic group having
from about 1 to about 6 carbon atoms.
[0057] Specific examples of this type of positively charged
phospholipid include, but are not limited to,
1,2-dipalmitoyl-sn-glycero-3-ethylethano- lamnine([DPePE),
1,2-dimyristoyl-sn-glycero-3-ethylphosphoethanolamine(DMe- PE),
1,2-distearoyl-sn -glycero-3-ethylphosphoethanolamineDS ePE),
1,2-dilauroyl-sn-glycero-3-ethylphosphoethanolamine (DLePE), and
1,2-dioleoyl-sn-glycero-3-ethylphosphoethanolamine (DOePE).
[0058] Other charged lipids suitable for use in the invention
include those described in U.S. Pat. No. 5,466,841 to Horrobin et
al. issued on Nov. 14, 1995, U.S. Pat. Nos. 5,698,721 and 5,902,802
to Heath issued Dec. 16, 1997 and May 11, 1999, respectively, and
U.S. Pat. No. 4,480,041 to Myles et al. issued Oct. 30, 1984, the
entire contents of all of which are incorporated herein by
reference.
[0059] The charged lipid and the therapeutic, prophylactic or
diagnostic agent can be present in the particles of the invention
at a charge ratio of lipid to active of from about 0.25:1 or more,
preferably from about 0.25:1 to about 1:0.25, for example, about
0.5:1 to about 1:0.5. Preferably the charge ratio is about 1:1.
When an excess of charge is present, it is preferred that the
excess charge is contributed by the lipid.
[0060] A suitable charge ratio can be determined as follows. First,
the number of charges present on both the bioactive agent and
lipid, at the conditions under which association of the two will
occur, prior to administration, should be determined. Next, the
equivalent weight of both the bioactive agent and lipid should be
determined. This can be carried out following the example below
employing insulin as the bioactive agent and DPePC as the charged
lipid at a pH of about 7.4.
1 Molecular Weight of Insulin: 5,800 g/mole Number of Negative
Charges on Insulin: 6 equivalent Equivalent Weight Per Charge:
5,800 .times. 1/6 = 967 g Molecular Weight of DPePC: 763 g/mole
Number of Negative Charges on DPePC: 1 equivalent Equivalent Weight
Per Charge: 763 .times. 1/1 = 763 g
[0061] Therefore, to obtain for example, a 1:1 charge ratio of
DPePC to insulin
[0062] 763 g DPePC is associated with 967 g insulin OR
[0063] 1 g DPePC is associated with 1.27 (967/763=1.27) g
insulin.
[0064] Alternatively,
[0065] 967 g insulin is associated with 763 g DPePC OR
[0066] 1 g insulin is associated with 0.79 (763/967=0.79) g
DPePC.
[0067] In molar terms,
[0068] 1 mole DPePC is associated with 1/6 mole insulin OR
[0069] 1 mole insulin is associated with 6 moles DPePC .
[0070] This analysis can be used to determine the amount of lipid
and active agent needed for any ratio desired and any combination
of bioactive agent and lipid.
[0071] The charged lipid can be present in the particles in an
amount ranging from about 1 to about 99% by weight. Preferably, the
charged lipid is present in the particles in an amount ranging from
about 10% to about 90% by weight.
[0072] The particles of the invention can also comprise
phospholipids, which are zwitterionic and therefore do not possess
an overall net charge. Such lipids, can assist in providing
particles with the proper characterisitics for inhalation. Such
phospholipids suitable for use in the invention include, but are
not limited to, a 1,2-diacyl-sn-glycero -3-phosphocholine and a
1,2-diacyl-sn-glycero-3-phosphoalkanolamine. These lipids can
preferably be present in the particles in an amount ranging from
about 10% to about 90% by weight. Preferably, these lipids can be
present in the particles in an amount ranging from abut 50% to
about 80% by weight.
[0073] The 1,2-diacyl-sn-glycero-3-phosphocholine phospholipids can
be represented by Formula V: 5
[0074] R.sub.1 and R.sub.2 are independently an aliphatic group
having from about 3 to about 24 carbon atoms, preferably from about
10 to about 20 carbon atoms. R.sub.4 is independently hydrogen, or
an aliphatic group having from about 1 to about 6 carbon atoms.
[0075] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine
phospholipids include, but are not limited to,
1,2-dipahnitoyl-sn-glycero- -3-phosphocholine (DPPC),
1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC),
1,2-dilaureoyl-sn-3-glycero -phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
[0076] The 1,2-diacyl-sn-glycero-3-phosphoalkanolamine
phospholipids can be represented by Formula VI: 6
[0077] wherein R.sub.1 and R.sub.2 are independently an aliphatic
group having from about 3 to about 24 carbon atoms, preferably,
from about 10 to about 20 carbon atoms and R.sub.4 is independently
hydrogen or an aliphatic group having from about 1 to about 6
carbon atoms.
[0078] Specific examples of this type of phospholipid include, but
are not limited to,
1,2-dipalmitoyl-sn-glycero-3-ethanolamine(DPPE),
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine(DMPE),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE),
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
[0079] Therapeutic, prophylactic or diagnostic agents, can also be
referred to herein as "bioactive agents", "medicaments" or "drugs".
It is understood that one or more bioactive agents can be present
in the particles of the invention. Hydrophilic as well as
hydrophobic agents can be used. The agent must be capable of
possessing an overall net charge. The amount of bioactive agent
present in the particles of the invention can be from about 0.1
weight % to about 95 weight %, for example, from about 5 to about
75%, such as from about 10 to about 50%. Particles in which the
drug is distributed throughout a particle are preferred.
[0080] Suitable bioactive agents include agents which can act
locally, systemically or a combination thereof. The term "bioactive
agent," as used herein, is an agent, or its pharmaceutically
acceptable salt, which when released in vivo, possesses the desired
biological activity, for example therapeutic, diagnostic and/or
prophylactic properties in vivo.
[0081] Examples of bioactive agent include, but are not limited to,
synthetic inorganic and organic compounds, proteins and peptides,
polysaccharides and other sugars, lipids, and DNA and RNA nucleic
acid sequences having therapeutic, prophylactic or diagnostic
activities. Agents with a wide range of molecular weight can be
used, for example, between 100 and 500,000 grams or more per
mole.
[0082] The agents can have a variety of biological activities, such
as vasoactive agents, neuroactive agents, hormones, anticoagulants,
immunomodulating agents, cytotoxic agents, prophylactic agents,
antibiotics, antivirals, antisense, antigens, antineoplastic agents
and antibodies.
[0083] Proteins, include complete proteins, muteins and active
fragments thereof, such as insulin, immunoglobulins, antibodies,
cytokines (e.g., lymphokines, monokines, chemokines), interleukins,
interferons (.beta.-IFN, .alpha.-IFN and .gamma.-IFN),
erythropoietin, nucleases, tumor necrosis factor, colony
stimulating factors, enzymes (e.g. superoxide dismutase, tissue
plasminogen activator), tumor suppressors, blood proteins, hormones
and hormone analogs (e.g., growth hormone, adrenocorticotropic
hormone and luteinizing hormone releasing hormone (LHRH)), vaccines
(e.g., tumoral, bacterial and viral antigens), antigens, blood
coagulation factors; growth factors; granulocyte colony-stimulating
factor ("G-CSF"); peptides include protein inhibitors, protein
antagonists, and protein agonists, calcitonin; nucleic acids
include, for example, antisense molecules, oligonucleotides, and
ribozymes. Polysaccharides, such as heparin, can also be
administered.
[0084] Bioactive agent for local delivery within the lung, include
such as agents as those for the treatment of asthma, chronic
obstructive pulmonary disease (COPD), emphysema, or cystic
fibrosis. For example, genes for the treatment of diseases such as
cystic fibrosis can be administered, as can beta agonists steroids,
anticholinergics, and leukotriene modifers for asthma.
[0085] Other specific bioactive agents include, estrone sulfate,
albuterol sulfate, parathyroid hormone-related peptide,
somatostatin, nicotine, clonidine, salicylate, cromolyn sodium,
salmeterol, formeterol, L-dopa, Carbidopa or a combination thereof,
gabapenatin, clorazepate, carbamazepine and diazepam.
[0086] Nucleic acid sequences include genes, antisense molecules
which can, for instance, bind to complementary DNA to inhibit
transcription, and ribozymes.
[0087] The particles can include any of a variety of diagnostic
agents to locally or systemically deliver the agents following
administration to a patient. For example, imaging agents which
include commercially available agents used in positron emission
tomography (PET), computer assisted tomography (CAT), single photon
emission computerized tomography, x-ray, fluoroscopy, and magnetic
resonance imaging (MRI) can be employed.
[0088] Examples of suitable materials for use as contrast agents in
MR.sub.1 include the gadolinium chelates currently available, such
as diethylene triamine pentacetic acid (DTPA) and gadopentotate
dimeglumine, as well as iron, magnesium, manganese, copper and
chromium.
[0089] Examples of materials useful for CAT and x-rays include
iodine based materials for intravenous administration, such as
ionic monomers typified by diatrizoate and iothalamate and ionic
dimers, for example, ioxagalte.
[0090] Diagnostic agents can be detected using standard techniques
available in the art and commercially available equipment.
[0091] The particles can further comprise a carboxylic acid which
is distinct from the agent and lipid. In one embodiment, the
carboxylic acid includes at least two carboxyl groups. Carboxylic
acids, include the salts thereof as well as combinations of two or
more carboxylic acids and/or salts thereof. In a preferred
embodiment, the carboxylic acid is a hydrophilic carboxylic acid or
salt thereof. Suitable carboxylic acids include but are not limited
to hydroxydicarboxylic acids, hydroxytricarboxilic acids and the
like. Citric acid and citrates, such as, for example sodium
citrate, are preferred. Combinations or mixtures of carboxylic
acids and/or their salts also can be employed.
[0092] The carboxylic acid can be present in the particles in an
amount ranging from about 0 to about 80% weight. Preferably, the
carboxylic acid can be present in the particles in an amount of
about 10 to about 20%.
[0093] The particles suitable for use in the invention can further
comprise a multivalent salt or its ionic components. As used
herein, a "multivalent" salt refers to salts having a ionic
component with a valency greater than one. For example, divalent
salts. In a preferred embodiment, the salt is a divalent salt. In
another preferred embodiment, the salt is a salt of an
alkaline-earth metal, such as, for example, calcium chloride. The
particles of the invention can also include mixtures or
combinations of salts and/or their ionic components.
[0094] The salt or its ionic components are present in the
particles in an amount ranging from about 0 to about 40%
weight.
[0095] The particles suitable for use in the invention can further
comprise an amino acid. In a preferred embodiment the amino acid is
hydrophobic. Suitable naturally occurring hydrophobic amino acids,
include but are not limited to, leucine, isoleucine, alanine,
valine, phenylalanine, glycine and tryptophan. Combinations of
hydrophobic amino acids can also be employed Non-naturally
occurring amino acids include, for example, beta-amino acids. Both
D, L configurations and racemic mixtures of hydrophobic amino acids
can be employed. Suitable hydrophobic amino acids can also include
amino acid derivatives or analogs. As used herein, an amino acid
analog includes the D or L configuration of an amino acid having
the following formula: --NH-- CHR--CO--, wherein R is an aliphatic
group, a substituted aliphatic group, a benzyl group, a substituted
benzyl group, an aromatic group or a substituted aromatic group and
wherein R does not correspond to the side chain of a
naturally-occurring amino acid. As used herein, aliphatic groups
include straight chained, branched or cyclic C1-C8 hydrocarbons
which are completely saturated, which contain one or two
heteroatoms such as nitrogen, oxygen or sulfur and/or which contain
one or more units of unsaturation. Aromatic or aryl groups include
carbocyclic aromatic groups such as phenyl and naphthyl and
heterocyclic aromatic groups such as imidazolyl, indolyl, thienyl,
furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzoftiranyl,
quinolinyl, isoquinolinyl and acridintyl.
[0096] Suitable substituents on an aliphatic, aromatic or benzyl
group include --OH, halogen (--Br, --Cl, --I and --F)
--O(aliphatic, substituted aliphatic, benzyl, substituted benzyl,
aryl or substituted aryl group), --CN, --NO.sub.2, --COOH,
--NH.sub.2, --NH(aliphatic group, substituted aliphatic, benzyl,
substituted benzyl, aryl or substituted aryl group), --N(aliphatic
group, substituted aliphatic, benzyl, substituted benzyl, aryl or
substituted aryl group).sub.2, --COO(aliphatic group, substituted
aliphatic, benzyl, substituted benzyl, aryl or substituted aryl
group), --CONH.sub.2, --CONH(aliphatic, substituted aliphatic
group, benzyl, substituted benzyl, aryl or substituted aryl
group)), --SH, --S(aliphatic, substituted aliphatic, benzyl,
substituted benzyl, aromatic or substituted aromatic group) and
--NH--C(.dbd.NH)--NH.sub.2. A substituted benzylic or aromatic
group can also have an aliphatic or substituted aliphatic group as
a substituent. A substituted aliphatic group can also have a
benzyl, substituted benzyl, aryl or substituted aryl group as a
substituent. A substituted aliphatic, substituted aromatic or
substituted benzyl group can have one or more substituents.
Modifying an amino acid substituent can increase, for example, the
lypophilicity or hydrophobicity of natural amino acids which are
hydrophilic.
[0097] A number of the suitable amino acids, amino acids analogs
and salts thereof can be obtained commercially. Others can be
synthesized by methods known in the art. Synthetic techniques are
described, for example, in Green and Wuts, "Protecting Groups in
Organic Synthesis", John Wiley and Sons, Chapters 5 and 7,
1991.
[0098] Hydrophobicity is generally defined with respect to the
partition of an amino acid between a nonpolar solvent and water.
Hydrophobic amino acids are those acids which show a preference for
the nonpolar solvent. Relative hydrophobicity of amino acids can be
expressed on a hydrophobicity scale on which glycine has the value
0.5. On such a scale, amino acids which have a preference for water
have values below 0.5 and those that have a preference for nonpolar
solvents have a value above 0.5. As used herein, the term
hydrophobic amino acid refers to an amino acid that, on the
hydrophobicity scale has a value greater or equal to 0.5, in other
words, has a tendency to partition in the nonpolar acid which is at
least equal to that of glycine.
[0099] Examples of amino acids which can be employed include, but
are not limited to: glycine, proline, alanine, cysteine,
methionine, valine, leucine, tyrosine, isoleucine, phenylalanine,
tryptophan. Preferred hydrophobic amino acids include leucine,
isoleucine, alanine, valine, phenylalanine, glycine and tryptophan.
Combinations of hydrophobic amino acids can also be employed.
Furthermore, combinations of hydrophobic and hydrophilic
(preferentially partitioning in water) amino acids, where the
overall combination is hydrophobic, can also be employed.
Combinations of one or more amino acids can also be employed.
[0100] The amino acid can be present in the particles of the
invention in an amount from about 0% to about 60 weight %.
Preferably, the amino acid can be present in the particles in an
amount ranging from about 5 to about 30 weight %. The salt of a
hydrophobic amino acid can be present in the particles of the
invention in an amount of from about 0% to about 60 weight %.
Preferably, the amino acid salt is present in the particles in an
amount ranging from about 5 to about 30 weight %. Methods of
forming and delivering particles which include an amino acid are
described in U.S. Pat. application Ser. No 09/382,959, filed on
Aug. 25, 1999, entitled Use of Simple Amino Acids to Form Porous
Particles During Spray Drying the entire teaching of which is
incorporated herein by reference.
[0101] In a further embodiment, the particles can also include
other materials such as, for example, buffer salts, dextran,
polysaccharides, lactose, trehalose, cyclodextrins, proteins,
peptides, polypeptides, fatty acids, fatty acid esters, inorganic
compounds, phosphates.
[0102] In one embodiment of the invention, the particles can
further comprise polymers. The use of polymers can further prolong
release. Biocompatible or biodegradable polymers are preferred.
Such polymers are described, for example, in U.S. Pat. No.
5,874,064, issued on Feb. 23, 1999 to Edwards et al., the teachings
of which are incorporated herein by reference in their
entirety.
[0103] In yet another embodiment, the particles include a
surfactant other than one of the charged lipids described above. As
used herein, the term "surfactant" refers to any agent which
preferentially absorbs to an interface between two immiscible
phases, such as the interface between water and an organic polymer
solution, a water/air interface or organic solvent/air interface.
Surfactants generally possess a hydrophilic moiety and a lipophilic
moiety, such that, upon absorbing to microparticles, they tend to
present moieties to the external environment that do not attract
similarly-coated particles, thus reducing particle agglomeration.
Surfactants may also promote absorption of a therapeutic or
diagnostic agent and increase bioavailability of the agent.
[0104] Suitable surfactants which can be employed in fabricating
the particles of the invention include but are not limited to
hexadecanol; fatty alcohols such as polyethylene glycol (PEG);
polyoxyethylene-9-laury- l ether; a surface active fatty acid, such
as palmitic acid or oleic acid; glycocholate; surfactin; a
poloxomer; a sorbitan fatty acid ester such as sorbitan trioleate
(Span 85); and tyloxapol.
[0105] The surfactant can be present in the particles in an amount
ranging from about 0 to about 60 weight %. Preferably, it can be
present in the particles in an amount ranging from about 5 to about
50 weight %.
[0106] It is understood that when the particles includes a
carboxylic acid, a multivalent salt, an amino acid, a surfactant or
any combination thereof that interaction between these components
of the particle and the charged lipid can occur.
[0107] The particles, also referred to herein as powder, can be in
the form of a dry powder suitable for inhalation. In a particular
embodiment, the particles can have a tap density of less than about
0.4 g/cm.sup.3. Particles which have a tap density of less than
about 0.4 g/cm.sup.3 are referred to herein as "aerodynamically
light particles". More preferred are particles having a tap density
less than about 0.1 g/cm.sup.3.
[0108] Aerodynamically light particles have a preferred size, e.g.,
a volume median geometric diameter (VMGD) of at least about 5
microns (.mu.m). In one embodiment, the VMGD is from about 5 .mu.m
to about 30 .mu.m. In another embodiment of the invention, the
particles have a VMGD ranging from about 9 Elm to about 30 Jim. In
other embodiments, the particles have a median diameter, mass
median diameter (MMD), a mass median envelope diameter (MMED) or a
mass median geometric diameter (MMGD) of at least 5.mu.m, for
example from about 5 .mu.m to about 30 .mu.m.
[0109] Aerodynamically light particles preferably have "mass median
aerodynamic diameter" (MMAD), also referred to herein as
"aerodynamic diameter", between about 1 .mu.m and about 5 .mu.m. In
one embodiment of the invention, the MMAD is between about 1 .mu.m
and about 3 .mu.m. In another embodiment, the MMAD is between about
3 .mu.m and about 5 .mu.m.
[0110] In another embodiment of the invention, the particles have
an envelope mass density, also referred to herein as "mass density"
of less than about 0.4 g/cm.sup.3. The envelope mass density of an
isotropic particle is defined as the mass of the particle divided
by the minimum sphere envelope volume within which it can be
enclosed.
[0111] Tap density can be measured by using instruments known to
those skilled in the art such as the Dual Platform Microprocessor
Controlled Tap Density Tester (Vankel, NC) or a GeoPyc.TM.
instrument (Micrometrics Instrument Corp., Norcross, Ga. 30093).
Tap density is a standard measure of the envelope mass density. Tap
density can be determined using the method of USP Bulk Density and
Tapped Density, United States Pharmacopia convention, Rockville,
Md., 10th Supplement, 4950-4951, 1999. Features which can
contribute to low tap density include irregular surface texture and
porous structure.
[0112] The diameter of the particles, for example, their VMGD, can
be measured using an electrical zone sensing instrument such as a
Multisizer iHe, (Coulter Electronic, Luton, Beds, England), or a
laser diffraction instrument (for example Helos, manufactured by
Sympatec, Princeton, N.J.). Other instruments for measuring
particle diameter are well known in the art. The diameter of
particles in a sample will range depending upon factors such as
particle composition and methods of synthesis. The distribution of
size of particles in a sample can be selected to permit optimal
deposition within targeted sites within the respiratory tract.
[0113] Experimentally, aerodynamic diameter can be determined by
employing a gravitational settling method, whereby the time for an
ensemble of particles to settle a certain distance is used to infer
directly the aerodynamic diameter of the particles. An indirect
method for measuring the mass median aerodynamic diameter (MMAD) is
the multi-stage liquid impinger (MSLI).
[0114] The aerodynamic diameter, d.sub.aer, can be calculated from
the equation:
d.sub.aer=d.sub.g{square root}.rho..sub.tap
[0115] where d.sub.g is the geometric diameter, for example the
MMGD and .rho. is the powder density.
[0116] Particles which have a tap density less than about 0.4
g/cm.sup.3, median diameters of at least about 5 .mu.m, and an
aerodynamic diameter of between about 1 Jim and about 5 .mu.m,
preferably between about 1 .mu.m and about 3 .mu.m, are more
capable of escaping inertial and gravitational deposition in the
oropharyngeal region, and are targeted to the airways or the deep
lung. The use of larger, more porous particles is advantageous
since they are able to aerosolize more efficiently than smaller,
denser aerosol particles such as those currently used for
inhalation therapies.
[0117] In comparison to smaller particles the larger
aerodynamically light particles, preferably having a VMGD of at
least about 5 .mu.m, also can potentially more successfully avoid
phagocytic engulfment by alveolar macrophages and clearance from
the lungs, due to size exclusion of the particles from the
phagocytes' cytosolic space. Phagocytosis of particles by alveolar
macrophages diminishes precipitously as particle diameter increases
beyond about 3 .mu.m. Kawaguchi, H., et al., Biomaterials 7: 61-66
(1986); Krenis, L. J. and Strauss, B., Proc. Soc. Exp. Med., 107:
748-750 (1961); and Rudt, S. and Muller, R. H., J. Contr. Rel., 22:
263-272 (1992). For particles of statistically isotropic shape,
such as spheres with rough surfaces, the particle envelope volume
is approximately equivalent to the volume of cytosolic space
required within a macrophage for complete particle
phagocytosis.
[0118] The particles may be fabricated with the appropriate
material, surface roughness, diameter and tap density for localized
delivery to selected regions of the respiratory tract such as the
deep lung or upper or central airways. For example, higher density
or larger particles may be used for upper airway delivery, or a
mixture of varying sized particles in a sample, provided with the
same or different therapeutic agent may be administered to target
different regions of the lung in one administration. Particles
having an aerodynamic diameter ranging from about 3 to about 5
.mu.m are preferred for delivery to the central and upper airways.
Particles having an aerodynamic diameter ranging from about 1 to
about 3 .mu.m are preferred for delivery to the deep lung.
[0119] Inertial impaction and gravitational settling of aerosols
are predominant deposition mechanisms in the airways and acini of
the lungs during normal breathing conditions. Edwards, D. A., J.
Aerosol Sci., 26: 293-317 (1995). The importance of both deposition
mechanisms increases in proportion to the mass of aerosols and not
to particle (or envelope) volume. Since the site of aerosol
deposition in the lungs is determined by the mass of the aerosol
(at least for particles of mean aerodynamic diameter greater than
approximately 1 .mu.m), diminishing the tap density by increasing
particle surface irregularities and particle porosity permits the
delivery of larger particle envelope volumes into the lungs, all
other physical parameters being equal.
[0120] The low tap density particles have a small aerodynamic
diameter in comparison to the actual envelope sphere diameter. The
aerodynamic diameter, d.sub.aer, is related to the envelope sphere
diameter, d (Gonda, I., "Physico-chemical principles in aerosol
delivery," in Topics in Pharmaceutical Sciences 1991 (eds. D. J. A.
Crommelin and K. K. Midha), pp. 95-117, Stuttgart: Medpharm
Scientific Publishers, 1992)), by the formula:
d.sub.aer=d{square root}p
[0121] where the envelope mass .rho. is in units of g/cm.sup.3.
Maximal deposition of monodispersed aerosol particles in the
alveolar region of the human lung (.about.60%) occurs for an
aerodynamic diameter of approximately d.sub.aer=3 .mu.m. Heyder, J.
et al., J. Aerosol Sci., 17: 811-825 (1986). Due to their small
envelope mass density, the actual diameter d of aerodynamically
light particles comprising a monodisperse inhaled powder that will
exhibit maximum deep-lung deposition is:
d=3/{square root}.mu.m (where .rho.<1 g/cm.sup.3);
[0122] where d is always greater than 3 .mu.m. For example,
aerodynamically light particles that display an envelope mass
density, .rho.=0.1 g/cm.sup.3, will exhibit a maximum deposition
for particles having envelope diameters as large as 9.5 .mu.m. The
increased particle size diminishes interparticle adhesion forces.
Visser, J., Powder Technology, 58: 1-10. Thus, large particle size
increases efficiency of aerosolization to the deep lung for
particles of low envelope mass density, in addition to contributing
to lower phagocytic losses.
[0123] The aerodyanamic diameter can be calculated to provide for
maximum deposition within the lungs, previously achieved by the use
of very small particles of less than about five microns in
diameter, preferably between about one and about three microns,
which are then subject to phagocytosis. Selection of particles
which have a larger diameter, but which are sufficiently light
(hence the characterization "aerodynamically light"), results in an
equivalent delivery to the lungs, but the larger size particles are
not phagocytosed. Improved delivery can be obtained by using
particles with a rough or uneven surface relative to those with a
smooth surface.
[0124] Suitable particles can be fabricated or separated, for
example by filtration or centrifugation, to provide a particle
sample with a preselected size distribution. For example, greater
than about 30%, 50%, 70%, or 80% of the particles in a sample can
have a diameter within a selected range of at least about 5 .mu.m.
The selected range within which a certain percentage of the
particles must fall may be for example, between about 5 and about
30 .mu.m, or optimally between about 5 and about 15 .mu.m. In one
preferred embodiment, at least a portion of the particles have a
diameter between about 9 and about 11 .mu.m. Optionally, the
particle sample also can be fabricated wherein at least about 90%,
or optionally about 95% or about 99%, have a diameter within the
selected range. The presence of the higher proportion of the
aerodynamically light, larger diameter particles in the particle
sample enhances the delivery of therapeutic or diagnostic agents
incorporated therein to the deep lung. Large diameter particles
generally mean particles having a median geometric diameter of at
least about 5 .mu.m.
[0125] The particles can be prepared by spray drying. For example,
a spray drying mixture, also referred to herein as "feed solution"
or "feed mixture", which includes the bioactive agent and one or
more charged lipids having a charge opposite to that of the active
agent upon association are fed to a spray dryer.
[0126] For example, when employing a protein active agent, the
agent may be dissolved in a buffer system above or below the pI of
the agent. Specifically, insulin for example may be dissolved in an
aqueous buffer system (e.g., citrate, phosphate, acetate, etc.) or
in 0.01 N HCl. The pH of the resultant solution then can be
adjusted to a desired value using an appropriate base solution
(e.g., 1 N NaOH). In one preferred embodiment, the pH may be
adjusted to about pH 7.4. At this pH insulin molecules have a net
negative charge (pI=5.5). In another embodiment, the pH may be
adjusted to about pH 4.0. At this pH insulin molecules have a net
positive charge (pI=5.5). Typically the cationic phospholipid is
dissolved in an organic solvent or combination of solvents. The two
solutions are then mixed together and the resulting mixture is
spray dried.
[0127] For a small molecule active agent, the agent may be
dissolved in a buffer system above or below the pKa of the
ionizable group(s). Specifically, albuterol sulfate or estrone
sulfate, for example, can be dissolved in an aqueous buffer system
(e.g., citrate, phosphate, acetate, etc.) or in sterile water for
irrigation. The pH of the resultant solution then can be adjusted
to a desired value using an appropriate acid or base solution. If
the pH is adjusted to about pH 3 to about pH 8 range, estrone
sulfate will possess one negative charge per molecule and albuterol
sulfate will possess one positive charge per molecule. Therefore,
charge interaction can be engineered by the choice of an
appropriate phospholipid. Typically the negatively charged or the
positively charged phospholipid is dissolved in an organic solvent
or combination of solvents and the two solutions are then mixed
together and the resulting mixture is spray dried.
[0128] Suitable organic solvents that can be present in the mixture
being spray dried include, but are not limited to, alcohols for
example, ethanol, methanol, propanol, isopropanol, butanols, and
others. Other organic solvents include, but are not limited to,
perfluorocarbons, dichloromethane, chloroform, ether, ethyl
acetate, methyl tert-butyl ether and others. Aqueous solvents that
can be present in the feed mixture include water and buffered
solutions. Both organic and aqueous solvents can be present in the
spray-drying mixture fed to the spray dryer. In one embodiment, an
ethanol water solvent is preferred with the ethanol:water ratio
ranging from about 50:50 to about 90:10. The mixture can have a,
acidic or alkaline pH. Optionally, a pH buffer can be included.
Preferably, the pH can range from about 3 to about 10.
[0129] The total amount of solvent or solvents being employed in
the mixture being spray dried generally is greater than 99 weight
percent. The amount of solids (drug, charged lipid and other
ingredients) present in the mixture being spray dried generally is
less than about 1.0 weight percent. Preferably, the amount of
solids in the mixture being spray dried ranges from about 0.05% to
about 0.5% by weight.
[0130] Using a mixture which includes an organic and an aqueous
solvent in the spray drying process allows for the combination of
hydrophilic and hydrophobic components, while not requiring the
formation of liposomes or other structures or complexes to
facilitate solubilization of the combination of such components
within the particles.
[0131] Suitable spray-drying techniques are described, for example,
by K. Masters in "Spray Drying Handbook", John Wiley & Sons,
New York, 1984. Generally, during spray-drying, heat from a hot gas
such as heated air or nitrogen is used to evaporate the solvent
from droplets formed by atomizing a continuous liquid feed. Other
spray-drying techniques are well known to those skilled in the art.
In a preferred embodiment, a rotary atomizer is employed. An
example of a suitable spray dryer using rotary atomization includes
the Mobile Minor spray dryer, manufactured by Niro, Denmark. The
hot gas can be, for example, air, nitrogen or argon.
[0132] Preferably, the particles of the invention are obtained by
spray drying using an inlet temperature between about 100.degree.
C. and about 400.degree. C. and an outlet temperature between about
50.degree. C. and about 130.degree. C.
[0133] The spray dried particles can be fabricated with a rough
surface texture to reduce particle agglomeration and improve
flowability of the powder. The spray-dried particle can be
fabricated with features which enhance aerosolization via dry
powder inhaler devices, and lead to lower deposition in the mouth,
throat and inhaler device.
[0134] The particles of the invention can be employed in
compositions suitable for drug delivery via the pulmonary system.
For example, such compositions can include the particles and a
pharmaceutically acceptable carrier for administration to a
patient, preferably for administration via inhalation. The
particles can be co-delivered with larger carrier particles, not
including a therapeutic agent, the latter possessing mass median
diameters for example in the range between about 50 .mu.m and about
100 1m. The particles can be administered alone or in any
appropriate pharmaceutically acceptable carrier, such as a liquid,
for example saline, or a powder, for administration to the
respiratory system.
[0135] Particles including a medicament, for example one or more of
the drugs listed above, are administered to the respiratory tract
of a patient in need of treatment, prophylaxis or diagnosis.
Administration of particles to the respiratory system can be by
means such as known in the art. For example, particles are
delivered from an inhalation device. In a preferred embodiment,
particles are administered via a dry powder inhaler (DPI).
Metered-dose-inhalers (MDI), nebulizers or instillation techniques
also can be employed.
[0136] Various suitable devices and methods of inhalation which can
be used to administer particles to a patient's respiratory tract
are known in the art. For example, suitable inhalers are described
in U.S. Pat. No. 4,069,819, issued Aug. 5, 1976 to Valentini, et
al., U.S. Pat. No.4,995,385 issued Feb. 26, 1991 to Valentini, et
al., and U.S. Pat. No. 5,997,848 issued Dec. 7, 1999 to Patton, et
al. Various suitable devices and methods of inhalation which can be
used to administer particles to a patient's respiratory tract are
known in the art. For example, suitable inhalers are described in
U.S. Pat. Nos. 4,995,385, and 4,069,819 issued to Valentini, et
al., U.S. Pat. No. 5,997,848 issued to Patton. Other examples
include, but are not limited to, the Spinhaler.RTM. (Fisons,
Loughborough, U.K.), Rotahaler(t (Glaxo-Wellcome, Research Triangle
Technology Park, N.C.), FlowCaps.RTM. (Hovione, Loures, Portugal),
Inhalator( (Boehringer-Ingelheim, Germany), and the Aerolizer.RTM.
(Novartis, Switzerland), the diskhaler (Glaxo-Wellcome, RTP, NC)
and others, such as known to those skilled in the art. Preferably,
the particles are administered as a dry powder via a dry powder
inhaler.
[0137] Preferably, particles administered to the respiratory tract
travel through the upper airways (oropharynx and larynx), the lower
airways which include the trachea followed by bifurcations into the
bronchi and bronchioli and through the terminal bronchioli which in
turn divide into respiratory bronchioli leading then to the
ultimate respiratory zone, the alveoli or the deep lung. In a
preferred embodiment of the invention, most of the mass of
particles deposits in the deep lung. In another embodiment of the
invention, delivery is primarily to the central airways. Delivery
to the upper airways can also be obtained.
[0138] In one embodiment of the invention, delivery to the
pulmonary system of particles is in a single, breath-actuated step,
as described in U.S. patent application, High Efficient Delivery of
a Large Therapeutic Mass Aerosol, application Ser. No. 09/591,307,
filed Jun. 9, 2000, which is incorporated herein by reference in
its entirety. In another embodiment of the invention, at least 50%
of the mass of the particles stored in the inhaler receptacle is
delivered to a subject's respiratory system in a single,
breath-activated step. In a further embodiment, at least 5
milligrams and preferably at least 10 milligrams of a medicament is
delivered by administering, in a single breath, to a subject's
respiratory tract particles enclosed in the receptacle. Amounts as
high as 15, 20, 25, 30, 35, 40 and 50 milligrams can be
delivered.
[0139] As used herein, the term "effective amount" means the amount
needed to achieve the desired therapeutic or diagnostic effect or
efficacy. The actual effective amounts of drug can vary according
to the specific drug or combination thereof being utilized, the
particular composition formulated, the mode of administration, and
the age, weight, condition of the patient, and severity of the
symptoms or condition being treated. Dosages for a particular
patient can be determined by one of ordinary skill in the art using
conventional considerations, (e.g. by means of an appropriate,
conventional pharmacological protocol). For example, effective
amounts of albuterol sulfate range from about 100 micrograms
(.mu.g) to about 10 milligrams (mg).
[0140] Aerosol dosage, formulations and delivery systems also may
be selected for a particular therapeutic application, as described,
for example, in Gonda, I. "Aerosols for delivery of therapeutic and
diagnostic agents to the respiratory tract," in Critical Reviews in
Therapeutic Drug Carrier Systems, 6: 273-313, 1990; and in Moren,
"Aerosol dosage forms and formulations," in: Aerosols in Medicine.
Principles, Diagnosis and Therapy, Moren, et al., Eds, Esevier,
Amsterdam, 1985.
[0141] Drug release rates can be described in terms of release
constants. The first order release constant can be expressed using
the following equations:
M.sub.(t)=M.sub.(.infin.)*(1-e.sup.-k*t) (1)
[0142] Where k is the first order release constant. M.sub.(.infin.)
is the total mass of drug in the drug delivery system, e.g. the dry
powder, and M.sub.(t) is the amount of drug mass released from dry
powders at time t.
[0143] Equations (1) may be expressed either in amount (i.e., mass)
of drug released or concentration of drug released in a specified
volume of release medium. For example, Equation (1) may be
expressed as:
C.sub.(t)=C.sub.(.infin.)*(1-e.sup.-k*t)
[0144] or
Release.sub.(t)=Release.sub.(.infin.)*(1-e.sup.-k*t) (2)
[0145] Where k is the first order release constant. C.sub.(.infin.)
is the maximum theoretical concentration of drug in the release
medium, and C.sub.(t) is the concentration of dmg being released
from dry powders to the release medium at time t.
[0146] Drug release rates in terms of first order release constant
can be calculated using the following equations:
k=-1n (M.sub.(.infin.)M.sub.(t))/M.sub.(.infin.)/t (3)
[0147] The release constants presented in Tables 4 and 8 employ
equation (2).
[0148] As used herein, the term "a" or "an" refers to one or
more.
[0149] The term "nominal dose" as used herein, refers to the total
mass of bioactive agent which is present in the mass of particles
targeted for administration and represents the maximum amount of
bioactive agent available for administration.
EXEMPLIFICATION
[0150] MATERIALS
[0151] Humulin L (human insulin zinc suspension) was obtained from
Lilly (100 U/mL)
[0152] MASS MEDIAN AERODYNAMIC DIAMETER-MMAD (.mu.m)
[0153] The mass median aerodynamic diameter was determined using an
Aerosizer/Aerodisperser (Amherst Process Instrument, Amherst,
Mass.). Approximately 2 mg of powder formulation was introduced
into the Aerodisperser and the aerodynamic size was determined by
time of flight measurements.
[0154] VOLUME MEDIAN GEOMETRIC DIAMETER-VMGD (.mu.m)
[0155] The volume median geometric diameter was measured using a
RODOS dry powder disperser (Sympatec, Princeton, N.J.) in
conjunction with a HELOS laser diffractometer (Sympatec). Powder
was introduced into the RODOS inlet and aerosolized by shear forces
generated by a compressed air stream regulated at 2 bar. The
aerosol cloud was subsequently drawn into the measuring zone of the
HELOS, where it scattered light from a laser beam and produced a
fraunhofer diffraction pattern used to infer the particle size
distribution and determine the median value.
[0156] Where noted, the volume median geometric diameter was
determined using a Coulter Multisizer II. Approximately 5-10 mg
powder formulation was added to 50 mL isoton II solution until the
coincidence of particles was between 5 and 8%.
[0157] DETERMINATION OF PLASMA INSULIN LEVELS
[0158] Quantification of insulin in rat plasma was performed using
a human insulin specific RIA kit (Linco Research, Inc., St.
Charles, Mo., catalog #HI-14K). The assay shows less than 0.1%
cross reactivity with rat insulin. The assay kit procedure was
modified to accommodate the low plasma volumes obtained from rats,
and had a sensitivity of approximately 5.mu.U/mL.
[0159] DETERMINATION OF ESTRONE --SULFATE PLASMA LEVELS
[0160] Quantification of estrone-sulfate in rat plasma was
performed using an estrone-sulfate RIA kit (Diagnostic Systems
Laboratories, Inc., Webster, Tex., catalog #DSL-C5400). The assay
kit procedure was modified to accommodate the low plasma volumes
obtained from rats and to correct for influence of the human serum
standard matrix, and had a sensitivity of approximately 0.025
ng/mL.
[0161] PREPARATION OF INSULIN FORMULATIONS
[0162] The powder formulations listed in Table 1 were prepared as
follows. Pre-spray drying solutions were prepared by dissolving the
lipid in ethanol and the insulin, leucine, and/or sodium citrate in
water. The ethanol solution was then mixed with the water solution
at a ratio of 60/40 ethanol water. Final total solute concentration
of the solution used for spray drying varied from 1 g/L to 3 g/L.
As an example, the DPPC/citrate/insulin (60/10/30) spray drying
solution was prepared by dissolving 600 mg DPPC in 600 mL of
ethanol, dissolving 100 mg of sodium citrate and 300 mg of insulin
in 400 mL of water and then mixing the two solutions to yield one
liter of cosolvent with a total solute concentration of 1 g/L
(w/v). Higher solute concentrations of 3 g/L (w/v) were prepared by
dissolving three times more of each solute in the same volumes of
ethanol and water.
[0163] The solution was then used to produce dry powders. A Nitro
Atomizer Portable Spray Dryer (Niro, Inc., Columbus, Md.) was used.
Compressed air with variable pressure (1 to 5 bar) ran a rotary
atomizer (2,000 to 30,000 rpm) located above the dryer. Liquid feed
with varying rate (20 to 66 mL/min) was pumped continuously by an
electronic metering pump (LMI, Model #A151-192s) to the atomizer.
Both the inlet and outlet temperatures were measured. The inlet
temperature was controlled manually; it could be varied between
100.degree. C. and 400.degree. C. and was established at 100, 110,
150, 175 or 200.degree. C., with a limit of control of 5.degree. C.
The outlet temperature was determined by the inlet temperature and
such factors as the gas and liquid feed rates (it varied between
50.degree. C. and 130.degree. C.). A container was tightly attached
to the cyclone for collecting the powder product.
2TABLE 1 POWDER COMPOSITION (%) FORMULATION NUMBER DPePC DSePC DPPG
DPPC Leucine Citrate Insulin 1.dagger. 70 10 20 2 70 20 10 3 70 10
20 4 50 50 5.dagger-dbl. 40 10 50 6 70 10 20 7 50 50 8 54.5 45.5 9
50 10 40 10 70 10 2 11 70 8 2 20 12.dagger. 40 10 50 13.dagger. 60
10 30 13A.dagger. 60 10 30 14.dagger-dbl. 70 20 15.dagger. 70 20 10
.dagger.Lots # 4-xxx-201002 (#1) , 4-XXX-201 065 (#12), 04-00024
(#13), 4-xxx-114068C (#13A) and 4-xxx-167113 (#15), which contain
the lipid DPPC, serve as negative controls. .dagger-dbl.Powder
formulation #5 was spray dried at pH = 4.0. .dagger-dbl.Powder
formulation #14 was spray dried at pH = 7.4.
[0164] The physical characteristic of the insulin containing
powders is set forth in Table 2. The MMAD and VMGD were determined
as detailed above.
3TABLE 2 COMPOSITIONS MMAD VMGD Density Formulations (% WEIGHT
BASIS) (.mu.m) .sctn. (.mu.m) .paragraph. (g/cc) .dagger-dbl.
Humulin R -- -- -- -- Humulin L -- -- -- -- Humulin U -- -- -- -- 1
DPPC/Leu/Insulin (Sigma) = 70/10/20 2.6 13.4 0.038 2 DSePC
(Avanti)/Leu/Insulin (Sigma) = 3.3 10.0 0.109 70/10/20 3 DSePC
(Avanti)/Leu/Insulin (Sigma) = 3.4 13.6 0.063 70/10/20 4 DPePC
(Avanti)/Insulin (Sigma) = 50/50 3.2 15.3 0.044 5 DPPG/Sodium
Citrate/Insulin = 40/10/50 3.9 11.6 0.113 6 DPePC
(Genzyme)/Leu/Insulin (BioBras) = 2.6 9.1 0.082 70/10/20 7 DPePC
(Avanti)/Insulin (BioBras) = 50/50 2.8 11.4 0.060 8 DPePC
(Genzyme)/Insulin (BioBras) = 2.8 12.6 0.049 54.5/45.5 9 DPePC
(Genzyme)/Leu/Insulin (BioBras) = 2.2 8.4 0.069 50/10/40 10 DPePC
(Avanti)/Leu/Insulin (BioBras) = 3.7 15.5 0.057 70/10/20 11 DPePC
(Avanti)/Leu/Sodium Citrate/ 2.6 15.3 0.029 Insulin (BioBras) =
70/8/2/20 12 DPPC/Sodium Citrate/Insulin = 40/10/50 3.5 11.6 0.091
13 DPPC/Insulin/Sodium Citrate = 60/30/10 1.9 8.0 0.056 .dagger.
Used as a control formulation for comparison in either in vitro or
in vivo studies. .sctn. Mass median aerodynamic diameter
.paragraph. Volumetric median geometric diameter at 2 bar pressure
.dagger-dbl. Determined using d.sub.aer = d.sub.g{square
root}.rho.
[0165] The data presented in Table 2 showing the physical
characteristics of the formulations comprising insulin are
predictive of the respirability of the formulations. That is, as
discussed above the large geometric diameters, small aerodynamic
diameters and low densities possessed by the powder prepared as
described herein render the particles respirable.
[0166] IN VIVO INSULIN EXPERIMENTS
[0167] The following experiment was performed to determine the rate
and extent of insulin absorption into the blood stream of rats
following pulmonary administration of dry powder formulations
comprising insulin to rats.
[0168] The nominal insulin dose administered was 100 .mu.g per rat.
To achieve the nominal doses, the total weight of powder
administered per rat ranged from 0.2 mg to 1 mg, depending on
percent composition of each powder. Male Sprague-Dawley rats were
obtained from Taconic Farms (Germantown, N.Y.). At the time of use,
the animals weighed 386 g in average (+5 g S.E.M.). The animals
were allowed free access to food and water.
[0169] The powders were delivered to the lungs using an insufflator
device for rats (PennCentury, Philadelphia, Pa.). The powder amount
was transferred into the insufflator sample chamber. The delivery
tube of the insufflator was then inserted through the mouth into
the trachea and advanced until the tip of the tube was about a
centimeter from the carina (first bifurcation). The volume of air
used to deliver the powder from the insufflator sample chamber was
3 mL, delivered from a 10 mL syringe. In order to maximize powder
delivery to the rat, the syringe was recharged and discharged two
more times for a total of three air discharges per powder dose.
[0170] The injectable insulin formulation Humulin L was
administered via subcutaneous injection, with an injection volume
of 7.2 .mu.L for a nominal dose of 25kg insulin. Catheters were
placed into the jugular veins of the rats the day prior to dosing.
At sampling times, blood samples were drawn from the jugular vein
catheters and immediately transferred to EDTA coated tubes.
Sampling times were 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hrs. after
powder administration. In some cases an additional sampling time
(12 hrs.) was included, and/or the 24 hr. time point omitted. After
centrifugation, plasma was collected from the blood samples. Plasma
samples were stored at 4.degree. C. if analysis was performed
within 24 hours or at -75.degree. C. if analysis would occur later
than 24 hours after collection. The plasma insulin concentration
was determined as described above.
[0171] Table 3 contains the insulin plasma levels quantified using
the assay described above.
4TABLE 3 PLASMA INSULIN CONCENTRATION (.mu.U/mL) .+-. S.E.M. Time
Humlin (hrs) 1 2 3 4 5 6 13A 14 L 15 0 5.0 .+-. 5.2 .+-. 5.0 .+-.
5.0 .+-. 5.3 .+-. 5.7 .+-. 5.0 .+-. 5.0 5.0 5.0 .+-. 0.0 0.2 0.0
0.0 0.2 0.7 0.0 0.0 0.0 0.0 0.25 1256.4 .+-. 61.6 .+-. 98.5 .+-.
518.2 .+-. 240.8 .+-. 206.8 .+-. 1097.7 .+-. 933.9 .+-. 269.1 .+-.
1101.9 .+-. 144.3 22.5 25.3 179.2 67.6 35.1 247.5 259.7 82.8 258.9
0.5 1335.8 .+-. 85.2 .+-. 136.7 .+-. 516.8 .+-. 326.2 .+-. 177.3
.+-. 893.5 .+-. 544.9 .+-. 459.9 .+-. 1005.4 .+-. 81.9 21.7 37.6
190.9 166.9 7.8 177.0 221.1 91.6 263.9 1 859.0 .+-. 85.4 .+-. 173.0
.+-. 497.0 .+-. 157.3 .+-. 170.5 .+-. 582.5 .+-. 229.6 .+-. 764.7
.+-. 387.5 .+-. 199.4 17.6 28.8 93.9 52.5 32.9 286.3 74.4 178.8
143.9 2 648.6 .+-. 94.8 .+-. 158.3 .+-. 496.5 .+-. 167.7 .+-. 182.2
.+-. 208.5 .+-. 129.8 .+-. 204.4 .+-. 343.8 171.1 25.0 39.1 104.9
70.5 75.0 78.3 45.7 36.7 95.3 4 277.6 .+-. 52.5 .+-. 98.0 .+-.
343.8 .+-. 144.8 .+-. 170.2 .+-. 34.9 .+-. 41.9 .+-. 32.1 .+-.
170.6 .+-. 86.8 9.1 24.3 66.7 43.8 56.3 5.4 28.7 22.6 79.9 6 104.0
.+-. 33.0 .+-. 58.7 .+-. 251.2 .+-. 95.7 .+-. 159.5 .+-. 12.3 .+-.
9.0 .+-. 11.1 .+-. 15.4 .+-. 43.1 10.7 4.1 68.4 27.3 43.4 2.4 2.9
7.5 4.5 8 54.4 .+-. 30.2 42.5 .+-. 63.2 .+-. 52.5 .+-. 94.8 .+-.
5.2 .+-. 5.0 .+-. 5.5 .+-. 6.5 .+-. 34.7 8.1 17.8 16.5 13.7 23.5
0.1 0.0 2.1 0.6 12 17.2 .+-. 6.5 24 5.0 .+-. 5.5 .+-. 0.0 0.3 n 5 5
6 6 6 6 8
[0172] The in vivo release data of Table 3 show that powder
formulations comprising insulin and positively charged lipids
(DPePC and DSePC) have significantly lower initial burst of insulin
than that seen with powder formulations comprising insulin and the
lipid DPPC (Formulations 1 and 13) and sustained elevated levels at
6 to 8 hours. FIG. 1 sets forth the release profile for insulin
from Formulations 2, 3, 6 and 15.
[0173] In addition, the use of charged lipids having a charge which
is the same of the active at neutral pH, can also be employed
provided that the preparation of the spray dried formulation is
conducted at a pH where the lipid and active agent possess overall
charges which are opposite and are therefore capable of charge
interaction. See, for example, Formulations 5 which employs the
negatively charged lipid DPPG. Formulation 5 was prepared and spray
dried at a pH of about 4.0. At this pH, DPPG is negatively charged
and insulin becomes positively charged (pI=5.5) thereby providing
for a charge interaction to occur. However, when the DPPG and
insulin are prepared and spray dried at pH=7.4 where both the DPPG
and insulin possess an overall negative charge, Formulation 14, the
proper environment for charge interaction to occur is not provided.
It is noted that Formulation 5 showed a significantly lower initial
burst of insulin (240.8.+-.67.6 .mu.U/mL) as compared to
Formulation 14 (933.9.+-.259.7 .mu.U/mL) with higher sustained
levels at 6 to 8 hours post treatment. FIG. 2 shows a comparison of
the in vivo release profile for Formulations 5, 14 and 13A ( lipid,
DPPC).
[0174] IN VITRO ANALYSIS OF INSULIN-CONTAINING FORMULATIONS
[0175] The in vitro release of insulin containing dry powder
formulations was performed as described by Gietz et al. in Eur. J
Pharm. Biopharm., 45:259-264 (1998), with several modifications.
Briefly, in 20 mL screw-capped glass scintillation vials about 10
mg of each dry powder formulation was mixed with 4 mL of warm
(37.degree. C.) 1% agarose solution using polystyrene stir bars.
The resulting mixture was then distributed in 1 mL aliquots to a
set of five fresh 20 mL glass scintillation vials. The dispersion
of dry powder in agarose was cooled in an ambient temperature
dessicator box protected from light to allow gelling. Release
studies were conducted on an orbital shaker at about 37.degree. C.
At predetermined time points, previous release medium (1.5 mL) was
removed and fresh release medium (1.5 mL) was added to each vial.
Typical time points for these studies were 5 minutes, 1, 2, 4, 6
and 24 hours. The release medium used consisted of 20 mM
4-(2-hydroxyethyl)-pipe- razine-1-ethanesulfonic acid (HEPES), 138
mM NaCl, 0.5% Pluronic (Synperonic PE/F68; to prevent insulin
filbrillation in the release medium); pH 7.4. A Pierce (Rockford,
Ill.) protein assay kit (See Anal Biochem, 150:76-85 (1985)) using
known concentrations of insulin standard was used to monitor
insulin concentrations in the release medium.
[0176] Table 4 summarizes the in vitro release data and first order
release constants for powder formulations of Table 1 comprising
insulin.
5TABLE 4 Maximum .dagger-dbl. Powder Cumulative Cumulative Release
Formula- % Insulin % Insulin at 24 hr First Order .dagger-dbl. tion
Released Released (Cumulative Release Number at 6 hr at 24 hr %)
Constants (hr.sup.-1) Humulin 92.67 .+-. 0.36 94.88 .+-. 0.22 91.6
.+-. 5.42 1.0105 .+-. 0.2602 R Humulin 19.43 .+-. 0.41 29.71 .+-.
0.28 36.7 .+-. 2.56 0.0924 .+-. 0.0183 L Humulin 5.17 .+-. 0.18
12.65 .+-. 0.43 46.6 .+-. 27.0 0.0158 .+-. 0.0127 U 2 31.50 .+-.
0.33 47.52 .+-. 0.43 48.22 .+-. 0.46 0.1749 .+-. 0.0038 3 26.34
.+-. 0.71 37.49 0.27 38.08 .+-. 0.72 0.1837 .+-. 0.0079 4 24.66
.+-. 0.20 31.58 .+-. 0.33 31.51 .+-. 1.14 0.2457 .+-. 0.0214 5
29.75 .+-. 0.17 35.28 .+-. 0.19 33.66 .+-. 2.48 0.4130 .+-. 0.0878
6 17.04 .+-. 0.71 24.71 .+-. 0.81 25.19 .+-. 0.52 0.1767 .+-.
0.0083 7 13.53 .+-. 0.19 19.12 .+-. 0.40 19.51 .+-. 0.48 0.1788
.+-. 0.0101 8 13.97 .+-. 0.27 17.81 .+-. 0.46 17.84 .+-. 0.55
0.2419 .+-. 0.0178 9 17.47 .+-. 0.38 22.17 .+-. 0.22 21.97 .+-.
0.64 0.2734 .+-. 0.0196 10 25.96 .+-. 0.31 34.94 .+-. 0.31 35.43
.+-. 0.90 0.2051 .+-. 0.0120 11 34.33 .+-. 0.51 47.21 .+-. 0.47
47.81 .+-. 0.85 0.1994 .+-. 0.0082 12 61.78 .+-. 0.33 68.56 .+-.
0.23 65.20 .+-. 3.34 0.5759 .+-. 0.0988 13 78.47 .+-. 0.40 85.75
.+-. 0.63 84.9 .+-. 3.81 0.5232 .+-. 0.0861 .dagger-dbl.
Release.sub.(t) = Release.sub.(mf)*(1 - e.sup.-k*t) .dagger. Used
as a control formulation.
[0177] The data presented in Table 4 show that for insulin
containing powder formulations employing the positively charged
lipid DPEPC (Formulations 4 and 6-11) and DSePC (Formulations 2 and
3), first order release constants similar to that observed with the
slow release injectable insulin formulation, Humulin L, can be
achieved. Further, the first order release constants of these same
formulations is significantly lower than that observed with the
fast release injectable insulin formulation, Humulin R. As such,
sustained release dry powder insulin formulations having varying
compositions of positively charged lipid can be formulated.
[0178] PREPARATION OF ESTRONE SULFATE-CONTAINING POWDER
FORMULATIONS
[0179] The estrone sulfate powder formulations listed in Table were
prepared as follows. Pre-spray drying solutions were prepared by
dissolving the lipin in ethanol and estrone sulfate and leucine in
water. The ethanol solution was then mixed with the water solution
at a ration 70/30 ethanol/water. Final total solute concentration
of the solution used for spray drying varied from 1 g/L to 3 g/L.
As an example, the DPePC/leucine/estrone sulfate (76/20/4) spray
drying solution was prepared by dissolving 760 mg of DPePC in 700
mL of ethanol, dissolving 200 mg of leucine and 40 mg of estrone
sulfate in 300 mL of water and then mixing the two solutions to
yield one liter of cosolvent with a total solute concentration of 1
g/L (w/v). Higher solute concentrations of, for example, 3 g/L (w/)
were prepared by dissolving three times more of each solute in the
same volumes of ethanol and water
[0180] The mixture was spray dried following the procedure
described above for the insulin containing powder formulation.
During spray drying, the feed rate was about 50 mL/min, the inlet
temperature ranged from about 110.degree. C. to about 120.degree.
C., and the outlet temperature was about 52.degree. C.
[0181] The physical characteristic of the estrone sulfate
containing powders is set forth in Table 5. The MMAD and VMGD were
determined as detailed above.
6TABLE 5 POWDER COMPOSITIONS DEN- FORMULATION (% WEIGHT MMAD VMGD
SITY NUMBER BASIS) (.mu.m) .sctn. (.mu.m) .paragraph. (g/cc)
.dagger-dbl. 16 DPePC 5.9 16.0 0.136 (Avanti)/Leucine/ Estrone
Sulfate (sodium salt) = 76/20/4 17 DPPC/Leucine/ 3.7 12.7 # 0.085
Estrone Sulfate (sodium salt) = 76/20/4 .dagger. Used as a control
for comparison for in vivo studies .sctn. Mass median aerodynamic
diameter .paragraph. Volumetric median geometric diameter at 2 bar
pressure # Measured using Coulter Multisizer .dagger-dbl.
Determined using d.sub.aer = d.sub.g{square root}.rho.
[0182] The data presented in Table 5 showing the physical
characteristics of the formulations comprising estrone sulfate are
predictive of the respirability of the formulations. That is, as
discussed above the large geometric diameters, small aerodynamic
diameters and low densities possessed by the powder prepared as
described herein render the particles respirable.
[0183] IN VIVO EXPERIMENTS-ESTRONE SULFATE CONTAINING POWDERS
[0184] The following experiment was performed to determine the rate
and extent of estrone sulfate absorption into the blood stream of
rats following pulmonary administration of dry powder formulations
comprising estrone sulfate.
[0185] The nominal estrone-sulfate dose administered was 40 .mu.g
per rat, in 1 mg of powder. Male Sprague-Dawley rats were obtained
from Taconic Farms (Germantown, N.Y.). At the time of use, the
animals weighed an average of 415 g (.+-.10 g S.E.M.). The animals
were allowed free access to food and water.
[0186] The powders were delivered to the lungs using an insufflator
device for rats (PennCentury, Philadelphia, Pa.). The powder amount
was transferred into the insufflator sample chamber. The delivery
tube of the insufflator was then inserted through the mouth into
the trachea and advanced until the tip of the tube was about a
centimeter from the carina (first bifurcation). The volume of air
used to deliver the powder from the insufflator sample chamber was
3 mL, delivered from a 10 mL syringe. In order to maximize powder
delivery to the rat, the syringe was recharged and discharged two
more times for a total of three air discharges per powder dose.
[0187] Catheters were placed into the jugular veins of the rats the
day prior to dosing. At sampling times, blood samples were drawn
from the jugular vein catheters and immediately transferred to EDTA
coated tubes. Sampling times were 0, 0.25, 0.5, 1, 2, 4, and 6
hours after powder administration. After centrifugation, plasma was
collected from the blood samples. Plasma samples were stored at
4.degree. C. if analysis was performed within 24 hours or at
-75.degree. C. if analysis would occur later than 24 hours after
collection.
[0188] Table 6 contains the estrone sulfate plasma levels
quantified using the assay described above.
7TABLE 6 PLASMA ESTRONE-SULFATE CONCENTRATION (ng/mL) .+-. S.E.M.
TIME (HRS) FORMULATION 16 FORMULATION 17 0 0.07 .+-. 0.02 0.08 .+-.
0.05 0.25 12.07 .+-. 1.96 22.26 .+-. 8.96 0.5 18.88 .+-. 2.21 23.39
.+-. 12.72 1 12.20 .+-. 3.31 10.59 .+-. 0.61 2 4.65 .+-. 0.77 3.45
.+-. 0.63 4 4.02 .+-. 1.42 0.86 .+-. 0.10 6 1.49 .+-. 0.48 0.33
.+-. 0.12 n 4 3
[0189] The results presented in Table 6 and depicted graphically in
FIG. 3, show that the formulation comprising DPePC (overall
positive charge) and estrone sulfate (negative charge) exhibited
sustained release of estrone sulfate when compared to the
formulation employing the lipid DPPC (no overall net charge) and
estrone sulfate. Specifically, at six hours post administration,
the plasma level of estrone sulfate for the DPePC containing
formulation was 1.49.+-.0.48 ng/mL as compared to 0.33.+-.0.12
ng/mL for the DPPC containing formulation.
[0190] PREPARATION OF ALBUTEROL-CONTAINING POWDER FORMULATIONS
[0191] The albuterol sulfate powder formulations listed in Table 7,
were prepared as follows. Pre-spray drying solutions were prepared
by dissolving the lipid in ethanol and albuterol sulfate and
leucine in water. The ethanol solution was then mixed with the
water solution at a ratio of 70/30 ethanol/water. Final total
solute concentration of the solution used for spray drying varied
from 1 g/L to 3 g/L. As an example, the DPPC/leucine/albuterol
sulfate (76/16/8) spray drying solution was prepared by dissolving
760 mg of DPPC in 700 mL of ethanol, dissolving 160 mg leucine and
870 mg of albuterol sulfate in 300 mL water and then mixing the two
solutions to yield one liter of cosolvent with a total solute
concentration of 1 g/L (w/v). Higher solute concentrations of 3 g/L
(w/v) were prepared by dissolving three times more of each solute
in the same volumes of ethnaol and water. The solution was
spray-dried as described above for the insulin containing
formulation. Specifically, the inlet temperature was from about
110.degree. C. to about 140.degree. C., and the outlet temperature
ranged from about 45-57.degree. C.
[0192] The physical characteristics of the albuterol sulfate
containing powders is set forth in Table 7. The MMAD and VMGD were
determined as detailed above.
8TABLE 7 POWDER COMPOSITIONS DEN- FORMULATION (% WEIGHT MMAD VMGD
SITY NUMBER BASIS) (.mu.m) .sctn. (.mu.m) .paragraph. g/cc
.dagger-dbl. 18 DSPC/Leucine/ 3.3 6.1 0.293 Albuterol Sulfate =
76/16/8 19 DSPG/Leucine/ 4.1 6.4 0.410 Albuterol Sulfate = 76/16/8
20 DPPC/Leucine/ 2.8 12.0 0.054 Albuterol Sulfate = 76/23/1 21
DPPG/Leucine/ 3.3 7.1 0.216 Albuterol Sulfate = 76/16/8 .dagger.
Used as a control for comparison in either in vitro or in vivo
studies .sctn. Mass median aerodynamic diameter .paragraph.
Volumetric median geometric diameter at 2 bar pressure .dagger-dbl.
Determined using d.sub.aer = d.sub.g{square root}.rho.
[0193] The data presented in Table 7 showing the physical
characteristics of the formulations comprising albuterol sulfate
are predictive of the respirability of the formulations. That is,
as discussed above the large geometric diameters, small aerodynamic
diameters and low densities possessed by the formulations prepared
as described herein render the formulations respirable.
[0194] IN VIVO TESTING OF ALBUTEROL SULFATE FORMULATIONS
[0195] A whole-body plethysmography method for evaluating pulmonary
function in guinea pigs was used to assess the sustained effects of
the albuterol sulfate formulations listed in Table 7.
[0196] The system used was the BUXCO whole-body unrestrained
plethysmograph system with BUXCO XA pulmonary function software
(BUXCO Electronics, Inc., Sharon, Conn.). The method was conducted
as described by Silbaugh S. A. and Mauderly, J. L., in American
Physiological Society, Vol. 84:1666-1669 (1984) and Chang, B. T.,
et al in Journal of Pharmacological and Toxicological Methods, Vol.
39(3):163-168 (1998). This method allows individual animals to be
challenged repeatedly over time with methacholine given by
nebulization. A calculated measurement of airway resistance based
on flow parameters, the enhanced pause PenH was used as a marker
for protection from methacholine-induced bronchoconstriction.
Baseline pulmonary function (airway hyperresponsiveness) values
were measured prior to any experimental treatment. Airway
hyperresponsiveness was then assessed in response to saline and
methacholine at various timepoints (2-3, 16 and 24 hours) following
administration of albuterol-sulfate formulations. Average PenH is
calculated from data collected between 4 and 9 minutes following
challenge with saline or methacholine. The percent of baseline PenH
at each timepoint is calculated for each experimental animal.
Values from animals that received the same albuterol sulfate
formulation were subsequently averaged to determine the mean group
response (.+-.standard error) at each timepoint.
[0197] The nominal dose of albuterol-sulfate administered was 50
.mu.g for the DPPG-based formulation (#21) and 25 .mu.g for the
DPPC-based formulation (#20). To achieve those nominal doses, the
total weights of powder administered were 0.625 mg and 2.5 mg,
respectively.
[0198] Male Hartley guinea pigs were obtained from Elm Hill
Breeding Labs (Chelmsford, Mass.). At the time of use, the animals
weighed an average of 363 g ).+-.5 g S.E.M.). The animals were
allowed free access to food and water. The powder amount was
transferred into the insufflator sample chamber (insufflation
device for guinea pigs, Penn Century, Philadelphia, Pa.). The
delivery tube of the insufflator was inserted through the mouth
into the trachea and advanced until the tip of the tube was about a
centimeter from the carina (first bifurcation). The volume of the
air used to deliver the powder from the insufflator sample chamber
was 3 mL, delivered from a 10 mL syringe. In order to maximize
powder delivery to the guinea pig, the syringe was recharged and
discharged two more times for a total of three air discharges per
powder dose. Methacholine challenges were performed at time points
2-3, 16 and 24 hours after administration.
[0199] FIG. 4 shows that the formulation comprising DPPG (overal
negative charge) and albuterol sulfate (overall positive charge)
provided sustained protection against methacholine-induced
bronchoconstriction when compared to the formulation comprising
DPPC (no overall net charge) and albuterol sulfate for at least 24
hours following administration.
[0200] In another experiment, as much as 200 .mu.g of albuterol
sulfate in a DPPC-based formulation did not provide prolonged
protection against induced bronchoconstriction.
[0201] IN VITRO RELEASE STUDIES-ALBUTEROL SULFATE
[0202] Controlled Release Studies of Albuterol Sulfate were
conducted using the COSTAR.TM. Brand Transwell Inserts, With
Plates, Sterile. The plates were equipped with 6 wells having an
area of 4.7cm.sup.2. The insert size was 24 mm, the pore size was
3.0 .mu.m. A predetermined amount of the powder to be tested
(approximately 10-15 mg) was placed into a HPMC Size #2 capsule.
The capsule was then placed inside an inhaler and the powder was
sprayed on the Transwell insert using an in-house vacuum system.
Formulations were run in triplicate.
[0203] After spraying, the insert was placed inside the Transwell
plate containing a volume of 1.8 mL of Phosphate Buffered Saline
(pH =7.4) which had previously been equilibrated at 37.degree. C.
for 30 minutes. The Transwell plate was hermetically sealed in
order to prevent evaporation of the buffer during the
experiment.
[0204] The Transwell Experiment was carried out in an incubator at
37.degree. C. on an orbital shaker at a speed of 100 min.sup.-1. At
specified time-points throughout the experiment, 1.8 mL of
phosphate buffered saline was removed from the Transwell plate. The
inserts were then placed into a new Transwell plate containing 1.8
mL of fresh phosphate buffered saline. Typical Transwell
experiments are conducted for 4 hours. Samples are withdrawn after
5 min., 15, min., 30, min., 1 h, 1.5 h, 2 h, 3 h, and 4 h.
[0205] The amount of albuterol sulfate in the PBS buffer sampled at
predetermined in vitro release time points was quantitated using a
RP-HPLC method with Phenomenex Luna 5.mu., C8(2), 250.times.4.6 mm
column (Torrance, CA) and VW detection at 275 nm.
[0206] Table 8 summarizes the in vitro release data and first order
release constants for the powder formulations of Table 7 comprising
albuterol sulfate. The first order release constants for the powder
formulation comprising DSPG (negatively charged) and albuterol
sulfate is about 4 time slower compared to the powder formulation
comprising DSPC (no net overall charge) and albuterol sulfate
(positive).
9TABLE 8 Cumulative First Powder % Maximum Order Formu-
Compositions Insulin Release Release lation (% weight Released at 4
hr Constants Number basis) at 4 hr (Cumulative %).dagger-dbl.
(hr.sup.-1).dagger-dbl. 18 DSPC/Leucine/ 106.21 .+-. 105.64 .+-.
0.20 29.7360 .+-. Albuterol Sulfate 1.73 0.7504 (sodium salt) =
76/16/8 19 DSPG/Leucine/ 97.44 .+-. 95.13 .+-. 1.39 7.9334 .+-.
Albuterol Sulfate 0.68 0.6877 (sodium salt) = 76/16/8
.dagger-dbl.Release.sub.(t) = Release.sub.(inf)*(1 - e.sup.-k*t)
.dagger.Used as a control formulation.
[0207] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *